William Weis

All Publications

• Multi-level force-dependent allosteric enhancement of alphaE-catenin binding to F-actin by vinculin. Journal of molecular biology Bax, N. A., Wang, A., Huang, D. L., Pokutta, S., Weis, W. I., Dunn, A. R. 2023: 167969

  Abstract

  Classical cadherins are transmembrane proteins whose extracellular domains link neighboring cells, and whose intracellular domains connect to the actin cytoskeleton via beta-catenin and alpha-catenin. The cadherin-catenin complex transmits forces that drive tissue morphogenesis and wound healing. In addition, tension-dependent changes in alphaE-catenin conformation enables it to recruit the actin-binding protein vinculin to cell-cell junctions, which contributes to junctional strengthening. How and whether multiple cadherin-complexes cooperate to reinforce cell-cell junctions in response to load remains poorly understood. Here, we used single-molecule optical trap measurements to examine how multiple cadherin-catenin complexes interact with F-actin under load, and how this interaction is influenced by the presence of vinculin. We show that force oriented toward the (-) end of the actin filament results in mean lifetimes 3-fold longer than when force was applied towards the barbed (+) end. We also measured force-dependent actin binding by a quaternary complex comprising the cadherin-catenin complex and the vinculin head region, which cannot itself bind actin. Binding lifetimes of this quaternary complex increased as additional complexes bound F-actin, but only when load was oriented toward the (-) end. In contrast, the cadherin-catenin complex alone did not show this form of cooperativity. These findings reveal multi-level, force-dependent regulation that enhances the strength of the association of multiple cadherin/catenin complexes with F-actin, conferring positive feedback that may strengthen the junction and polarize F-actin to facilitate the emergence of higher-order cytoskeletal organization.

  View details for DOI 10.1016/j.jmb.2023.167969

  View details for PubMedID 36682678

• PI(4,5)P2-stimulated positive feedback drives the recruitment of Dishevelled to Frizzled in Wnt-β-catenin signaling. Science signaling Mahoney, J. P., Bruguera, E. S., Vasishtha, M., Killingsworth, L. B., Kyaw, S., Weis, W. I. 2022; 15 (748): eabo2820

  Abstract

  In the Wnt-β-catenin pathway, Wnt binding to Frizzled (Fzd) and LRP5 or LRP6 (LRP5/6) co-receptors inhibits the degradation of the transcriptional coactivator β-catenin by recruiting the cytosolic effector Dishevelled (Dvl). Polymerization of Dvl at the plasma membrane recruits the β-catenin destruction complex, enabling the phosphorylation of LRP5/6, a key step in inhibiting β-catenin degradation. Using purified Fzd proteins reconstituted in lipid nanodiscs, we investigated the factors that promote the recruitment of Dvl to the plasma membrane. We found that the affinity of Fzd for Dvl was not affected by Wnt ligands, in contrast to other members of the GPCR superfamily for which the binding of extracellular ligands affects the affinity for downstream transducers. Instead, Fzd-Dvl binding was enhanced by increased concentration of the lipid PI(4,5)P2, which is generated by Dvl-associated lipid kinases in response to Wnt and which is required for LRP5/6 phosphorylation. Moreover, binding to Fzd did not promote Dvl DEP domain dimerization, which has been proposed to be required for signaling downstream of Fzd. Our findings suggest a positive feedback loop in which Wnt-stimulated local PI(4,5)P2 production enhances Dvl recruitment and further PI(4,5)P2 production to support Dvl polymerization, LRP5/6 phosphorylation, and β-catenin stabilization.

  View details for DOI 10.1126/scisignal.abo2820

  View details for PubMedID 35998232

• Mechanism of the cadherin-catenin F-actin catch bond interaction. eLife Wang, A., Dunn, A. R., Weis, W. I. 2022; 11

  Abstract

  Mechanotransduction at cell-cell adhesions is crucial for the structural integrity, organization, and morphogenesis of epithelia. At cell-cell junctions, ternary E-cadherin/beta-catenin/alphaE-catenin complexes sense and transmit mechanical load by binding to F-actin. The interaction with F-actin, described as a two-state catch bond, is weak in solution but is strengthened by applied force due to force-dependent transitions between weak and strong actin-binding states. Here, we provide direct evidence from optical trapping experiments that the catch bond property principally resides in the alphaE-catenin actin-binding domain (ABD). Consistent with our previously proposed model, deletion of the first helix of the five-helix ABD bundle enables stable interactions with F-actin under minimal load that are well-described by a single-state slip bond, even when alphaE-catenin is complexed with beta-catenin and E-cadherin. Our data argue for a conserved catch bond mechanism for adhesion proteins with structurally similar ABDs. We also demonstrate that a stably bound ABD strengthens load-dependent binding interactions between a neighboring complex and F-actin, but the presence of the other alphaE-catenin domains weakens this effect. These results provide mechanistic insight to the cooperative binding of the cadherin-catenin complex to F-actin, which regulate dynamic cytoskeletal linkages in epithelial tissues.

  View details for DOI 10.7554/eLife.80130

  View details for PubMedID 35913118

• Reconstitution of purified membrane protein dimers in lipid nanodiscs with defined stoichiometry and orientation using a split GFP tether. The Journal of biological chemistry Bruguera, E. S., Mahoney, J. P., Weis, W. I. 1800: 101628

  Abstract

  Many membrane proteins function as dimers or larger oligomers, including transporters, channels, certain signaling receptors and adhesion molecules. In some cases, the interactions between individual proteins may be weak and/or dependent on specific lipids, such that detergent solubilization used for biochemical and structural studies disrupts functional oligomerization. Solubilized membrane protein oligomers can be captured in lipid nanodiscs, but this is an inefficient process that can produce stoichiometrically and topologically heterogeneous preparations. Here we describe a technique to obtain purified, homogeneous membrane protein dimers in nanodiscs using a split GFP tether. Complementary split GFP tags associate to tether the co-expressed dimers and control both stoichiometry and orientation within the nanodiscs, as assessed by quantitative western blotting and negative stain electron microscopy. The split GFP tether confers several advantages over other methods: it is highly stable in solution and in SDS-PAGE, which facilitates screening of dimer expression and purification by fluorescence, and also provides a dimer-specific purification handle for use with GFP nanobody-conjugated resin. We used this method to purify a Frizzled-4 (Fzd4) homodimer and a Fzd4/low-density lipoprotein receptor-related protein 6 (LRP6) heterodimer in nanodiscs. These examples demonstrate the utility and flexibility of this method, which enables subsequent mechanistic molecular and structural studies of membrane protein pairs.

  View details for DOI 10.1016/j.jbc.2022.101628

  View details for PubMedID 35074428

• The C-terminal actin-binding domain of talin forms an asymmetric catch bond with F-actin. Proceedings of the National Academy of Sciences of the United States of America Owen, L. M., Bax, N. A., Weis, W. I., Dunn, A. R. 2022; 119 (10): e2109329119

  Abstract

  SignificanceTalin is a mechanosensitive adaptor protein that links integrins to the actin cytoskeleton at cell-extracellular matrix adhesions. Although the C-terminal actin-binding domain ABS3 of talin is required for function, it binds weakly to actin in solution. We show that ABS3 binds actin strongly only when subjected to mechanical forces comparable to those generated by the cytoskeleton. Moreover, the interaction between ABS3 and actin depends strongly on the direction of force in a manner predicted to organize actin to facilitate adhesion growth and efficient cytoskeletal force generation. These characteristics can explain how force sensing by talin helps to nucleate adhesions precisely when and where they are required to transmit force between the cytoskeleton and the extracellular matrix.

  View details for DOI 10.1073/pnas.2109329119

  View details for PubMedID 35245171

• Lattice micropatterning for cryo-electron tomography studies of cell-cell contacts. Journal of structural biology Engel, L., Vasquez, C. G., Montabana, E. A., Sow, B. M., Walkiewicz, M. P., Weis, W. I., Dunn, A. R. 2021: 107791

  Abstract

  Cryo-electron tomography is the highest resolution tool available for structural analysis of macromolecular complexes within their native cellular environments. At present, data acquisition suffers from low throughput, in part due to the low probability of positioning a cell such that the subcellular structure of interest is on a region of the electron microscopy (EM) grid that is suitable for imaging. Here, we photo-micropatterned EM grids to optimally position endothelial cells so as to enable high-throughput imaging of cell-cell contacts. Lattice micropatterned grids increased the average distance between intercellular contacts and the thicker cell nuclei such that the regions of interest were sufficiently thin for direct imaging. We observed a diverse array of membranous and cytoskeletal structures at intercellular contacts, demonstrating the utility of this technique in enhancing the rate of data acquisition for cellular cryo-electron tomography studies.

  View details for DOI 10.1016/j.jsb.2021.107791

  View details for PubMedID 34520869

• Distinct intramolecular interactions regulate autoinhibition of vinculin binding in alphaT-catenin and alphaE-catenin. The Journal of biological chemistry Heier, J. A., Pokutta, S., Dale, I. W., Kim, S. K., Hinck, A. P., Weis, W. I., Kwiatkowski, A. V. 2021: 100582

  Abstract

  alpha-Catenin binds directly to beta-catenin and connects the cadherin-catenin complex to the actin cytoskeleton. Tension regulates alpha-catenin conformation. Actomyosin-generated force stretches the middle(M)-region to relieve autoinhibition and reveal a binding site for the actin-binding protein vinculin. It is not known whether the intramolecular interactions that regulate alphaE(epithelial)-catenin binding are conserved across the alpha-catenin family. Here, we describe the biochemical properties of alphaT(testes)-catenin, an alpha-catenin isoform critical for cardiac function, and how intramolecular interactions regulate vinculin binding autoinhibition. Isothermal titration calorimetry (ITC) showed that alphaT-catenin binds the beta-catenin/N-cadherin complex with a similar low nanomolar affinity to that of alphaE-catenin. Limited proteolysis revealed that the alphaT-catenin M-region adopts a more open conformation than alphaE-catenin. The alphaT-catenin M-region binds the vinculin N-terminus with low nanomolar affinity, indicating that the isolated alphaT-catenin M-region is not autoinhibited and thereby distinct from alphaE-catenin. However, the alphaT-catenin head (N- and M-regions) binds vinculin 1000-fold more weakly (low micromolar affinity), indicating that the N-terminus regulates M-region binding to vinculin. In cells, alphaT-catenin recruitment of vinculin to cell-cell contacts requires the actin-binding domain and actomyosin-generated tension, indicating that force regulates vinculin binding. Together, our results show that the alphaT-catenin N-terminus is required to maintain M-region autoinhibition and modulate vinculin binding. We postulate that the unique molecular properties of alphaT-catenin allow it to function as a scaffold for building specific adhesion complexes.

  View details for DOI 10.1016/j.jbc.2021.100582

  View details for PubMedID 33771561

• Structural analysis of carbohydrate binding by the macrophage mannose receptor CD206. The Journal of biological chemistry Feinberg, H., Jegouzo, S. A., Lasanajak, Y., Smith, D. F., Drickamer, K., Weis, W. I., Taylor, M. E. 2021: 100368

  Abstract

  The human mannose receptor expressed on macrophages and hepatic endothelial cells scavenges released lysosomal enzymes, glycopeptide fragments of collagen, and pathogenic micro-organisms and thus reduces damage following tissue injury. The receptor binds mannose, fucose or N-acetylglucosamine (GlcNAc) residues on these targets. C-type carbohydrate-recognition domain 4 (CRD4) of the receptor contains the site for Ca2+-dependent interaction with sugars. To investigate the details of CRD4 binding, glycan array screening was used to identify oligosaccharide ligands. The strongest signals were for glycans that contain either Manalpha1-2Man constituents or fucose in various linkages. The mechanisms of binding to monosaccharides and oligosaccharide sub-structures present in many of these ligands were examined in multiple crystal structures of CRD4. Binding of mannose residues to CRD4 results primarily from interaction of the equatorial 3- and 4-OH groups with a conserved principal Ca2+ common to almost all sugar-binding C-type CRDs. In the Manalpha1-2Man complex, supplementary interactions with the reducing mannose residue explain the enhanced affinity for this disaccharide. Bound GlcNAc also interacts with the principal Ca2+ through equatorial 3- and 4-OH groups, whereas fucose residues can bind in several orientations, through either the 2- and 3-OH groups or the 3- and 4-OH groups. Secondary contacts with additional sugars in fucose-containing oligosaccharides, such as the Lewis-a trisaccharide, provide enhanced affinity for these glycans. These results explain many of the biologically important interactions of the mannose receptor with both mammalian glycoproteins and microbes such as yeast, and suggest additional classes of ligands that have not been previously identified.

  View details for DOI 10.1016/j.jbc.2021.100368

  View details for PubMedID 33545173

• A resolution record for cryoEM. Faculty reviews Ashmore, J., Carragher, B., Rosenthal, P. B., Weis, W. 1800; 10: 64

  Abstract

  Cryo electron microscopy (cryoEM) is a fast-growing technique for structure determination. Two recent papers report the first atomic resolution structure of a protein obtained by averaging images of frozen-hydrated biomolecules. They both describe maps of symmetric apoferritin assemblies, a common test specimen, in unprecedented detail. New instrument improvements, different in the two studies, have contributed better images, and image analysis can extract structural information sufficient to resolve individual atomic positions. While true atomic resolution maps will not be routine for most proteins, the studies suggest structures determined by cryoEM will continue to improve, increasing their impact on biology and medicine.

  View details for DOI 10.12703/r-01-000002

  View details for PubMedID 35088060

• Author Correction: Structural insights into mu-opioid receptor activation. Nature Huang, W., Manglik, A., Venkatakrishnan, A. J., Laeremans, T., Feinberg, E. N., Sanborn, A. L., Kato, H. E., Livingston, K. E., Thorsen, T. S., Kling, R. C., Granier, S., Gmeiner, P., Husbands, S. M., Traynor, J. R., Weis, W. I., Steyaert, J., Dror, R. O., Kobilka, B. K. 2020

  Abstract

  An amendment to this paper has been published and can be accessed via a link at the top of the paper.

  View details for DOI 10.1038/s41586-020-2542-z

  View details for PubMedID 32724208

• Limited Dishevelled/Axin oligomerization determines efficiency of Wnt/β-catenin signal transduction. eLife Kan, W. n., Enos, M. D., Korkmazhan, E. n., Muennich, S. n., Chen, D. H., Gammons, M. V., Vasishtha, M. n., Bienz, M. n., Dunn, A. R., Skiniotis, G. n., Weis, W. I. 2020; 9

  Abstract

  In Wnt/β-catenin signaling, the transcriptional coactivator β-catenin is regulated by its phosphorylation in a complex that includes the scaffold protein Axin and associated kinases. Wnt binding to its coreceptors activates the cytosolic effector Dishevelled (Dvl), leading to the recruitment of Axin and the inhibition of β-catenin phosphorylation. This process requires interaction of homologous DIX domains present in Dvl and Axin, but is mechanistically undefined. We show that Dvl DIX forms antiparallel, double-stranded oligomers in vitro, and that Dvl in cells forms oligomers typically <10 molecules at endogenous expression levels. Axin DIX (DAX) forms small single-stranded oligomers, but its self-association is stronger than that of DIX. DAX caps the ends of DIX oligomers, such that a DIX oligomer has at most four DAX binding sites. The relative affinities and stoichiometry of the DIX-DAX interaction provide a mechanism for efficient inhibition of β-catenin phosphorylation upon Axin recruitment to the Wnt receptor complex.

  View details for DOI 10.7554/eLife.55015

  View details for PubMedID 32297861

• Structural basis of αE-catenin-F-actin catch bond behavior. eLife Xu, X. P., Pokutta, S. n., Torres, M. n., Swift, M. F., Hanein, D. n., Volkmann, N. n., Weis, W. I. 2020; 9

  Abstract

  Cell-cell and cell-matrix junctions transmit mechanical forces during tissue morphogenesis and homeostasis. α-Catenin links cell-cell adhesion complexes to the actin cytoskeleton, and mechanical load strengthens its binding to F-actin in a direction-sensitive manner. Specifically, optical trap experiments revealed that force promotes a transition between weak and strong actin-bound states. Here, we describe the cryo-electron microscopy structure of the F-actin-bound αE-catenin actin-binding domain, which in solution forms a 5-helix bundle. In the actin-bound structure, the first helix of the bundle dissociates and the remaining four helices and connecting loops rearrange to form the interface with actin. Deletion of the first helix produces strong actin binding in the absence of force, suggesting that the actin-bound structure corresponds to the strong state. Our analysis explains how mechanical force applied to αE-catenin or its homolog vinculin favors the strongly bound state, and the dependence of catch bond strength on the direction of applied force.

  View details for DOI 10.7554/eLife.60878

  View details for PubMedID 32915141

• Extracellular matrix micropatterning technology for whole cell cryogenic electron microscopy studies. Journal of micromechanics and microengineering : structures, devices, and systems Engel, L., Gaietta, G., Dow, L. P., Swift, M. F., Pardon, G., Volkmann, N., Weis, W. I., Hanein, D., Pruitt, B. L. 2019; 29 (11)

  Abstract

  Cryogenic electron tomography is the highest resolution tool available for structural analysis of macromolecular organization inside cells. Micropatterning of extracellular matrix (ECM) proteins is an established in vitro cell culture technique used to control cell shape. Recent traction force microscopy studies have shown correlation between cell morphology and the regulation of force transmission. However, it remains unknown how cells sustain increased strain energy states and localized stresses at the supramolecular level. Here, we report a technology to enable direct observation of mesoscale organization in epithelial cells under morphological modulation, using a maskless protein photopatterning method (PRIMO) to confine cells to ECM micropatterns on electron microscopy substrates. These micropatterned cell culture substrates can be used in mechanobiology research to correlate changes in nanometer-scale organization at cell-cell and cell-ECM contacts to strain energy states and traction stress distribution in the cell.

  View details for DOI 10.1088/1361-6439/ab419a

  View details for PubMedID 32879557

  View details for PubMedCentralID PMC7457726

• Binding partner- and force-promoted changes in alphaE-catenin conformation probed by native cysteine labeling. Scientific reports Terekhova, K., Pokutta, S., Kee, Y. S., Li, J., Tajkhorshid, E., Fuller, G., Dunn, A. R., Weis, W. I. 2019; 9 (1): 15375

  Abstract

  Adherens Junctions (AJs) are cell-cell adhesion complexes that sense and propagate mechanical forces by coupling cadherins to the actin cytoskeleton via beta-catenin and the F-actin binding protein alphaE-catenin. When subjected to mechanical force, the cadherincatenin complex can tightly link to F-actin through alphaE-catenin, and also recruits the F-actin-binding protein vinculin. In this study, labeling of native cysteines combined with mass spectrometry revealed conformational changes in alphaE-catenin upon binding to the E-cadherinbeta-catenin complex, vinculin and F-actin. A method to apply physiologically meaningful forces in solution revealed force-induced conformational changes in alphaE-catenin when bound to F-actin. Comparisons of wild-type alphaE-catenin and a mutant with enhanced vinculin affinity using cysteine labeling and isothermal titration calorimetry provide evidence for allosteric coupling of the N-terminal beta-catenin-binding and the middle (M) vinculin-binding domain of alphaE-catenin. Cysteine labeling also revealed possible crosstalk between the actin-binding domain and the rest of the protein. The data provide insight into how binding partners and mechanical stress can regulate the conformation of full-length alphaE-catenin, and identify the M domain as a key transmitter of conformational changes.

  View details for DOI 10.1038/s41598-019-51816-3

  View details for PubMedID 31653927

• Solving the structure of Lgl2, a difficult blind test of unsupervised structure determination PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ufimtsev, I. S., Almagor, L., Weis, W. I., Levitt, M. 2019; 116 (22): 10819–23

  View details for DOI 10.1073/pnas.1821513116

  View details for Web of Science ID 000469280300039

• Structural insights into the aPKC regulatory switch mechanism of the human cell polarity protein lethal giant larvae 2 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Almagor, L., Ufimtsev, I. S., Ayer, A., Li, J., Weis, W. I. 2019; 116 (22): 10804–12

  View details for DOI 10.1073/pnas.1821514116

  View details for Web of Science ID 000469280300037

• Structural insights into the activation of metabotropic glutamate receptors (vol 566, pg 79, 2019) NATURE Koehl, A., Hu, H., Feng, D., Sun, B., Zhang, Y., Robertson, M. J., Chu, M., Kobilka, T., Laeremans, T., Steyaert, J., Tarrasch, J., Dutta, S., Fonseca, R., Weis, W. I., Mathiesen, J. M., Skiniotis, G., Kobilka, B. K. 2019; 567 (7747): E10

  View details for DOI 10.1038/s41586-019-0983-z

  View details for Web of Science ID 000461126600009

• Author Correction: Structural insights into the activation of metabotropic glutamate receptors. Nature Koehl, A., Hu, H., Feng, D., Sun, B., Zhang, Y., Robertson, M. J., Chu, M., Kobilka, T. S., Laeremans, T., Steyaert, J., Tarrasch, J., Dutta, S., Fonseca, R., Weis, W. I., Mathiesen, J. M., Skiniotis, G., Kobilka, B. K. 2019

  Abstract

  The surname of author Toon Laeremans was misspelled 'Laermans'. This error has been corrected online.

  View details for PubMedID 30814739

• Resolving indexing ambiguities in X-ray free-electron laser diffraction patterns. Acta crystallographica. Section D, Structural biology Uervirojnangkoorn, M., Lyubimov, A. Y., Zhou, Q., Weis, W. I., Brunger, A. T. 2019; 75 (Pt 2): 234–41

  Abstract

  Processing X-ray free-electron laser (XFEL) diffraction images poses challenges, as an XFEL pulse is powerful enough to destroy or damage the diffracting volume and thereby yields only one diffraction image per volume. Moreover, the crystal is stationary during the femtosecond pulse, so reflections are generally only partially recorded. Therefore, each XFEL diffraction image must be scaled individually and, ideally, corrected for partiality prior to merging. An additional complication may arise owing to indexing ambiguities when the symmetry of the Bravais lattice is higher than that of the space group, or when the unit-cell dimensions are similar to each other. Here, an automated method is presented that diagnoses these indexing ambiguities based on the Brehm-Diederichs algorithm [Brehm & Diederichs (2014), Acta Cryst. D70, 101-109] and produces a consistent indexing choice for the large majority of diffraction images. This method was applied to an XFEL diffraction data set measured from crystals of the neuronal SNARE-complexin-1-synaptotagmin-1 complex. After correcting the indexing ambiguities, substantial improvements were observed in the merging statistics and the atomic model refinement R values. This method should be a useful addition to the arsenal of tools for the processing of XFEL diffraction data sets.

  View details for PubMedID 30821711

• Structure of a Signaling Cannabinoid Receptor 1-G Protein Complex CELL Kumar, K., Shalev-Benami, M., Robertson, M. J., Hu, H., Banister, S. D., Hollingsworth, S. A., Latorraca, N. R., Kato, H. E., Hilger, D., Maeda, S., Weis, W. I., Farrens, D. L., Dror, R. O., Malhotra, S. V., Kobilka, B. K., Skiniotis, G. 2019; 176 (3): 448-+

  View details for DOI 10.1016/j.cell.2018.11.040

  View details for Web of Science ID 000456526100007

• CD23 is a glycan-binding receptor in some mammalian species. The Journal of biological chemistry Jégouzo, S. A., Feinberg, H. n., Morrison, A. n., Holder, A. n., May, A. n., Huang, Z. n., Jiang, L. n., Lasanajak, Y. n., Smith, D. F., Werling, D. n., Drickamer, K. n., Weis, W. I., Taylor, M. E. 2019

  Abstract

  CD23, the low affinity IgE receptor found on B lymphocytes and other cells, contains a C-terminal lectin-like domain that resembles C-type carbohydrate-recognition domains (CRDs) found in many glycan-binding receptors. In most mammalian species, the CD23 residues required to form a sugar-binding site are present, although binding of CD23 to IgE does not involve sugars. Solid-phase binding competition assays, glycoprotein blotting experiments and glycan array analysis employing the lectin-like domains of cow and mouse CD23 demonstrate that they bind to mannose, N-acetylglucosamine, glucose, and fucose and to glycoproteins that bear these sugars in nonreducing terminal positions. Crystal structures of the cow CRD in the presence of α-methyl mannoside and GlcNAcβ1-2Man reveal that a range of oligosaccharide ligands can be accommodated in an open binding site in which most interactions are with a single terminal sugar residue. Although mouse CD23 shows a pattern of monosaccharide and glycoprotein binding similar to cow CD23, the binding is weaker. In contrast, no sugar binding was observed in similar experiments with human CD23. The absence of sugar-binding activity correlates with accumulation of mutations in the CD23 gene in the primate lineage leading to humans, resulting in loss of key sugar-binding residues. These results are consistent with a role for CD23 in many species as a receptor for potentially pathogenic micro-organisms as well as IgE. However, the ability of CD23 to bind several different ligands varies between species, suggesting that it has distinct functions in different organisms.

  View details for DOI 10.1074/jbc.RA119.010572

  View details for PubMedID 31488546

• Processing simultaneously collected MAD data from two closely spaced (90 eV) wavelengths measured at an X-ray free-electron laser Mendez, D., Weis, W., Brunger, A., Wakatsuki, S., Sauter, N. INT UNION CRYSTALLOGRAPHY. 2019: A244

  View details for DOI 10.1107/S0108767319097617

  View details for Web of Science ID 000549524100240

• Publisher Correction: Structural insights into binding specificity, efficacy and bias of a beta2AR partial agonist. Nature chemical biology Masureel, M., Zou, Y., Picard, L., van der Westhuizen, E., Mahoney, J. P., Rodrigues, J. P., Mildorf, T. J., Dror, R. O., Shaw, D. E., Bouvier, M., Pardon, E., Steyaert, J., Sunahara, R. K., Weis, W. I., Zhang, C., Kobilka, B. K. 2018

  Abstract

  In the version of this paper originally published, the structure for epinephrine shown in Figure 1a was redrawn with an extra carbon. The structure has been replaced in the HTML and PDF versions of the article. The original and corrected versions of the structure are shown below.

  View details for PubMedID 30504785

• Structural insights into binding specificity, efficacy and bias of a beta2AR partial agonist. Nature chemical biology Masureel, M., Zou, Y., Picard, L., van der Westhuizen, E., Mahoney, J. P., Rodrigues, J. P., Mildorf, T. J., Dror, R. O., Shaw, D. E., Bouvier, M., Pardon, E., Steyaert, J., Sunahara, R. K., Weis, W. I., Zhang, C., Kobilka, B. K. 2018; 14 (11): 1059–66

  Abstract

  Salmeterol is a partial agonist for the beta2 adrenergic receptor (beta2AR) and the first long-acting beta2AR agonist to be widely used clinically for the treatment of asthma and chronic obstructive pulmonary disease. Salmeterol's safety and mechanism of action have both been controversial. To understand its unusual pharmacological action and partial agonism, we obtained the crystal structure of salmeterol-bound beta2AR in complex with an active-state-stabilizing nanobody. The structure reveals the location of the salmeterol exosite, where sequence differences between beta1AR and beta2AR explain the high receptor-subtype selectivity. A structural comparison with the beta2AR bound to the full agonist epinephrine reveals differences in the hydrogen-bond network involving residues Ser2045.43 and Asn2936.55. Mutagenesis and biophysical studies suggested that these interactions lead to a distinct active-state conformation that is responsible for the partial efficacy of G-protein activation and the limited beta-arrestin recruitment for salmeterol.

  View details for PubMedID 30327561

• The Molecular Basis of G Protein-Coupled Receptor Activation. Annual review of biochemistry Weis, W. I., Kobilka, B. K. 2018; 87: 897–919

  Abstract

  G protein-coupled receptors (GPCRs) mediate the majority of cellular responses to external stimuli. Upon activation by a ligand, the receptor binds to a partner heterotrimeric G protein and promotes exchange of GTP for GDP, leading to dissociation of the G protein into alpha and betagamma subunits that mediate downstream signals. GPCRs can also activate distinct signaling pathways through arrestins. Active states of GPCRs form by small rearrangements of the ligand-binding, or orthosteric, site that are amplified into larger conformational changes. Molecular understanding of the allosteric coupling between ligand binding and G protein or arrestin interaction is emerging from structures of several GPCRs crystallized in inactive and active states, spectroscopic data, and computer simulations. The coupling is loose, rather than concerted, and agonist binding does not fully stabilize the receptor in an active conformation. Distinct intermediates whose populations are shifted by ligands of different efficacies underlie the complex pharmacology of GPCRs.

  View details for PubMedID 29925258

• Structure of the µ-opioid receptor-Gi protein complex. Nature Koehl, A., Hu, H., Maeda, S., Zhang, Y., Qu, Q., Paggi, J. M., Latorraca, N. R., Hilger, D., Dawson, R., Matile, H., Schertler, G. F., Granier, S., Weis, W. I., Dror, R. O., Manglik, A., Skiniotis, G., Kobilka, B. K. 2018

  Abstract

  The mu-opioid receptor (muOR) is a G-protein-coupled receptor (GPCR) and the target of most clinically and recreationally used opioids. The induced positive effects of analgesia and euphoria are mediated by muOR signalling through the adenylyl cyclase-inhibiting heterotrimeric G protein Gi. Here we present the 3.5A resolution cryo-electron microscopy structure of the muOR bound to the agonist peptide DAMGO and nucleotide-free Gi. DAMGO occupies the morphinan ligand pocket, with its Nterminus interacting with conserved receptor residues and its Cterminus engaging regions important for opioid-ligand selectivity. Comparison of the muOR-Gi complex to previously determined structures of other GPCRs bound to the stimulatory G protein Gs reveals differences in the position of transmembrane receptor helix 6 and in the interactions between the G protein alpha-subunit and the receptor core. Together, these results shed light on the structural features that contribute to the Gi protein-coupling specificity of the OR.

  View details for PubMedID 29899455

• Analysis of a vinculin homolog in a sponge (phylum Porifera) reveals that vertebrate-like cell adhesions emerged early in animal evolution. The Journal of biological chemistry Miller, P. W., Pokutta, S., Mitchell, J. M., Chodaparambil, J. V., Clarke, D. N., Nelson, W., Weis, W. I., Nichols, S. A. 2018

  Abstract

  The evolution of cell adhesion mechanisms in animals facilitated the assembly of organized multicellular tissues. Studies in traditional animal models have revealed two predominant adhesion structures, the adherens junction (AJ) and focal adhesions (FAs), which are involved in the attachment of neighboring cells to each other and to the secreted extracellular matrix (ECM), respectively. The AJ (containing cadherins and catenins) and FAs (comprising integrins, talin, and paxillin) differ in protein composition, but both junctions contain the actin-binding protein vinculin. The near ubiquity of these structures in animals suggests that AJ and FAs evolved early, possibly coincident with multicellularity. However, a challenge to this perspective is that previous studies of sponges-a divergent animal lineage-indicate that their tissues are organized primarily by an alternative, sponge-specific cell adhesion mechanism called "aggregation factor." In this study, we examined the structure, biochemical properties, and tissue localization of a vinculin ortholog in the sponge Oscarella pearsei (Op). Our results indicate that Op vinculin localizes to both cell-cell and cell-ECM contacts and has biochemical and structural properties similar to those of vertebrate vinculin. We propose that Op vinculin played a role in cell adhesion and tissue organization in the last common ancestor of sponges and other animals. These findings provide compelling evidence that sponge tissues are indeed organized like epithelia in other animals and support the notion that AJ- and FA-like structures extend to the earliest periods of animal evolution.

  View details for PubMedID 29880641

• Epithelial barrier dysfunction in desmoglein-1 deficiency. The Journal of allergy and clinical immunology Polivka, L. n., Hadj-Rabia, S. n., Bal, E. n., Leclerc-Mercier, S. n., Madrange, M. n., Hamel, Y. n., Bonnet, D. n., Mallet, S. n., Lepidi, H. n., Ovaert, C. n., Barbet, P. n., Dupont, C. n., Neven, B. n., Munnich, A. n., Godsel, L. M., Campeotto, F. n., Weil, R. n., Laplantine, E. n., Marchetto, S. n., Borg, J. P., Weis, W. I., Casanova, J. L., Puel, A. n., Green, K. J., Bodemer, C. n., Smahi, A. n. 2018

  View details for PubMedID 29705242

• Vinculin forms a directionally asymmetric catch bond with F-actin SCIENCE Huang, D. L., Bax, N. A., Buckley, C. D., Weis, W. I., Dunn, A. R. 2017; 357 (6352): 703–6

  Abstract

  Vinculin is an actin-binding protein thought to reinforce cell-cell and cell-matrix adhesions. However, how mechanical load affects the vinculin-F-actin bond is unclear. Using a single-molecule optical trap assay, we found that vinculin forms a force-dependent catch bond with F-actin through its tail domain, but with lifetimes that depend strongly on the direction of the applied force. Force toward the pointed (-) end of the actin filament resulted in a bond that was maximally stable at 8 piconewtons, with a mean lifetime (12 seconds) 10 times as long as the mean lifetime when force was applied toward the barbed (+) end. A computational model of lamellipodial actin dynamics suggests that the directionality of the vinculin-F-actin bond could establish long-range order in the actin cytoskeleton. The directional and force-stabilized binding of vinculin to F-actin may be a mechanism by which adhesion complexes maintain front-rear asymmetry in migrating cells.

  View details for PubMedID 28818948

• Mechanism of pathogen recognition by human dectin-2. The Journal of biological chemistry Feinberg, H., Jégouzo, S. A., Rex, M. J., Drickamer, K., Weis, W. I., Taylor, M. E. 2017; 292 (32): 13402-13414

  Abstract

  Dectin-2, a C-type lectin on macrophages and other cells of the innate immune system, functions in response to pathogens, particularly fungi. The carbohydrate-recognition domain (CRD) in dectin-2 is linked to a transmembrane sequence that interacts with the common Fc receptor γ subunit to initiate immune signaling. The molecular mechanism by which dectin-2 selectively binds to pathogens has been investigated by characterizing the CRD expressed in a bacterial system. Competition binding studies indicated that the CRD binds to monosaccharides with modest affinity and that affinity was greatly enhanced for mannose-linked α1-2 or α1-4 to a second mannose residue. Glycan array analysis confirmed selective binding of the CRD to glycans that contain Manα1-2Man epitopes. Crystals of the CRD in complex with a mammalian-type high-mannose Man9GlcNAc2 oligosaccharide exhibited interaction with Manα1-2Man on two different termini of the glycan, with the reducing-end mannose residue ligated to Ca2+ in a primary binding site and the nonreducing terminal mannose residue occupying an adjacent secondary site. Comparison of the binding sites in DC-SIGN and langerin, two other pathogen-binding receptors of the innate immune system, revealed why these two binding sites accommodate only terminal Manα1-2Man structures, whereas dectin-2 can bind Manα1-2Man in internal positions in mannans and other polysaccharides. The specificity and geometry of the dectin-2-binding site provide the molecular mechanism for binding of dectin-2 to fungal mannans and also to bacterial lipopolysaccharides, capsular polysaccharides, and lipoarabinomannans that contain the Manα1-2Man disaccharide unit.

  View details for DOI 10.1074/jbc.M117.799080

  View details for PubMedID 28652405

  View details for PubMedCentralID PMC5555199

• Structural and functional characterization of Caenorhabditis elegans alpha-catenin reveals constitutive binding to beta-catenin and F-actin JOURNAL OF BIOLOGICAL CHEMISTRY Kang, H., Bang, I., Jin, K. S., Lee, B., Lee, J., Shao, X., Heier, J. A., Kwiatkowski, A. V., Nelson, W. J., Hardin, J., Weis, W. I., Choi, H. 2017; 292 (17): 7077-7086

  Abstract

  Intercellular epithelial junctions formed by classical cadherins, β-catenin, and the actin-binding protein α-catenin link the actin cytoskeletons of adjacent cells into a structural continuum. These assemblies transmit forces through the tissue and respond to intracellular and extracellular signals. However, the mechanisms of junctional assembly and regulation are poorly understood. Studies of cadherin-catenin assembly in a number of metazoans have revealed both similarities and unexpected differences in the biochemical properties of the cadherin·catenin complex that likely reflect the developmental and environmental requirements of different tissues and organisms. Here, we report the structural and biochemical characterization of HMP-1, the Caenorhabditis elegans α-catenin homolog, and compare it with mammalian α-catenin. HMP-1 shares overall similarity in structure and actin-binding properties, but displayed differences in conformational flexibility and allosteric regulation from mammalian α-catenin. HMP-1 bound filamentous actin with an affinity in the single micromolar range, even when complexed with the β-catenin homolog HMP-2 or when present in a complex of HMP-2 and the cadherin homolog HMR-1, indicating that HMP-1 binding to F-actin is not allosterically regulated by the HMP-2·HMR-1 complex. The middle (i.e. M) domain of HMP-1 appeared to be less conformationally flexible than mammalian α-catenin, which may underlie the dampened effect of HMP-2 binding on HMP-1 actin-binding activity compared with that of the mammalian homolog. In conclusion, our data indicate that HMP-1 constitutively binds β-catenin and F-actin, and although the overall structure and function of HMP-1 and related α-catenins are similar, the vertebrate proteins appear to be under more complex conformational regulation.

  View details for DOI 10.1074/jbc.M116.769778

  View details for Web of Science ID 000400478300018

  View details for PubMedCentralID PMC5409474

• a-catenin reveals constitutive binding to ß-catenin and F-actin. journal of biological chemistry Kang, H., Bang, I., Jin, K. S., Lee, B., Lee, J., Shao, X., Heier, J. A., Kwiatkowski, A. V., Nelson, W. J., Hardin, J., Weis, W. I., Choi, H. 2017; 292 (17): 7077-7086

  Abstract

  Intercellular epithelial junctions formed by classical cadherins, β-catenin, and the actin-binding protein α-catenin link the actin cytoskeletons of adjacent cells into a structural continuum. These assemblies transmit forces through the tissue and respond to intracellular and extracellular signals. However, the mechanisms of junctional assembly and regulation are poorly understood. Studies of cadherin-catenin assembly in a number of metazoans have revealed both similarities and unexpected differences in the biochemical properties of the cadherin·catenin complex that likely reflect the developmental and environmental requirements of different tissues and organisms. Here, we report the structural and biochemical characterization of HMP-1, the Caenorhabditis elegans α-catenin homolog, and compare it with mammalian α-catenin. HMP-1 shares overall similarity in structure and actin-binding properties, but displayed differences in conformational flexibility and allosteric regulation from mammalian α-catenin. HMP-1 bound filamentous actin with an affinity in the single micromolar range, even when complexed with the β-catenin homolog HMP-2 or when present in a complex of HMP-2 and the cadherin homolog HMR-1, indicating that HMP-1 binding to F-actin is not allosterically regulated by the HMP-2·HMR-1 complex. The middle (i.e. M) domain of HMP-1 appeared to be less conformationally flexible than mammalian α-catenin, which may underlie the dampened effect of HMP-2 binding on HMP-1 actin-binding activity compared with that of the mammalian homolog. In conclusion, our data indicate that HMP-1 constitutively binds β-catenin and F-actin, and although the overall structure and function of HMP-1 and related α-catenins are similar, the vertebrate proteins appear to be under more complex conformational regulation.

  View details for DOI 10.1074/jbc.M116.769778

  View details for PubMedID 28298447

• Cell-cell adhesion in metazoans relies on evolutionarily conserved features of the α-catenin·β-catenin-binding interface. The Journal of biological chemistry Shao, X. n., Kang, H. n., Loveless, T. n., Lee, G. R., Seok, C. n., Weis, W. I., Choi, H. J., Hardin, J. n. 2017; 292 (40): 16477–90

  Abstract

  Stable tissue integrity during embryonic development relies on the function of the cadherin·catenin complex (CCC). The Caenorhabditis elegans CCC is a useful paradigm for analyzing in vivo requirements for specific interactions among the core components of the CCC, and it provides a unique opportunity to examine evolutionarily conserved mechanisms that govern the interaction between α- and β-catenin. HMP-1, unlike its mammalian homolog α-catenin, is constitutively monomeric, and its binding affinity for HMP-2/β-catenin is higher than that of α-catenin for β-catenin. A crystal structure shows that the HMP-1·HMP-2 complex forms a five-helical bundle structure distinct from the structure of the mammalian α-catenin·β-catenin complex. Deletion analysis based on the crystal structure shows that the first helix of HMP-1 is necessary for binding HMP-2 avidly in vitro and for efficient recruitment of HMP-1 to adherens junctions in embryos. HMP-2 Ser-47 and Tyr-69 flank its binding interface with HMP-1, and we show that phosphomimetic mutations at these two sites decrease binding affinity of HMP-1 to HMP-2 by 40-100-fold in vitro. In vivo experiments using HMP-2 S47E and Y69E mutants showed that they are unable to rescue hmp-2(zu364) mutants, suggesting that phosphorylation of HMP-2 on Ser-47 and Tyr-69 could be important for regulating CCC formation in C. elegans Our data provide novel insights into how cadherin-dependent cell-cell adhesion is modulated in metazoans by conserved elements as well as features unique to specific organisms.

  View details for PubMedID 28842483

  View details for PubMedCentralID PMC5633108

• Mechanism of intracellular allosteric β2AR antagonist revealed by X-ray crystal structure. Nature Liu, X. n., Ahn, S. n., Kahsai, A. W., Meng, K. C., Latorraca, N. R., Pani, B. n., Venkatakrishnan, A. J., Masoudi, A. n., Weis, W. I., Dror, R. O., Chen, X. n., Lefkowitz, R. J., Kobilka, B. K. 2017; 548 (7668): 480–84

  Abstract

  G-protein-coupled receptors (GPCRs) pose challenges for drug discovery efforts because of the high degree of structural homology in the orthosteric pocket, particularly for GPCRs within a single subfamily, such as the nine adrenergic receptors. Allosteric ligands may bind to less-conserved regions of these receptors and therefore are more likely to be selective. Unlike orthosteric ligands, which tonically activate or inhibit signalling, allosteric ligands modulate physiologic responses to hormones and neurotransmitters, and may therefore have fewer adverse effects. The majority of GPCR crystal structures published to date were obtained with receptors bound to orthosteric antagonists, and only a few structures bound to allosteric ligands have been reported. Compound 15 (Cmpd-15) is an allosteric modulator of the β2 adrenergic receptor (β2AR) that was recently isolated from a DNA-encoded small-molecule library. Orthosteric β-adrenergic receptor antagonists, known as beta-blockers, are amongst the most prescribed drugs in the world and Cmpd-15 is the first allosteric beta-blocker. Cmpd-15 exhibits negative cooperativity with agonists and positive cooperativity with inverse agonists. Here we present the structure of the β2AR bound to a polyethylene glycol-carboxylic acid derivative (Cmpd-15PA) of this modulator. Cmpd-15PA binds to a pocket formed primarily by the cytoplasmic ends of transmembrane segments 1, 2, 6 and 7 as well as intracellular loop 1 and helix 8. A comparison of this structure with inactive- and active-state structures of the β2AR reveals the mechanism by which Cmpd-15 modulates agonist binding affinity and signalling.

  View details for PubMedID 28813418

• Structure of photosystem II and substrate binding at room temperature NATURE Young, I. D., Ibrahim, M., Chatterjee, R., Gul, S., Fuller, F. D., Koroidov, S., Brewster, A. S., Tran, R., Alonso-Mori, R., Kroll, T., Michels-Clark, T., Laksmono, H., Sierra, R. G., Stan, C. A., Hussein, R., Zhang, M., Douthit, L., Kubin, M., de Lichtenberg, C., Long Vo Pham, L. V., Nilsson, H., Cheah, M. H., Shevela, D., Saracini, C., Bean, M. A., Seuffert, I., Sokaras, D., Weng, T., Pastor, E., Weninger, C., Fransson, T., Lassalle, L., Braeuer, P., Aller, P., Docker, P. T., Andi, B., Orville, A. M., Glownia, J. M., Nelson, S., Sikorski, M., Zhu, D., Hunter, M. S., Lane, T. J., Aquila, A., Koglin, J. E., Robinson, J., Liang, M., Boutet, S., Lyubimov, A. Y., Uervirojnangkoorn, M., Moriarty, N. W., Liebschner, D., Afonine, P. V., Waterman, D. G., Evans, G., Wernet, P., Dobbek, H., Weis, W. I., Brunger, A. T., Zwart, P. H., Adams, P. D., Zouni, A., Messinger, J., Bergmann, U., Sauter, N. K., Kern, J., Yachandra, V. K., Yano, J. 2016; 540 (7633): 453-?

  Abstract

  Light-induced oxidation of water by photosystem II (PS II) in plants, algae and cyanobacteria has generated most of the dioxygen in the atmosphere. PS II, a membrane-bound multi-subunit pigment protein complex, couples the one-electron photochemistry at the reaction centre with the four-electron redox chemistry of water oxidation at the Mn4CaO5 cluster in the oxygen-evolving complex (OEC). Under illumination, the OEC cycles through five intermediate S-states (S0 to S4), in which S1 is the dark-stable state and S3 is the last semi-stable state before O-O bond formation and O2 evolution. A detailed understanding of the O-O bond formation mechanism remains a challenge, and will require elucidation of both the structures of the OEC in the different S-states and the binding of the two substrate waters to the catalytic site. Here we report the use of femtosecond pulses from an X-ray free electron laser (XFEL) to obtain damage-free, room temperature structures of dark-adapted (S1), two-flash illuminated (2F; S3-enriched), and ammonia-bound two-flash illuminated (2F-NH3; S3-enriched) PS II. Although the recent 1.95 Å resolution structure of PS II at cryogenic temperature using an XFEL provided a damage-free view of the S1 state, measurements at room temperature are required to study the structural landscape of proteins under functional conditions, and also for in situ advancement of the S-states. To investigate the water-binding site(s), ammonia, a water analogue, has been used as a marker, as it binds to the Mn4CaO5 cluster in the S2 and S3 states. Since the ammonia-bound OEC is active, the ammonia-binding Mn site is not a substrate water site. This approach, together with a comparison of the native dark and 2F states, is used to discriminate between proposed O-O bond formation mechanisms.

  View details for DOI 10.1038/nature20161

  View details for Web of Science ID 000389716800046

  View details for PubMedID 27871088

  View details for PubMedCentralID PMC5201176

• Advances in X-ray free electron laser (XFEL) diffraction data processing applied to the crystal structure of the synaptotagmin-1 / SNARE complex. eLife Lyubimov, A. Y., Uervirojnangkoorn, M., Zeldin, O. B., Zhou, Q., Zhao, M., Brewster, A. S., Michels-Clark, T., Holton, J. M., Sauter, N. K., Weis, W. I., Brunger, A. T. 2016; 5

  Abstract

  X-ray free electron lasers (XFELs) reduce the effects of radiation damage on macromolecular diffraction data and thereby extend the limiting resolution. Previously, we adapted classical post-refinement techniques to XFEL diffraction data to produce accurate diffraction data sets from a limited number of diffraction images (Uervirojnangkoorn et al., 2015), and went on to use these techniques to obtain a complete data set from crystals of the synaptotagmin-1 / SNARE complex and to determine the structure at 3.5 Å resolution (Zhou et al., 2015). Here, we describe new advances in our methods and present a reprocessed XFEL data set of the synaptotagmin-1 / SNARE complex. The reprocessing produced small improvements in electron density maps and the refined atomic model. The maps also contained more information than those of a lower resolution (4.1 Å) synchrotron data set. Processing a set of simulated XFEL diffraction images revealed that our methods yield accurate data and atomic models.

  View details for DOI 10.7554/eLife.18740

  View details for PubMedID 27731796

  View details for PubMedCentralID PMC5094853

• Binding Sites for Acylated Trehalose Analogs of Glycolipid Ligands on an Extended Carbohydrate Recognition Domain of the Macrophage Receptor Mincle JOURNAL OF BIOLOGICAL CHEMISTRY Feinberg, H., Rambaruth, N. D., Jegouzo, S. A., Jacobsen, K. M., Djurhuus, R., Poulsen, T. B., Weis, W. I., Taylor, M. E., Drickamer, K. 2016; 291 (40): 21222-?

  Abstract

  The macrophage receptor mincle binds to trehalose dimycolate on the surface of Mycobacterium tuberculosis Signaling initiated by this interaction leads to cytokine production, which underlies the ability of mycobacteria to evade the immune system and also to function as adjuvants. In previous work the mechanism for binding of the sugar headgroup of trehalose dimycolate to mincle has been elucidated, but the basis for enhanced binding to glycolipid ligands, in which hydrophobic substituents are attached to the 6-hydroxyl groups, has been the subject of speculation. In the work reported here, the interaction of trehalose derivatives with bovine mincle has been probed with a series of synthetic mimics of trehalose dimycolate in binding assays, in structural studies by x-ray crystallography, and by site-directed mutagenesis. Binding studies reveal that, rather than reflecting specific structural preference, the apparent affinity of mincle for ligands with hydrophobic substituents correlates with their overall size. Structural and mutagenesis analysis provides evidence for interaction of the hydrophobic substituents with multiple different portions of the surface of mincle and confirms the presence of three Ca(2+)-binding sites. The structure of an extended portion of the extracellular domain of mincle, beyond the minimal C-type carbohydrate recognition domain, also constrains the way the binding domains may interact on the surface of macrophages.

  View details for DOI 10.1074/jbc.M116.749515

  View details for Web of Science ID 000385406200035

  View details for PubMedID 27542410

  View details for PubMedCentralID PMC5076529

• Characterization of the Cadherin-Catenin Complex of the Sea Anemone Nematostella vectensis and Implications for the Evolution of Metazoan Cell-Cell Adhesion MOLECULAR BIOLOGY AND EVOLUTION Clarke, D. N., Miller, P. W., Lowe, C. J., Weis, W. I., Nelson, W. J. 2016; 33 (8): 2016-2029

  Abstract

  The cadherin-catenin complex (CCC) mediates cell-cell adhesion in bilaterian animals by linking extracellular cadherin-based adhesions to the actin cytoskeleton. However, it is unknown whether the basic organization of the complex is conserved across all metazoans. We tested whether protein interactions and actin-binding properties of the CCC are conserved in a nonbilaterian animal, the sea anemone Nematostella vectensis We demonstrated that N. vectensis has a complete repertoire of cadherin-catenin proteins, including two classical cadherins, one α-catenin, and one β-catenin. Using size-exclusion chromatography and multi-angle light scattering, we showed that α-catenin and β-catenin formed a heterodimer that bound N. vectensis Cadherin-1 and -2. Nematostella vectensis α-catenin bound F-actin with equivalent affinity as either a monomer or an α/β-catenin heterodimer, and its affinity for F-actin was, in part, regulated by a novel insert between the N- and C-terminal domains. Nematostella vectensis α-catenin inhibited Arp2/3 complex-mediated nucleation of actin filaments, a regulatory property previously thought to be unique to mammalian αE-catenin. Thus, despite significant differences in sequence, the key interactions of the CCC are conserved between bilaterians and cnidarians, indicating that the core function of the CCC as a link between cell adhesions and the actin cytoskeleton is ancestral in the eumetazoans.

  View details for DOI 10.1093/molbev/msw084

  View details for Web of Science ID 000380105900011

  View details for PubMedID 27189570

  View details for PubMedCentralID PMC4948710

• Allosteric nanobodies reveal the dynamic range and diverse mechanisms of G-protein-coupled receptor activation NATURE Staus, D. P., Strachan, R. T., Manglik, A., Pani, B., Kahsai, A. W., Kim, T. H., Wingler, L. M., Ahn, S., Chatterjee, A., Masoudi, A., Kruse, A. C., Pardon, E., Steyaert, J., Weis, W. I., Prosser, R. S., Kobilka, B. K., Costa, T., Lefkowitz, R. J. 2016; 535 (7612): 448-?

  Abstract

  G-protein-coupled receptors (GPCRs) modulate many physiological processes by transducing a variety of extracellular cues into intracellular responses. Ligand binding to an extracellular orthosteric pocket propagates conformational change to the receptor cytosolic region to promote binding and activation of downstream signalling effectors such as G proteins and β-arrestins. It is well known that different agonists can share the same binding pocket but evoke unique receptor conformations leading to a wide range of downstream responses (‘efficacy’). Furthermore, increasing biophysical evidence, primarily using the β2-adrenergic receptor (β2AR) as a model system, supports the existence of multiple active and inactive conformational states. However, how agonists with varying efficacy modulate these receptor states to initiate cellular responses is not well understood. Here we report stabilization of two distinct β2AR conformations using single domain camelid antibodies (nanobodies)—a previously described positive allosteric nanobody (Nb80) and a newly identified negative allosteric nanobody (Nb60). We show that Nb60 stabilizes a previously unappreciated low-affinity receptor state which corresponds to one of two inactive receptor conformations as delineated by X-ray crystallography and NMR spectroscopy. We find that the agonist isoprenaline has a 15,000-fold higher affinity for β2AR in the presence of Nb80 compared to the affinity of isoprenaline for β2AR in the presence of Nb60, highlighting the full allosteric range of a GPCR. Assessing the binding of 17 ligands of varying efficacy to the β2AR in the absence and presence of Nb60 or Nb80 reveals large ligand-specific effects that can only be explained using an allosteric model which assumes equilibrium amongst at least three receptor states. Agonists generally exert efficacy by stabilizing the active Nb80-stabilized receptor state (R80). In contrast, for a number of partial agonists, both stabilization of R80 and destabilization of the inactive, Nb60-bound state (R60) contribute to their ability to modulate receptor activation. These data demonstrate that ligands can initiate a wide range of cellular responses by differentially stabilizing multiple receptor states.

  View details for DOI 10.1038/nature18636

  View details for Web of Science ID 000380344200046

  View details for PubMedID 27409812

  View details for PubMedCentralID PMC4961583

• 25 Years of Tension over Actin Binding to the Cadherin Cell Adhesion Complex: The Devil is in the Details. Trends in cell biology Nelson, W. J., Weis, W. I. 2016; 26 (7): 471-473

  Abstract

  Over the past 25 years, there has been a conceptual (re)evolution in understanding how the cadherin cell adhesion complex, which contains F-actin-binding proteins, binds to the actin cytoskeleton. There is now good synergy between structural, biochemical, and cell biological results that the cadherin-catenin complex binds to F-actin under force.

  View details for DOI 10.1016/j.tcb.2016.04.010

  View details for PubMedID 27166091

• IOTA: integration optimization, triage and analysis tool for the processing of XFEL diffraction images JOURNAL OF APPLIED CRYSTALLOGRAPHY Lyubimov, A. Y., Uervirojnangkoorn, M., Zeldin, O. B., Brewster, A. S., Murray, T. D., Sauter, N. K., Berger, J. M., Weis, W. I., Brunger, A. T. 2016; 49: 1057-1064

  Abstract

  Serial femtosecond crystallography (SFX) uses an X-ray free-electron laser to extract diffraction data from crystals not amenable to conventional X-ray light sources owing to their small size or radiation sensitivity. However, a limitation of SFX is the high variability of the diffraction images that are obtained. As a result, it is often difficult to determine optimal indexing and integration parameters for the individual diffraction images. Presented here is a software package, called IOTA, which uses a grid-search technique to determine optimal spot-finding parameters that can in turn affect the success of indexing and the quality of integration on an image-by-image basis. Integration results can be filtered using a priori information about the Bravais lattice and unit-cell dimensions and analyzed for unit-cell isomorphism, facilitating an improvement in subsequent data-processing steps.

  View details for DOI 10.1107/S1600576716006683

  View details for PubMedID 27275148

• Crystal structures of the M1 and M4 muscarinic acetylcholine receptors. Nature Thal, D. M., Sun, B., Feng, D., Nawaratne, V., Leach, K., Felder, C. C., Bures, M. G., Evans, D. A., Weis, W. I., Bachhawat, P., Kobilka, T. S., Sexton, P. M., Kobilka, B. K., Christopoulos, A. 2016; 531 (7594): 335-340

  Abstract

  Muscarinic M1-M5 acetylcholine receptors are G-protein-coupled receptors that regulate many vital functions of the central and peripheral nervous systems. In particular, the M1 and M4 receptor subtypes have emerged as attractive drug targets for treatments of neurological disorders, such as Alzheimer's disease and schizophrenia, but the high conservation of the acetylcholine-binding pocket has spurred current research into targeting allosteric sites on these receptors. Here we report the crystal structures of the M1 and M4 muscarinic receptors bound to the inverse agonist, tiotropium. Comparison of these structures with each other, as well as with the previously reported M2 and M3 receptor structures, reveals differences in the orthosteric and allosteric binding sites that contribute to a role in drug selectivity at this important receptor family. We also report identification of a cluster of residues that form a network linking the orthosteric and allosteric sites of the M4 receptor, which provides new insight into how allosteric modulation may be transmitted between the two spatially distinct domains.

  View details for DOI 10.1038/nature17188

  View details for PubMedID 26958838

  View details for PubMedCentralID PMC4915387

• Purification, crystallization and initial crystallographic analysis of the alpha-catenin homologue HMP-1 from Caenorhabditis elegans ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS Kang, H., Bang, I., Weis, W. I., Choi, H. 2016; 72: 234-239

  Abstract

  Adherens junctions transmit mechanical force between cells. In these junctions, β-catenin binds to cadherins and to the N-terminal domain of α-catenin, which in turn binds to actin filaments via its C-terminal domain. The middle (M) domain of α-catenin plays an important role in responding to mechanical tension. The nematode Caenorhabditis elegans contains α- and β-catenin homologues called HMP-1 and HMP-2, respectively, but HMP-1 behaves differently from its mammalian homologue. Thus, structural and biochemical studies of HMP-1 have been initiated to understand the mechanism of HMP-1 and the evolution of α-catenin. The N-terminal domain of HMP-1 in complex with the minimal HMP-1-binding region of HMP-2 was purified and crystallized. These crystals diffracted to 1.6 Å resolution and belonged to space group P3121, with unit-cell parameters a = b = 57.1, c = 155.4 Å. The M domain of HMP-1 was also purified and crystallized. The M-domain crystals diffracted to 2.4 Å resolution and belonged to space group P212121, with unit-cell parameters a = 72.8, b = 81.5, c = 151.4 Å. Diffraction data were collected and processed from each crystal, and the structures were solved by molecular replacement.

  View details for DOI 10.1107/S2053230X16001862

  View details for Web of Science ID 000371619200011

  View details for PubMedID 26919528

  View details for PubMedCentralID PMC4774883

• A Small-Molecule Antagonist of the beta-Catenin/TCF4 Interaction Blocks the Self-Renewal of Cancer Stem Cells and Suppresses Tumorigenesis CANCER RESEARCH Fang, L., Zhu, Q., Neuenschwander, M., Specker, E., Wulf-Goldenberg, A., Weis, W. I., von Kries, J. P., Birchmeier, W. 2016; 76 (4): 891-901

  Abstract

  Wnt/β-catenin signaling is a highly conserved pathway essential for embryogenesis and tissue homeostasis. However, deregulation of this pathway can initiate and promote human malignancies, especially of the colon and head and neck. Therefore, Wnt/β-catenin signaling represents an attractive target for cancer therapy. We performed high-throughput screening using AlphaScreen and ELISA techniques to identify small molecules that disrupt the critical interaction between β-catenin and the transcription factor TCF4 required for signal transduction. We found that compound LF3, a 4-thioureido-benzenesulfonamide derivative, robustly inhibited this interaction. Biochemical assays revealed clues that the core structure of LF3 was essential for inhibition. LF3 inhibited Wnt/β-catenin signals in cells with exogenous reporters and in colon cancer cells with endogenously high Wnt activity. LF3 also suppressed features of cancer cells related to Wnt signaling, including high cell motility, cell-cycle progression, and the overexpression of Wnt target genes. However, LF3 did not cause cell death or interfere with cadherin-mediated cell-cell adhesion. Remarkably, the self-renewal capacity of cancer stem cells was blocked by LF3 in concentration-dependent manners, as examined by sphere formation of colon and head and neck cancer stem cells under nonadherent conditions. Finally, LF3 reduced tumor growth and induced differentiation in a mouse xenograft model of colon cancer. Collectively, our results strongly suggest that LF3 is a specific inhibitor of canonical Wnt signaling with anticancer activity that warrants further development for preclinical and clinical studies as a novel cancer therapy. Cancer Res; 76(4); 891-901. ©2015 AACR.

  View details for DOI 10.1158/0008-5472.CAN-15-1519

  View details for PubMedID 26645562

• Structure of the Intermediate Filament-Binding Region of Desmoplakin PLOS ONE Kang, H., Weiss, T. M., Bang, I., Weis, W. I., Choi, H. 2016; 11 (1)

  Abstract

  Desmoplakin (DP) is a cytoskeletal linker protein that connects the desmosomal cadherin/plakoglobin/plakophilin complex to intermediate filaments (IFs). The C-terminal region of DP (DPCT) mediates IF binding, and contains three plakin repeat domains (PRDs), termed PRD-A, PRD-B and PRD-C. Previous crystal structures of PRDs B and C revealed that each is formed by 4.5 copies of a plakin repeat (PR) and has a conserved positively charged groove on its surface. Although PRDs A and B are linked by just four amino acids, B and C are separated by a 154 residue flexible linker, which has hindered crystallographic analysis of the full DPCT. Here we present the crystal structure of a DPCT fragment spanning PRDs A and B, and elucidate the overall architecture of DPCT by small angle X-ray scattering (SAXS) analysis. The structure of PRD-A is similar to that of PRD-B, and the two domains are arranged in a quasi-linear arrangement, and separated by a 4 amino acid linker. Analysis of the B-C linker region using secondary structure prediction and the crystal structure of a homologous linker from the cytolinker periplakin suggests that the N-terminal ~100 amino acids of the linker form two PR-like motifs. SAXS analysis of DPCT indicates an elongated but non-linear shape with Rg = 51.5 Å and Dmax = 178 Å. These data provide the first structural insights into an IF binding protein containing multiple PRDs and provide a foundation for studying the molecular basis of DP-IF interactions.

  View details for DOI 10.1371/journal.pone.0147641

  View details for Web of Science ID 000369527800163

  View details for PubMedID 26808545

  View details for PubMedCentralID PMC4726743

• Purification and Structural Analysis of Desmoplakin. Methods in enzymology Choi, H. J., Weis, W. I. 2016; 569: 197–213

  Abstract

  Desmoplakin (DP) is an obligate component of desmosomes, where it links the desmosomal cadherin/plakoglobin/plakophilin assembly to intermediate filaments. DP contains a large amino-terminal domain (DPNT) that binds to the cadherin/plakoglobin/plakophilin complex, a central coiled-coil domain that dimerizes the molecule, and a C-terminal domain (DPCT) that binds to intermediate filaments. DPNT contains a plakin domain, comprising a set of spectrin-like repeats. DPCT contains three plakin repeat domains, each formed by 4.5 repeats of a sequence motif known as a plakin repeat that bind to intermediate filaments. Here, we review purification, biochemical characterization, and structural analysis of the DPNT plakin domain and the DPCT plakin repeat domains.

  View details for PubMedID 26778560

• High-density grids for efficient data collection from multiple crystals. Acta crystallographica. Section D, Structural biology Baxter, E. L., Aguila, L., Alonso-Mori, R., Barnes, C. O., Bonagura, C. A., Brehmer, W., Brunger, A. T., Calero, G., Caradoc-Davies, T. T., Chatterjee, R., DeGrado, W. F., Fraser, J. S., Ibrahim, M., Kern, J., Kobilka, B. K., Kruse, A. C., Larsson, K. M., Lemke, H. T., Lyubimov, A. Y., Manglik, A., McPhillips, S. E., Norgren, E., Pang, S. S., Soltis, S. M., Song, J., Thomaston, J., Tsai, Y., Weis, W. I., Woldeyes, R. A., Yachandra, V., Yano, J., Zouni, A., Cohen, A. E. 2016; 72: 2-11

  Abstract

  Higher throughput methods to mount and collect data from multiple small and radiation-sensitive crystals are important to support challenging structural investigations using microfocus synchrotron beamlines. Furthermore, efficient sample-delivery methods are essential to carry out productive femtosecond crystallography experiments at X-ray free-electron laser (XFEL) sources such as the Linac Coherent Light Source (LCLS). To address these needs, a high-density sample grid useful as a scaffold for both crystal growth and diffraction data collection has been developed and utilized for efficient goniometer-based sample delivery at synchrotron and XFEL sources. A single grid contains 75 mounting ports and fits inside an SSRL cassette or uni-puck storage container. The use of grids with an SSRL cassette expands the cassette capacity up to 7200 samples. Grids may also be covered with a polymer film or sleeve for efficient room-temperature data collection from multiple samples. New automated routines have been incorporated into the Blu-Ice/DCSS experimental control system to support grids, including semi-automated grid alignment, fully automated positioning of grid ports, rastering and automated data collection. Specialized tools have been developed to support crystallization experiments on grids, including a universal adaptor, which allows grids to be filled by commercial liquid-handling robots, as well as incubation chambers, which support vapor-diffusion and lipidic cubic phase crystallization experiments. Experiments in which crystals were loaded into grids or grown on grids using liquid-handling robots and incubation chambers are described. Crystals were screened at LCLS-XPP and SSRL BL12-2 at room temperature and cryogenic temperatures.

  View details for DOI 10.1107/S2059798315020847

  View details for PubMedID 26894529

  View details for PubMedCentralID PMC4756618

• Reevaluating alpha E-catenin monomer and homodimer functions by characterizing E-cadherin/alpha E-catenin chimeras JOURNAL OF CELL BIOLOGY Bianchini, J. M., Kitt, K. N., Gloerich, M., Pokutta, S., Weis, W. I., Nelson, W. J. 2015; 210 (7): 1065-1074

  View details for DOI 10.1083/jcb.201411080

  View details for Web of Science ID 000362624000006

• Reevaluating αE-catenin monomer and homodimer functions by characterizing E-cadherin/αE-catenin chimeras. The Journal of cell biology Bianchini, J. M., Kitt, K. N., Gloerich, M., Pokutta, S., Weis, W. I., Nelson, W. J. 2015; 210 (7): 1065-74

  Abstract

  As part of the E-cadherin-β-catenin-αE-catenin complex (CCC), mammalian αE-catenin binds F-actin weakly in the absence of force, whereas cytosolic αE-catenin forms a homodimer that interacts more strongly with F-actin. It has been concluded that cytosolic αE-catenin homodimer is not important for intercellular adhesion because E-cadherin/αE-catenin chimeras thought to mimic the CCC are sufficient to induce cell-cell adhesion. We show that, unlike αE-catenin in the CCC, these chimeras homodimerize, bind F-actin strongly, and inhibit the Arp2/3 complex, all of which are properties of the αE-catenin homodimer. To more accurately mimic the junctional CCC, we designed a constitutively monomeric chimera, and show that E-cadherin-dependent cell adhesion is weaker in cells expressing this chimera compared with cells in which αE-catenin homodimers are present. Our results demonstrate that E-cadherin/αE-catenin chimeras used previously do not mimic αE-catenin in the native CCC, and imply that both CCC-bound monomer and cytosolic homodimer αE-catenin are required for strong cell-cell adhesion.

  View details for DOI 10.1083/jcb.201411080

  View details for PubMedID 26416960

  View details for PubMedCentralID PMC4586751

• Architecture of the synaptotagmin-SNARE machinery for neuronal exocytosis. Nature Zhou, Q., Lai, Y., Bacaj, T., Zhao, M., Lyubimov, A. Y., Uervirojnangkoorn, M., Zeldin, O. B., Brewster, A. S., Sauter, N. K., Cohen, A. E., Soltis, S. M., Alonso-Mori, R., Chollet, M., Lemke, H. T., Pfuetzner, R. A., Choi, U. B., Weis, W. I., Diao, J., Südhof, T. C., Brunger, A. T. 2015; 525 (7567): 62-67

  Abstract

  Synaptotagmin-1 and neuronal SNARE proteins have central roles in evoked synchronous neurotransmitter release; however, it is unknown how they cooperate to trigger synaptic vesicle fusion. Here we report atomic-resolution crystal structures of Ca(2+)- and Mg(2+)-bound complexes between synaptotagmin-1 and the neuronal SNARE complex, one of which was determined with diffraction data from an X-ray free-electron laser, leading to an atomic-resolution structure with accurate rotamer assignments for many side chains. The structures reveal several interfaces, including a large, specific, Ca(2+)-independent and conserved interface. Tests of this interface by mutagenesis suggest that it is essential for Ca(2+)-triggered neurotransmitter release in mouse hippocampal neuronal synapses and for Ca(2+)-triggered vesicle fusion in a reconstituted system. We propose that this interface forms before Ca(2+) triggering, moves en bloc as Ca(2+) influx promotes the interactions between synaptotagmin-1 and the plasma membrane, and consequently remodels the membrane to promote fusion, possibly in conjunction with other interfaces.

  View details for DOI 10.1038/nature14975

  View details for PubMedID 26280336

• Structural insights into µ-opioid receptor activation. Nature Huang, W., Manglik, A., Venkatakrishnan, A. J., Laeremans, T., Feinberg, E. N., Sanborn, A. L., Kato, H. E., Livingston, K. E., Thorsen, T. S., Kling, R. C., Granier, S., Gmeiner, P., Husbands, S. M., Traynor, J. R., Weis, W. I., Steyaert, J., Dror, R. O., Kobilka, B. K. 2015; 524 (7565): 315-321

  Abstract

  Activation of the μ-opioid receptor (μOR) is responsible for the efficacy of the most effective analgesics. To shed light on the structural basis for μOR activation, here we report a 2.1 Å X-ray crystal structure of the murine μOR bound to the morphinan agonist BU72 and a G protein mimetic camelid antibody fragment. The BU72-stabilized changes in the μOR binding pocket are subtle and differ from those observed for agonist-bound structures of the β2-adrenergic receptor (β2AR) and the M2 muscarinic receptor. Comparison with active β2AR reveals a common rearrangement in the packing of three conserved amino acids in the core of the μOR, and molecular dynamics simulations illustrate how the ligand-binding pocket is conformationally linked to this conserved triad. Additionally, an extensive polar network between the ligand-binding pocket and the cytoplasmic domains appears to play a similar role in signal propagation for all three G-protein-coupled receptors.

  View details for DOI 10.1038/nature14886

  View details for PubMedID 26245379

• A Novel Mechanism for Binding of Galactose-terminated Glycans by the C-type Carbohydrate Recognition Domain in Blood Dendritic Cell Antigen 2. journal of biological chemistry Jégouzo, S. A., Feinberg, H., Dungarwalla, T., Drickamer, K., Weis, W. I., Taylor, M. E. 2015; 290 (27): 16759-16771

  Abstract

  Blood dendritic cell antigen 2 (BDCA-2; also designated CLEC4C or CD303) is uniquely expressed on plasmacytoid dendritic cells. Stimulation of BDCA-2 with antibodies leads to an anti-inflammatory response in these cells, but the natural ligands for the receptor are not known. The C-type carbohydrate recognition domain in the extracellular portion of BDCA-2 contains a signature motif typical of C-type animal lectins that bind mannose, glucose, or GlcNAc, yet it has been reported that BDCA-2 binds selectively to galactose-terminated, biantennary N-linked glycans. A combination of glycan array analysis and binding competition studies with monosaccharides and natural and synthetic oligosaccharides have been used to define the binding epitope for BDCA-2 as the trisaccharide Galβ1-3/4GlcNAcβ1-2Man. X-ray crystallography and mutagenesis studies show that mannose is ligated to the conserved Ca(2+) in the primary binding site that is characteristic of C-type carbohydrate recognition domains, and the GlcNAc and galactose residues make additional interactions in a wide, shallow groove adjacent to the primary binding site. As predicted from these studies, BDCA-2 binds to IgG, which bears galactose-terminated glycans that are not commonly found attached to other serum glycoproteins. Thus, BDCA-2 has the potential to serve as a previously unrecognized immunoglobulin Fc receptor.

  View details for DOI 10.1074/jbc.M115.660613

  View details for PubMedID 25995448

  View details for PubMedCentralID PMC4505424

• The linac coherent light source single particle imaging road map STRUCTURAL DYNAMICS Aquila, A., Barty, A., Bostedt, C., Boutet, S., Carini, G., Deponte, D., DRELL, P., Doniach, S., Downing, K. H., Earnest, T., Elmlund, H., Elser, V., Guehr, M., Hajdu, J., Hastings, J., Hau-Riege, S. P., Huang, Z., Lattman, E. E., Maia, F. R., Marchesini, S., Ourmazd, A., Pellegrini, C., Santra, R., Schlichting, I., Schroer, C., Spence, J. C., Vartanyants, I. A., Wakatsuki, S., Weis, W. I., Williams, G. J. 2015; 2 (4)

  Abstract

  Intense femtosecond x-ray pulses from free-electron laser sources allow the imaging of individual particles in a single shot. Early experiments at the Linac Coherent Light Source (LCLS) have led to rapid progress in the field and, so far, coherent diffractive images have been recorded from biological specimens, aerosols, and quantum systems with a few-tens-of-nanometers resolution. In March 2014, LCLS held a workshop to discuss the scientific and technical challenges for reaching the ultimate goal of atomic resolution with single-shot coherent diffractive imaging. This paper summarizes the workshop findings and presents the roadmap toward reaching atomic resolution, 3D imaging at free-electron laser sources.

  View details for DOI 10.1063/1.4918726

  View details for Web of Science ID 000360649200003

  View details for PubMedCentralID PMC4711616

• Munc18a Does Not Alter Fusion Rates Mediated by Neuronal SNAREs, Synaptotagmin, and Complexin. journal of biological chemistry Zhang, Y., Diao, J., Colbert, K. N., Lai, Y., Pfuetzner, R. A., Padolina, M. S., Vivona, S., Ressl, S., Cipriano, D. J., Choi, U. B., Shah, N., Weis, W. I., Brunger, A. T. 2015; 290 (16): 10518-10534

  Abstract

  Sec1/Munc18 (SM) proteins are essential for membrane trafficking, but their molecular mechanism remains unclear. Using a single vesicle-vesicle content-mixing assay with reconstituted neuronal SNAREs, synaptotagmin-1, and complexin-1, we show that the neuronal SM protein Munc18a/nSec1 has no effect on the intrinsic kinetics of both spontaneous fusion and Ca(2+)-triggered fusion between vesicles that mimic synaptic vesicles and the plasma membrane. However, wild type Munc18a reduced vesicle association ∼50% when the vesicles bearing the t-SNAREs syntaxin-1A and SNAP-25 were preincubated with Munc18 for 30 min. Single molecule experiments with labeled SNAP-25 indicate that the reduction of vesicle association is a consequence of sequestration of syntaxin-1A by Munc18a and subsequent release of SNAP-25 (i.e. Munc18a captures syntaxin-1A via its high affinity interaction). Moreover, a phosphorylation mimic mutant of Munc18a with reduced affinity to syntaxin-1A results in less reduction of vesicle association. In summary, Munc18a does not directly affect fusion, although it has an effect on the t-SNARE complex, depending on the presence of other factors and experimental conditions. Our results suggest that Munc18a primarily acts at the prefusion stage.

  View details for DOI 10.1074/jbc.M114.630772

  View details for PubMedID 25716318

  View details for PubMedCentralID PMC4400359

• A Conserved Phosphorylation Switch Controls the Interaction between Cadherin and beta-Catenin In Vitro and In Vivo DEVELOPMENTAL CELL Choi, H., Loveless, T., Lynch, A. M., Bang, I., Hardin, J., Weis, W. I. 2015; 33 (1): 82-93

  Abstract

  In metazoan adherens junctions, β-catenin links the cytoplasmic tail of classical cadherins to the F-actin-binding protein α-catenin. Phosphorylation of a Ser/Thr-rich region in the cadherin tail dramatically enhances affinity for β-catenin and promotes cell-cell adhesion in cell culture systems, but its importance has not been demonstrated in vivo. Here, we identify a critical phosphorylated serine in the C. elegans cadherin HMR-1 required for strong binding to the β-catenin homolog HMP-2. Ablation of this phosphoserine interaction produces developmental defects that resemble full loss-of-function (Hammerhead and Humpback) phenotypes. Most metazoans possess a single gene for β-catenin, which is also a transcriptional coactivator in Wnt signaling. Nematodes and planaria, however, have a set of paralogous β-catenins; for example, C. elegans HMP-2 functions only in cell-cell adhesion, whereas SYS-1 mediates transcriptional activation through interactions with POP-1/Tcf. Our structural data define critical sequence differences responsible for the unique ligand specificities of these two proteins.

  View details for DOI 10.1016/j.devcel.2015.02.005

  View details for Web of Science ID 000352454200009

  View details for PubMedID 25850673

  View details for PubMedCentralID PMC4390766

• Enabling X-ray Free Electron Laser Crystallography for Challenging Biological Systems from a Limited Number of Crystals ELIFE Uervirojnangkoorn, M., Zeldin, O. B., Lyubimov, A. Y., Hattne, J., Brewster, A. S., Sauter, N. K., Brunger, A. T., Weis, W. I. 2015; 4

  Abstract

  There is considerable potential for X-ray free electron lasers (XFELs) to enable determination of macromolecular crystal structures that are difficult to solve using current synchrotron sources. Prior XFEL studies often involved the collection of thousands to millions of diffraction images, in part due to limitations of data processing methods. We implemented a data processing system based on classical post-refinement techniques, adapted to specific properties of XFEL diffraction data. When applied to XFEL data from three different proteins collected using various sample delivery systems and XFEL beam parameters, our method improved the quality of the diffraction data as well as the resulting refined atomic models and electron density maps. Moreover, the number of observations for a reflection necessary to assemble an accurate data set could be reduced to a few observations. These developments will help expand the applicability of XFEL crystallography to challenging biological systems, including cases where sample is limited.

  View details for Web of Science ID 000351865600006

  View details for PubMedID 25781634

• Data Exploration Toolkit for serial diffraction experiments ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY Zeldin, O. B., Brewster, A. S., Hattne, J., Uervirojnangkoorn, M., Lyubimov, A. Y., Zhou, Q., Zhao, M., Weis, W. I., Sauter, N. K., Brunger, A. T. 2015; 71: 352-356

  Abstract

  Ultrafast diffraction at X-ray free-electron lasers (XFELs) has the potential to yield new insights into important biological systems that produce radiation-sensitive crystals. An unavoidable feature of the `diffraction before destruction' nature of these experiments is that images are obtained from many distinct crystals and/or different regions of the same crystal. Combined with other sources of XFEL shot-to-shot variation, this introduces significant heterogeneity into the diffraction data, complicating processing and interpretation. To enable researchers to get the most from their collected data, a toolkit is presented that provides insights into the quality of, and the variation present in, serial crystallography data sets. These tools operate on the unmerged, partial intensity integration results from many individual crystals, and can be used on two levels: firstly to guide the experimental strategy during data collection, and secondly to help users make informed choices during data processing.

  View details for DOI 10.1107/S1399004714025875

  View details for Web of Science ID 000349439900018

  View details for PubMedID 25664746

  View details for PubMedCentralID PMC4321488

• Three aSNAP and 10 ATP Molecules Are Used in SNARE Complex Disassembly by N-ethylmaleimide-sensitive Factor (NSF). journal of biological chemistry Shah, N., Colbert, K. N., Enos, M. D., Herschlag, D., Weis, W. I. 2015; 290 (4): 2175-2188

  Abstract

  The fusion of intracellular membranes is driven by the formation of a highly stable four-helix bundle of SNARE proteins embedded in the vesicle and target membranes. N-Ethylmaleimide sensitive factor recycles SNAREs after fusion by binding to the SNARE complex through an adaptor protein, αSNAP, and using the energy of ATP hydrolysis to disassemble the complex. Although only a single molecule of αSNAP binds to a soluble form of the SNARE complex, we find that three molecules of αSNAP are used for SNARE complex disassembly. We describe an engineered αSNAP trimer that supports more efficient SNARE complex disassembly than monomeric αSNAP. Using the trimerized αSNAP, we find that N-ethylmaleimide-sensitive factor hydrolyzes 10 ATP molecules on average to disassemble a single SNARE complex.

  View details for DOI 10.1074/jbc.M114.620849

  View details for PubMedID 25492864

  View details for PubMedCentralID PMC4303669

• Studying epithelial morphogenesis in Dictyostelium. Methods in molecular biology (Clifton, N.J.) Dickinson, D. J., Nelson, W. J., Weis, W. I. 2015; 1189: 267-281

  Abstract

  The discovery of polarized epithelial tissue in the social amoeba Dictyostelium discoideum establishes this classical model organism as a novel system for the study of epithelial polarity and morphogenesis. D. discoideum grows as single cells and is easily maintained in cell culture. Starvation of the cells triggers a multicellular developmental process that culminates with the formation of a fruiting body, whose normal morphogenesis is dependent on a polarized epithelium located at the apex of the developing structure. Here, we discuss techniques for genetic manipulation and imaging of multicellular D. discoideum, with a focus on methods that have facilitated the study of the epithelial tissue in this organism.

  View details for DOI 10.1007/978-1-4939-1164-6_18

  View details for PubMedID 25245700

• Mapping the conformational landscape of a dynamic enzyme by multitemperature and XFEL crystallography. eLife Keedy, D. A., Kenner, L. R., Warkentin, M., Woldeyes, R. A., Hopkins, J. B., Thompson, M. C., Brewster, A. S., Van Benschoten, A. H., Baxter, E. L., Uervirojnangkoorn, M., McPhillips, S. E., Song, J., Alonso-Mori, R., Holton, J. M., Weis, W. I., Brunger, A. T., Soltis, S. M., Lemke, H., Gonzalez, A., Sauter, N. K., Cohen, A. E., van den Bedem, H., Thorne, R. E., Fraser, J. S. 2015; 4

  Abstract

  Determining the interconverting conformations of dynamic proteins in atomic detail is a major challenge for structural biology. Conformational heterogeneity in the active site of the dynamic enzyme cyclophilin A (CypA) has been previously linked to its catalytic function, but the extent to which the different conformations of these residues are correlated is unclear. Here we compare the conformational ensembles of CypA by multitemperature synchrotron crystallography and fixed-target X-ray free-electron laser (XFEL) crystallography. The diffraction-before-destruction nature of XFEL experiments provides a radiation-damage-free view of the functionally important alternative conformations of CypA, confirming earlier synchrotron-based results. We monitored the temperature dependences of these alternative conformations with eight synchrotron datasets spanning 100-310 K. Multiconformer models show that many alternative conformations in CypA are populated only at 240 K and above, yet others remain populated or become populated at 180 K and below. These results point to a complex evolution of conformational heterogeneity between 180--240 K that involves both thermal deactivation and solvent-driven arrest of protein motions in the crystal. The lack of a single shared conformational response to temperature within the dynamic active-site network provides evidence for a conformation shuffling model, in which exchange between rotamer states of a large aromatic ring in the middle of the network shifts the conformational ensemble for the other residues in the network. Together, our multitemperature analyses and XFEL data motivate a new generation of temperature- and time-resolved experiments to structurally characterize the dynamic underpinnings of protein function.

  View details for DOI 10.7554/eLife.07574

  View details for PubMedID 26422513

  View details for PubMedCentralID PMC4721965

• Goniometer-based femtosecond crystallography with X-ray free electron lasers PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Cohen, A. E., Soltis, S. M., Gonzalez, A., Aguila, L., Alonso-Mori, R., Barnes, C. O., Baxter, E. L., Brehmer, W., Brewster, A. S., Brunger, A. T., Calero, G., Chang, J. F., Chollet, M., Ehrensberger, P., Eriksson, T. L., Feng, Y., Hattne, J., Hedman, B., Hollenbeck, M., Holton, J. M., Keable, S., Kobilka, B. K., Kovaleva, E. G., Kruse, A. C., Lemke, H. T., Lin, G., Lyubimov, A. Y., Manglik, A., Mathews, I. I., McPhillips, S. E., Nelson, S., Peters, J. W., Sauter, N. K., Smith, C. A., Song, J., Stevenson, H. P., Tsai, Y., Uervirojnangkoorn, M., Vinetsky, V., Wakatsuki, S., Weis, W. I., Zadvornyy, O. A., Zeldin, O. B., Zhu, D., Hodgson, K. O. 2014; 111 (48): 17122-17127

  Abstract

  The emerging method of femtosecond crystallography (FX) may extend the diffraction resolution accessible from small radiation-sensitive crystals and provides a means to determine catalytically accurate structures of acutely radiation-sensitive metalloenzymes. Automated goniometer-based instrumentation developed for use at the Linac Coherent Light Source enabled efficient and flexible FX experiments to be performed on a variety of sample types. In the case of rod-shaped Cpl hydrogenase crystals, only five crystals and about 30 min of beam time were used to obtain the 125 still diffraction patterns used to produce a 1.6-Å resolution electron density map. For smaller crystals, high-density grids were used to increase sample throughput; 930 myoglobin crystals mounted at random orientation inside 32 grids were exposed, demonstrating the utility of this approach. Screening results from cryocooled crystals of β2-adrenoreceptor and an RNA polymerase II complex indicate the potential to extend the diffraction resolution obtainable from very radiation-sensitive samples beyond that possible with undulator-based synchrotron sources.

  View details for DOI 10.1073/pnas.1418733111

  View details for Web of Science ID 000345920800042

  View details for PubMedID 25362050

  View details for PubMedCentralID PMC4260607

• Modified T4 Lysozyme Fusion Proteins Facilitate G Protein-Coupled Receptor Crystallogenesis STRUCTURE Thorsen, T. S., Matt, R., Weis, W. I., Kobilka, B. K. 2014; 22 (11): 1657-1664

  Abstract

  G protein-coupled receptors (GPCRs) mediate the majority of cellular responses to hormones and neurotransmitters. Most GPCR crystal structures have been obtained using a fusion protein strategy where the flexible third intracellular loop is replaced by T4 lysozyme (T4L). However, wild-type T4L may not be ideally suited for all GPCRs because of its size and the inherent flexibility between the N- and C-terminal subdomains. Here we report two modified T4L variants, designed to address flexibility and size, that can be used to optimize crystal quality or promote alternative packing interactions. These variants were tested on the M3 muscarinic receptor (M3). The original M3-T4L fusion protein produced twinned crystals that yielded a 3.4 Å structure from a 70 crystal data set. We replaced T4L with the modified T4L variants. Both T4L variants yielded M3 muscarinic receptor crystals with alternate lattices that were not twinned, including one that was solved at 2.8 Å resolution.

  View details for DOI 10.1016/j.str.2014.08.022

  View details for Web of Science ID 000344934300013

  View details for PubMedCentralID PMC4408211

• Mechano-Transduction: From Molecules to Tissues PLOS BIOLOGY Pruitt, B. L., Dunn, A. R., Weis, W. I., Nelson, W. J. 2014; 12 (11)

  Abstract

  External forces play complex roles in cell organization, fate, and homeostasis. Changes in these forces, or how cells respond to them, can result in abnormal embryonic development and diseases in adults. How cells sense and respond to these mechanical stimuli requires an understanding of the biophysical principles that underlie changes in protein conformation and result in alterations in the organization and function of cells and tissues. Here, we discuss mechano-transduction as it applies to protein conformation, cellular organization, and multi-cell (tissue) function.

  View details for DOI 10.1371/journal.pbio.1001996

  View details for Web of Science ID 000345627300009

  View details for PubMedID 25405923

  View details for PubMedCentralID PMC4236045

• The minimal cadherin-catenin complex binds to actin filaments under force SCIENCE Buckley, C. D., Tan, J., Anderson, K. L., Hanein, D., Volkmann, N., Weis, W. I., Nelson, W. J., Dunn, A. R. 2014; 346 (6209): 600-?

  Abstract

  Linkage between the adherens junction (AJ) and the actin cytoskeleton is required for tissue development and homeostasis. In vivo findings indicated that the AJ proteins E-cadherin, β-catenin, and the filamentous (F)-actin binding protein αE-catenin form a minimal cadherin-catenin complex that binds directly to F-actin. Biochemical studies challenged this model because the purified cadherin-catenin complex does not bind F-actin in solution. Here, we reconciled this difference. Using an optical trap-based assay, we showed that the minimal cadherin-catenin complex formed stable bonds with an actin filament under force. Bond dissociation kinetics can be explained by a catch-bond model in which force shifts the bond from a weakly to a strongly bound state. These results may explain how the cadherin-catenin complex transduces mechanical forces at cell-cell junctions.

  View details for DOI 10.1126/science.1254211

  View details for Web of Science ID 000343799700039

• Structural and thermodynamic characterization of cadherin·ß-catenin·a-catenin complex formation. journal of biological chemistry Pokutta, S., Choi, H., Ahlsen, G., Hansen, S. D., Weis, W. I. 2014; 289 (19): 13589-13601

  Abstract

  The classical cadherin·β-catenin·α-catenin complex mediates homophilic cell-cell adhesion and mechanically couples the actin cytoskeletons of adjacent cells. Although α-catenin binds to β-catenin and to F-actin, β-catenin significantly weakens the affinity of α-catenin for F-actin. Moreover, α-catenin self-associates into homodimers that block β-catenin binding. We investigated quantitatively and structurally αE- and αN-catenin dimer formation, their interaction with β-catenin and the cadherin·β-catenin complex, and the effect of the α-catenin actin-binding domain on β-catenin association. The two α-catenin variants differ in their self-association properties: at physiological temperatures, αE-catenin homodimerizes 10× more weakly than does αN-catenin but is kinetically trapped in its oligomeric state. Both αE- and αN-catenin bind to β-catenin with a Kd of 20 nM, and this affinity is increased by an order of magnitude when cadherin is bound to β-catenin. We describe the crystal structure of a complex representing the full β-catenin·αN-catenin interface. A three-dimensional model of the cadherin·β-catenin·α-catenin complex based on these new structural data suggests mechanisms for the enhanced stability of the ternary complex. The C-terminal actin-binding domain of α-catenin has no influence on the interactions with β-catenin, arguing against models in which β-catenin weakens actin binding by stabilizing inhibitory intramolecular interactions between the actin-binding domain and the rest of α-catenin.

  View details for DOI 10.1074/jbc.M114.554709

  View details for PubMedID 24692547

  View details for PubMedCentralID PMC4036364

• Structural and Thermodynamic Characterization of Cadherin center dot beta-Catenin center dot alpha-Catenin Complex Formation JOURNAL OF BIOLOGICAL CHEMISTRY Pokutta, S., Choi, H., Ahlsen, G., Hansen, S. D., Weis, W. I. 2014; 289 (19): 13589-13601

  Abstract

  The classical cadherin·β-catenin·α-catenin complex mediates homophilic cell-cell adhesion and mechanically couples the actin cytoskeletons of adjacent cells. Although α-catenin binds to β-catenin and to F-actin, β-catenin significantly weakens the affinity of α-catenin for F-actin. Moreover, α-catenin self-associates into homodimers that block β-catenin binding. We investigated quantitatively and structurally αE- and αN-catenin dimer formation, their interaction with β-catenin and the cadherin·β-catenin complex, and the effect of the α-catenin actin-binding domain on β-catenin association. The two α-catenin variants differ in their self-association properties: at physiological temperatures, αE-catenin homodimerizes 10× more weakly than does αN-catenin but is kinetically trapped in its oligomeric state. Both αE- and αN-catenin bind to β-catenin with a Kd of 20 nM, and this affinity is increased by an order of magnitude when cadherin is bound to β-catenin. We describe the crystal structure of a complex representing the full β-catenin·αN-catenin interface. A three-dimensional model of the cadherin·β-catenin·α-catenin complex based on these new structural data suggests mechanisms for the enhanced stability of the ternary complex. The C-terminal actin-binding domain of α-catenin has no influence on the interactions with β-catenin, arguing against models in which β-catenin weakens actin binding by stabilizing inhibitory intramolecular interactions between the actin-binding domain and the rest of α-catenin.

  View details for DOI 10.1074/jbc.M114.554709

  View details for Web of Science ID 000335522800053

  View details for PubMedCentralID PMC4036364

• Molecular functions of the TLE tetramerization domain in Wnt target gene repression. EMBO journal Chodaparambil, J. V., Pate, K. T., Hepler, M. R., Tsai, B. P., Muthurajan, U. M., Luger, K., Waterman, M. L., Weis, W. I. 2014; 33 (7): 719-731

  Abstract

  Wnt signaling activates target genes by promoting association of the co-activator β-catenin with TCF/LEF transcription factors. In the absence of β-catenin, target genes are silenced by TCF-mediated recruitment of TLE/Groucho proteins, but the molecular basis for TLE/TCF-dependent repression is unclear. We describe the unusual three-dimensional structure of the N-terminal Q domain of TLE1 that mediates tetramerization and binds to TCFs. We find that differences in repression potential of TCF/LEFs correlates with their affinities for TLE-Q, rather than direct competition between β-catenin and TLE for TCFs as part of an activation-repression switch. Structure-based mutation of the TLE tetramer interface shows that dimers cannot mediate repression, even though they bind to TCFs with the same affinity as tetramers. Furthermore, the TLE Q tetramer, not the dimer, binds to chromatin, specifically to K20 methylated histone H4 tails, suggesting that the TCF/TLE tetramer complex promotes structural transitions of chromatin to mediate repression.

  View details for DOI 10.1002/embj.201387188

  View details for PubMedID 24596249

• Drugging a Stem Cell Compartment Using Wnt3a Protein as a Therapeutic PLOS ONE Dhamdhere, G. R., Fang, M. Y., Jiang, J., Lee, K., Cheng, D., Olveda, R. C., Liu, B., Mulligan, K. A., Carlson, J. C., Ransom, R. C., Weis, W. I., Helms, J. A. 2014; 9 (1)

  Abstract

  The therapeutic potential of Wnt proteins has long been recognized but challenges associated with in vivo stability and delivery have hindered their development as drug candidates. By exploiting the hydrophobic nature of the protein we provide evidence that exogenous Wnt3a can be delivered in vivo if it is associated with a lipid vesicle. Recombinant Wnt3a associates with the external surface of the lipid membrane; this association stabilizes the protein and leads to prolonged activation of the Wnt pathway in primary cells. We demonstrate the consequences of Wnt pathway activation in vivo using a bone marrow engraftment assay. These data provide validation for the development of WNT3A as a therapeutic protein.

  View details for DOI 10.1371/journal.pone.0083650

  View details for Web of Science ID 000329462700008

  View details for PubMedID 24400074

  View details for PubMedCentralID PMC3882211

• Crystal structure reveals conservation of amyloid-ß conformation recognized by 3D6 following humanization to bapineuzumab. Alzheimer's research & therapy Feinberg, H., Saldanha, J. W., Diep, L., Goel, A., Widom, A., Veldman, G. M., Weis, W. I., Schenk, D., Basi, G. S. 2014; 6 (3): 31-?

  Abstract

  Immunotherapy targeting amyloid-β peptide is under active clinical investigation for treatment of Alzheimer's disease (AD). Among the hypotheses being investigated for impact on clinical outcome are the preferred epitope or conformation of amyloid-β to target for treatment, and the mechanism of action underlying immunotherapy. Bapineuzumab (humanized 3D6), a neo-epitope specific antibody recognizing amyloid-β1-5 with strong preference for an exposed Asp residue at the N-terminus of the peptide, has undergone advanced clinical testing for treatment of AD.To gain further insight into the epitope conformation, we interrogated structural details of amino-terminal epitopes in amyloid-β using x-ray crystallography of 3D6Fab:amyloid-β complexes. Humanization of 3D6 was carried out using standard procedures integrating recombinant methods, sequence informatics, and homology modeling predictions to identify important mouse framework residues for retention in the finished humanized product.Here we report the crystal structure of a recombinant Fab fragment of 3D6 in complex with amyloid-β1-7 solved at 2.0 Å resolution. The N-terminus of amyloid-β is bound to 3D6 as a 310 helix. The amino-terminal Asp residue is buried deepest in the antibody binding pocket, with the Cβ atom of residue 6 visible at the entrance to the binding pocket near the surface of the antibody. We further evaluate homology model based predictions used to guide humanization of 3D6 to bapineuzumab, with actual structure of the Fab. The structure of the Fab:amyloid-β complex validates design of the humanized antibody, and confirms the amyloid-β epitope recognized by 3D6 as previously mapped by ELISA.The conformation of amyloid-β antigen recognized by 3D6 is novel and distinct from other antibodies recognizing N-terminal epitopes. Our result provides the first report demonstrating structural conservation of antigen contact residues, and conformation of antigen recognized, between the parent murine antibody and its humanized version.

  View details for DOI 10.1186/alzrt261

  View details for PubMedID 25024748

  View details for PubMedCentralID PMC4095729

• Structural basis of GSK-3 inhibition by N-terminal phosphorylation and by the Wnt receptor LRP6. eLife Stamos, J. L., Chu, M. L., Enos, M. D., Shah, N., Weis, W. I. 2014; 3

  View details for DOI 10.7554/eLife.01998

  View details for PubMedID 24642411

• Common Polymorphisms in Human Langerin Change Specificity for Glycan Ligands JOURNAL OF BIOLOGICAL CHEMISTRY Feinberg, H., Rowntree, T. J., Tan, S. L., Drickamer, K., Weis, W. I., Taylor, M. E. 2013; 288 (52): 36762-36771

  Abstract

  Langerin, a C-type lectin on Langerhans cells, mediates carbohydrate-dependent uptake of pathogens in the first step of antigen presentation to the adaptive immune system. Langerin binds a diverse range of carbohydrates including high mannose structures, fucosylated blood group antigens, and glycans with terminal 6-sulfated galactose. Mutagenesis and quantitative binding assays indicate that salt bridges between the sulfate group and two lysine residues compensate for the nonoptimal binding of galactose at the primary Ca(2+) site. A commonly occurring single nucleotide polymorphism (SNP) in human langerin results in change of one of these lysine residues, Lys-313, to isoleucine. Glycan array screening reveals that this amino acid change abolishes binding to oligosaccharides with terminal 6SO4-Gal and enhances binding to oligosaccharides with terminal GlcNAc residues. Structural analysis shows that enhanced binding to GlcNAc may result from Ile-313 packing against the N-acetyl group. The K313I polymorphism is tightly linked to another SNP that results in the change N288D, which reduces affinity for glycan ligands by destabilizing the Ca(2+)-binding site. Langerin with Asp-288 and Ile-313 shows no binding to 6SO4-Gal-terminated glycans and increased binding to GlcNAc-terminated structures, but overall decreased binding to glycans. Altered langerin function in individuals with the linked N288D and K313I polymorphisms may affect susceptibility to infection by microorganisms.

  View details for DOI 10.1074/jbc.M113.528000

  View details for Web of Science ID 000329189700001

  View details for PubMedID 24217250

• Activation and allosteric modulation of a muscarinic acetylcholine receptor NATURE Kruse, A. C., Ring, A. M., Manglik, A., Hu, J., Hu, K., Eitel, K., Huebner, H., Pardon, E., Valant, C., Sexton, P. M., Christopoulos, A., Felder, C. C., Gmeiner, P., Steyaert, J., Weis, W. I., Garcia, K. C., Wess, J., Kobilka, B. K. 2013; 504 (7478): 101-?

  Abstract

  Despite recent advances in crystallography and the availability of G-protein-coupled receptor (GPCR) structures, little is known about the mechanism of their activation process, as only the β2 adrenergic receptor (β2AR) and rhodopsin have been crystallized in fully active conformations. Here we report the structure of an agonist-bound, active state of the human M2 muscarinic acetylcholine receptor stabilized by a G-protein mimetic camelid antibody fragment isolated by conformational selection using yeast surface display. In addition to the expected changes in the intracellular surface, the structure reveals larger conformational changes in the extracellular region and orthosteric binding site than observed in the active states of the β2AR and rhodopsin. We also report the structure of the M2 receptor simultaneously bound to the orthosteric agonist iperoxo and the positive allosteric modulator LY2119620. This structure reveals that LY2119620 recognizes a largely pre-formed binding site in the extracellular vestibule of the iperoxo-bound receptor, inducing a slight contraction of this outer binding pocket. These structures offer important insights into the activation mechanism and allosteric modulation of muscarinic receptors.

  View details for DOI 10.1038/nature12735

  View details for Web of Science ID 000327851700039

  View details for PubMedID 24256733

  View details for PubMedCentralID PMC4020789

• alpha E-catenin actin-binding domain alters actin filament conformation and regulates binding of nucleation and disassembly factors MOLECULAR BIOLOGY OF THE CELL Hansen, S. D., Kwiatkowski, A. V., Ouyang, C., Liu, H., Pokutta, S., Watkins, S. C., Volkmann, N., Hanein, D., Weis, W. I., Mullins, R. D., Nelson, W. J. 2013; 24 (23): 3710-3720

  Abstract

  The actin-binding protein αE-catenin may contribute to transitions between cell migration and cell-cell adhesion that depend on remodeling the actin cytoskeleton, but the underlying mechanisms are unknown. We show that the αE-catenin actin-binding domain (ABD) binds cooperatively to individual actin filaments and that binding is accompanied by a conformational change in the actin protomer that affects filament structure. αE-catenin ABD binding limits barbed-end growth, especially in actin filament bundles. αE-catenin ABD inhibits actin filament branching by the Arp2/3 complex and severing by cofilin, both of which contact regions of the actin protomer that are structurally altered by αE-catenin ABD binding. In epithelial cells, there is little correlation between the distribution of αE-catenin and the Arp2/3 complex at developing cell-cell contacts. Our results indicate that αE-catenin binding to filamentous actin favors assembly of unbranched filament bundles that are protected from severing over more dynamic, branched filament arrays.

  View details for DOI 10.1091/mbc.E13-07-0388

  View details for Web of Science ID 000328125100011

  View details for PubMedID 24068324

  View details for PubMedCentralID PMC3842997

• Evolution and Cell Physiology. 3. Using Dictyostelium discoideum to investigate mechanisms of epithelial polarity AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY Weis, W. I., Nelson, W. J., Dickinson, D. J. 2013; 305 (11): C1091-C1095

  Abstract

  In Metazoa, a polarized epithelium forms a single-cell-layered barrier that separates the outside from the inside of the organism. In tubular epithelia, the apical side of the cell is constricted relative to the basal side, forming a wedge-shaped cell that can pack into a tube. Apical constriction is mediated by actomyosin activity. In higher animals, apical actomyosin is connected between cells by specialized cell-cell junctions that contain a classical cadherin, the Wnt signaling protein β-catenin, and the actin-binding protein α-catenin. The molecular mechanisms that lead to selective accumulation of myosin at the apical surface of cells are poorly understood. We found that the nonmetazoan Dictyostelium discoideum forms a polarized epithelium that surrounds the stalk tube at the tip of the multicellular fruiting body. Although D. discoideum lacks a cadherin homolog, it expresses homologs of β- and α-catenin. Both catenins are essential for formation of the tip epithelium, polarized protein secretion, and proper multicellular morphogenesis. Myosin localizes apically in tip epithelial cells, and it appears that constriction of this epithelial tube is required for proper morphogenesis. Localization of myosin II is controlled by the protein IQGAP1 and its binding partners cortexillins I and II, which function downstream of α- and β-catenin to exclude myosin from the basolateral cortex and promote apical accumulation of myosin. These studies show that the function of catenins in cell polarity predates the evolution of Wnt signaling and classical cadherins, and that apical localization of myosin is a morphogenetic mechanism conserved from nonmetazoans to vertebrates.

  View details for DOI 10.1152/ajpcell.00233.2013

  View details for Web of Science ID 000327844000001

  View details for PubMedID 24067914

  View details for PubMedCentralID PMC3882382

• Applications of molecular replacement to G protein-coupled receptors ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY Kruse, A. C., Manglik, A., Kobilka, B. K., Weis, W. I. 2013; 69: 2287-2292

  Abstract

  G protein-coupled receptors (GPCRs) are a large class of integral membrane proteins involved in regulating virtually every aspect of human physiology. Despite their profound importance in human health and disease, structural information regarding GPCRs has been extremely limited until recently. With the advent of a variety of new biochemical and crystallographic techniques, the structural biology of GPCRs has advanced rapidly, offering key molecular insights into GPCR activation and signal transduction. To date, almost all GPCR structures have been solved using molecular-replacement techniques. Here, the unique aspects of molecular replacement as applied to individual GPCRs and to signaling complexes of these important proteins are discussed.

  View details for DOI 10.1107/S090744491301322X

  View details for Web of Science ID 000326648900016

  View details for PubMedID 24189241

  View details for PubMedCentralID PMC3817703

• Adrenaline-activated structure of ß2-adrenoceptor stabilized by an engineered nanobody. Nature Ring, A. M., Manglik, A., Kruse, A. C., Enos, M. D., Weis, W. I., Garcia, K. C., Kobilka, B. K. 2013; 502 (7472): 575-579

  Abstract

  G-protein-coupled receptors (GPCRs) are integral membrane proteins that have an essential role in human physiology, yet the molecular processes through which they bind to their endogenous agonists and activate effector proteins remain poorly understood. So far, it has not been possible to capture an active-state GPCR bound to its native neurotransmitter. Crystal structures of agonist-bound GPCRs have relied on the use of either exceptionally high-affinity agonists or receptor stabilization by mutagenesis. Many natural agonists such as adrenaline, which activates the β2-adrenoceptor (β2AR), bind with relatively low affinity, and they are often chemically unstable. Using directed evolution, we engineered a high-affinity camelid antibody fragment that stabilizes the active state of the β2AR, and used this to obtain crystal structures of the activated receptor bound to multiple ligands. Here we present structures of the active-state human β2AR bound to three chemically distinct agonists: the ultrahigh-affinity agonist BI167107, the high-affinity catecholamine agonist hydroxybenzyl isoproterenol, and the low-affinity endogenous agonist adrenaline. The crystal structures reveal a highly conserved overall ligand recognition and activation mode despite diverse ligand chemical structures and affinities that range from 100 nM to ∼80 pM. Overall, the adrenaline-bound receptor structure is similar to the others, but it has substantial rearrangements in extracellular loop three and the extracellular tip of transmembrane helix 6. These structures also reveal a water-mediated hydrogen bond between two conserved tyrosines, which appears to stabilize the active state of the β2AR and related GPCRs.

  View details for DOI 10.1038/nature12572

  View details for PubMedID 24056936

  View details for PubMedCentralID PMC3822040

• Mechanism for recognition of an unusual mycobacterial glycolipid by the macrophage receptor mincle. journal of biological chemistry Feinberg, H., Jégouzo, S. A., Rowntree, T. J., Guan, Y., Brash, M. A., Taylor, M. E., Weis, W. I., Drickamer, K. 2013; 288 (40): 28457-28465

  Abstract

  Binding of the macrophage lectin mincle to trehalose dimycolate, a key glycolipid virulence factor on the surface of Mycobacterium tuberculosis and Mycobacterium bovis, initiates responses that can lead both to toxicity and to protection of these pathogens from destruction. Crystallographic structural analysis, site-directed mutagenesis, and binding studies with glycolipid mimics have been used to define an extended binding site in the C-type carbohydrate recognition domain (CRD) of bovine mincle that encompasses both the headgroup and a portion of the attached acyl chains. One glucose residue of the trehalose Glcα1-1Glcα headgroup is liganded to a Ca(2+) in a manner common to many C-type CRDs, whereas the second glucose residue is accommodated in a novel secondary binding site. The additional contacts in the secondary site lead to a 36-fold higher affinity for trehalose compared with glucose. An adjacent hydrophobic groove, not seen in other C-type CRDs, provides a docking site for one of the acyl chains attached to the trehalose, which can be targeted with small molecule analogs of trehalose dimycolate that bind with 52-fold higher affinity than trehalose. The data demonstrate how mincle bridges between the surfaces of the macrophage and the mycobacterium and suggest the possibility of disrupting this interaction. In addition, the results may provide a basis for design of adjuvants that mimic the ability of mycobacteria to stimulate a response to immunization that can be employed in vaccine development.

  View details for DOI 10.1074/jbc.M113.497149

  View details for PubMedID 23960080

  View details for PubMedCentralID PMC3789947

• Danio rerio alpha E-catenin Is a Monomeric F-actin Binding Protein with Distinct Properties from Mus musculus alpha E-catenin JOURNAL OF BIOLOGICAL CHEMISTRY Miller, P. W., Pokutta, S., Ghosh, A., Almo, S. C., Weis, W. I., Nelson, W. J., Kwiatkowski, A. V. 2013; 288 (31): 22324-22332

  Abstract

  It is unknown whether homologs of the cadherin/catenin complex have conserved structures and functions across the Metazoa. Mammalian αE-catenin is an allosterically regulated actin-binding protein that binds the cadherin/β-catenin complex as a monomer and whose dimerization potentiates F-actin association. We tested whether these functional properties are conserved in another vertebrate, the zebrafish Danio rerio. Here we show, despite 90% sequence identity, that D. rerio and M. musculus αE-catenin have striking functional differences. We demonstrate that D. rerio αE-catenin is monomeric using size exclusion chromatography, native-PAGE, and small angle X-ray scattering. D. rerio αE-catenin binds F-actin in cosedimentation assays as a monomer and as an α/β-catenin heterodimer complex. D. rerio αE-catenin also bundles F-actin as shown by negative stained transmission electron microscopy, and does not inhibit Arp2/3 complex-mediated actin nucleation in bulk polymerization assays. Thus, core properties of α-catenin function - F-actin and β-catenin binding - are conserved between mouse and zebrafish. We speculate that unique regulatory properties have evolved to match specific developmental requirements.

  View details for DOI 10.1074/jbc.M113.458406

  View details for Web of Science ID 000330596300014

• Syntaxin1a variants lacking an N-peptide or bearing the LE mutation bind to Munc18a in a closed conformation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Colbert, K. N., Hattendorf, D. A., Weiss, T. M., Burkhardt, P., Fasshauer, D., Weis, W. I. 2013; 110 (31): 12637-12642

  Abstract

  In neurons, soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins drive the fusion of synaptic vesicles to the plasma membrane through the formation of a four-helix SNARE complex. Members of the Sec1/Munc18 protein family regulate membrane fusion through interactions with the syntaxin family of SNARE proteins. The neuronal protein Munc18a interacts with a closed conformation of the SNARE protein syntaxin1a (Syx1a) and with an assembled SNARE complex containing Syx1a in an open conformation. The N-peptide of Syx1a (amino acids 1-24) has been implicated in the transition of Munc18a-bound Syx1a to Munc18a-bound SNARE complex, but the underlying mechanism is not understood. Here we report the X-ray crystal structures of Munc18a bound to Syx1a with and without its native N-peptide (Syx1aΔN), along with small-angle X-ray scattering (SAXS) data for Munc18a bound to Syx1a, Syx1aΔN, and Syx1a L165A/E166A (LE), a mutation thought to render Syx1a in a constitutively open conformation. We show that all three complexes adopt the same global structure, in which Munc18a binds a closed conformation of Syx1a. We also identify a possible structural connection between the Syx1a N-peptide and SNARE domain that might be important for the transition of closed-to-open Syx1a in SNARE complex assembly. Although the role of the N-peptide in Munc18a-mediated SNARE complex assembly remains unclear, our results demonstrate that the N-peptide and LE mutation have no effect on the global conformation of the Munc18a-Syx1a complex.

  View details for DOI 10.1073/pnas.1303753110

  View details for Web of Science ID 000322441500041

  View details for PubMedID 23858467

• Structural Studies of Wnts and Identification of an LRP6 Binding Site STRUCTURE Chu, M. L., Ahn, V. E., Choi, H., Daniels, D. L., Nusse, R., Weis, W. I. 2013; 21 (7): 1235-1242

  Abstract

  Wnts are secreted growth factors that have critical roles in cell fate determination and stem cell renewal. The Wnt/β-catenin pathway is initiated by binding of a Wnt protein to a Frizzled (Fzd) receptor and a coreceptor, LDL receptor-related protein 5 or 6 (LRP5/6). We report the 2.1 Å resolution crystal structure of a Drosophila WntD fragment encompassing the N-terminal domain and the linker that connects it to the C-terminal domain. Differences in the structures of WntD and Xenopus Wnt8, including the positions of a receptor-binding β hairpin and a large solvent-filled cavity in the helical core, indicate conformational plasticity in the N-terminal domain that may be important for Wnt-Frizzled specificity. Structure-based mutational analysis of mouse Wnt3a shows that the linker between the N- and C-terminal domains is required for LRP6 binding. These findings provide important insights into Wnt function and evolution.

  View details for DOI 10.1016/j.str.2013.05.006

  View details for Web of Science ID 000321681600020

  View details for PubMedID 23791946

• Danio rerio αE-catenin is a Monomeric F-actin Binding Protein With Distinct Properties from Mus musculus αE-catenin. The Journal of biological chemistry Miller, P. W., Pokutta, S., Ghosh, A., Almo, S. C., Weis, W. I., Nelson, W. J., Kwiatkowski, A. V. 2013

  Abstract

  It is unknown whether homologs of the cadherin/catenin complex have conserved structures and functions across the Metazoa. Mammalian αE-catenin is an allosterically regulated actin-binding protein that binds the cadherin/β-catenin complex as a monomer and whose dimerization potentiates F-actin association. We tested whether these functional properties are conserved in another vertebrate, the zebrafish Danio rerio. Here we show, despite 90% sequence identity, that D. rerio and M. musculus αE-catenin have striking functional differences. We demonstrate that D. rerio αE-catenin is monomeric using size exclusion chromatography, native-PAGE, and small angle X-ray scattering. D. rerio αE-catenin binds F-actin in cosedimentation assays as a monomer and as an α/β-catenin heterodimer complex. D. rerio αE-catenin also bundles F-actin as shown by negative stained transmission electron microscopy, and does not inhibit Arp2/3 complex-mediated actin nucleation in bulk polymerization assays. Thus, core properties of α-catenin function - F-actin and β-catenin binding - are conserved between mouse and zebrafish. We speculate that unique regulatory properties have evolved to match specific developmental requirements.

  View details for DOI 10.1074/jbc.M113.458406

  View details for PubMedID 23788645

• Structure of active ß-arrestin-1 bound to a G-protein-coupled receptor phosphopeptide. Nature Shukla, A. K., Manglik, A., Kruse, A. C., Xiao, K., Reis, R. I., Tseng, W., Staus, D. P., Hilger, D., Uysal, S., Huang, L., Paduch, M., Tripathi-Shukla, P., Koide, A., Koide, S., Weis, W. I., Kossiakoff, A. A., Kobilka, B. K., Lefkowitz, R. J. 2013; 497 (7447): 137-141

  Abstract

  The functions of G-protein-coupled receptors (GPCRs) are primarily mediated and modulated by three families of proteins: the heterotrimeric G proteins, the G-protein-coupled receptor kinases (GRKs) and the arrestins. G proteins mediate activation of second-messenger-generating enzymes and other effectors, GRKs phosphorylate activated receptors, and arrestins subsequently bind phosphorylated receptors and cause receptor desensitization. Arrestins activated by interaction with phosphorylated receptors can also mediate G-protein-independent signalling by serving as adaptors to link receptors to numerous signalling pathways. Despite their central role in regulation and signalling of GPCRs, a structural understanding of β-arrestin activation and interaction with GPCRs is still lacking. Here we report the crystal structure of β-arrestin-1 (also called arrestin-2) in complex with a fully phosphorylated 29-amino-acid carboxy-terminal peptide derived from the human V2 vasopressin receptor (V2Rpp). This peptide has previously been shown to functionally and conformationally activate β-arrestin-1 (ref. 5). To capture this active conformation, we used a conformationally selective synthetic antibody fragment (Fab30) that recognizes the phosphopeptide-activated state of β-arrestin-1. The structure of the β-arrestin-1-V2Rpp-Fab30 complex shows marked conformational differences in β-arrestin-1 compared to its inactive conformation. These include rotation of the amino- and carboxy-terminal domains relative to each other, and a major reorientation of the 'lariat loop' implicated in maintaining the inactive state of β-arrestin-1. These results reveal, at high resolution, a receptor-interacting interface on β-arrestin, and they indicate a potentially general molecular mechanism for activation of these multifunctional signalling and regulatory proteins.

  View details for DOI 10.1038/nature12120

  View details for PubMedID 23604254

  View details for PubMedCentralID PMC3654799

• Characterization of the Biophysical Origins of Mechanical Homeostasis at Cellular Adhesions 57th Annual Meeting of the Biophysical-Society Mekhdjian, A. H., Morimatsu, M., Borghi, N., Pruitt, B. L., Weis, W. I., Nelson, W. J., Dunn, A. R. CELL PRESS. 2013: 491A–491A

  View details for Web of Science ID 000316074304491

• Roles of cadherins and catenins in cell-cell adhesion and epithelial cell polarity. Progress in molecular biology and translational science Nelson, W. J., Dickinson, D. J., Weis, W. I. 2013; 116: 3-23

  Abstract

  A simple epithelium is the building block of all metazoans and a multicellular stage of a nonmetazoan. It comprises a closed monolayer of quiescent cells that surround a luminal space. Cells are held together by cell-cell adhesion complexes and surrounded by extracellular matrix. These extracellular contacts are required for the formation of a polarized organization of plasma membrane proteins that regulate the directional absorption and secretion of ions, proteins, and other solutes. While advances have been made in understanding how proteins are sorted to different plasma membrane domains, less is known about how cell-cell adhesion is regulated and linked to the development of epithelial cell polarity and regulation of homeostasis.

  View details for DOI 10.1016/B978-0-12-394311-8.00001-7

  View details for PubMedID 23481188

• The evolutionary origin of epithelial cell-cell adhesion mechanisms. Current topics in membranes Miller, P. W., Clarke, D. N., Weis, W. I., Lowe, C. J., Nelson, W. J. 2013; 72: 267-311

  Abstract

  A simple epithelium forms a barrier between the outside and the inside of an organism, and is the first organized multicellular tissue found in evolution. We examine the relationship between the evolution of epithelia and specialized cell-cell adhesion proteins comprising the classical cadherin/β-catenin/α-catenin complex (CCC). A review of the divergent functional properties of the CCC in metazoans and non-metazoans, and an updated phylogenetic coverage of the CCC using recent genomic data reveal: (1) The core CCC likely originated before the last common ancestor of unikonts and their closest bikont sister taxa. (2) Formation of the CCC may have constrained sequence evolution of the classical cadherin cytoplasmic domain and β-catenin in metazoa. (3) The α-catenin-binding domain in β-catenin appears to be the favored mutation site for disrupting β-catenin function in the CCC. (4) The ancestral function of the α/β-catenin heterodimer appears to be an actin-binding module. In some metazoan groups, more complex functions of α-catenin were gained by sequence divergence in the non-actin-binding (N-, M-) domains. (5) Allosteric regulation of α-catenin may have evolved for more complex regulation of the actin cytoskeleton.

  View details for DOI 10.1016/B978-0-12-417027-8.00008-8

  View details for PubMedID 24210433

  View details for PubMedCentralID PMC4118598

• The beta-Catenin Destruction Complex COLD SPRING HARBOR PERSPECTIVES IN BIOLOGY Stamos, J. L., Weis, W. I. 2013; 5 (1)

  Abstract

  The Wnt/β-catenin pathway is highly regulated to insure the correct temporal and spatial activation of its target genes. In the absence of a Wnt stimulus, the transcriptional coactivator β-catenin is degraded by a multiprotein "destruction complex" that includes the tumor suppressors Axin and adenomatous polyposis coli (APC), the Ser/Thr kinases GSK-3 and CK1, protein phosphatase 2A (PP2A), and the E3-ubiquitin ligase β-TrCP. The complex generates a β-TrCP recognition site by phosphorylation of a conserved Ser/Thr-rich sequence near the β-catenin amino terminus, a process that requires scaffolding of the kinases and β-catenin by Axin. Ubiquitinated β-catenin is degraded by the proteasome. The molecular mechanisms that underlie several aspects of destruction complex function are poorly understood, particularly the role of APC. Here we review the molecular mechanisms of destruction complex function and discuss several potential roles of APC in β-catenin destruction.

  View details for DOI 10.1101/cshperspect.a007898

  View details for Web of Science ID 000315983600008

  View details for PubMedID 23169527

• High-resolution crystal structure of human protease-activated receptor 1 NATURE Zhang, C., Srinivasan, Y., Arlow, D. H., Fung, J. J., Palmer, D., Zheng, Y., Green, H. F., Pandey, A., Dror, R. O., Shaw, D. E., Weis, W. I., Coughlin, S. R., Kobilka, B. K. 2012; 492 (7429): 387-?

  Abstract

  Protease-activated receptor 1 (PAR1) is the prototypical member of a family of G-protein-coupled receptors that mediate cellular responses to thrombin and related proteases. Thrombin irreversibly activates PAR1 by cleaving the amino-terminal exodomain of the receptor, which exposes a tethered peptide ligand that binds the heptahelical bundle of the receptor to affect G-protein activation. Here we report the 2.2 Å resolution crystal structure of human PAR1 bound to vorapaxar, a PAR1 antagonist. The structure reveals an unusual mode of drug binding that explains how a small molecule binds virtually irreversibly to inhibit receptor activation by the tethered ligand of PAR1. In contrast to deep, solvent-exposed binding pockets observed in other peptide-activated G-protein-coupled receptors, the vorapaxar-binding pocket is superficial but has little surface exposed to the aqueous solvent. Protease-activated receptors are important targets for drug development. The structure reported here will aid the development of improved PAR1 antagonists and the discovery of antagonists to other members of this receptor family.

  View details for DOI 10.1038/nature11701

  View details for Web of Science ID 000312488200047

  View details for PubMedID 23222541

  View details for PubMedCentralID PMC3531875

• N-Terminal T4 Lysozyme Fusion Facilitates Crystallization of a G Protein Coupled Receptor PLOS ONE Zou, Y., Weis, W. I., Kobilka, B. K. 2012; 7 (10)

  Abstract

  A highly crystallizable T4 lysozyme (T4L) was fused to the N-terminus of the β(2) adrenergic receptor (β(2)AR), a G-protein coupled receptor (GPCR) for catecholamines. We demonstrate that the N-terminal fused T4L is sufficiently rigid relative to the receptor to facilitate crystallogenesis without thermostabilizing mutations or the use of a stabilizing antibody, G protein, or protein fused to the 3rd intracellular loop. This approach adds to the protein engineering strategies that enable crystallographic studies of GPCRs alone or in complex with a signaling partner.

  View details for DOI 10.1371/journal.pone.0046039

  View details for Web of Science ID 000309580800006

  View details for PubMedID 23056231

  View details for PubMedCentralID PMC3464249

• An epithelial tissue in Dictyostelium challenges the traditional origin of metazoan multicellularity BIOESSAYS Dickinson, D. J., Nelson, W. J., Weis, W. I. 2012; 34 (10): 833-840

  Abstract

  We hypothesize that aspects of animal multicellularity originated before the divergence of metazoans from fungi and social amoebae. Polarized epithelial tissues are a defining feature of metazoans and contribute to the diversity of animal body plans. The recent finding of a polarized epithelium in the non-metazoan social amoeba Dictyostelium discoideum demonstrates that epithelial tissue is not a unique feature of metazoans, and challenges the traditional paradigm that multicellularity evolved independently in social amoebae and metazoans. An alternative view, presented here, is that the common ancestor of social amoebae, fungi, and animals spent a portion of its life cycle in a multicellular state and possessed molecular machinery necessary for forming an epithelial tissue. Some descendants of this ancestor retained multicellularity, while others reverted to unicellularity. This hypothesis makes testable predictions regarding tissue organization in close relatives of metazoans and provides a novel conceptual framework for studies of early animal evolution.

  View details for DOI 10.1002/bies.201100187

  View details for Web of Science ID 000308712600008

  View details for PubMedID 22930590

  View details for PubMedCentralID PMC3517009

• alpha-Catenin and IQGAP Regulate Myosin Localization to Control Epithelial Tube Morphogenesis in Dictyostelium DEVELOPMENTAL CELL Dickinson, D. J., Robinson, D. N., Nelson, W. J., Weis, W. I. 2012; 23 (3): 533-546

  Abstract

  Apical actomyosin activity in animal epithelial cells influences tissue morphology and drives morphogenetic movements during development. The molecular mechanisms leading to myosin II accumulation at the apical membrane and its exclusion from other membranes are poorly understood. We show that in the nonmetazoan Dictyostelium discoideum, myosin II localizes apically in tip epithelial cells that surround the stalk, and constriction of this epithelial tube is required for proper morphogenesis. IQGAP1 and its binding partner cortexillin I function downstream of α- and β-catenin to exclude myosin II from the basolateral cortex and promote apical accumulation of myosin II. Deletion of IQGAP1 or cortexillin compromises epithelial morphogenesis without affecting cell polarity. These results reveal that apical localization of myosin II is a conserved morphogenetic mechanism from nonmetazoans to vertebrates and identify a hierarchy of proteins that regulate the polarity and organization of an epithelial tube in a simple model organism.

  View details for DOI 10.1016/j.devcel.2012.06.008

  View details for Web of Science ID 000308776400011

  View details for PubMedID 22902739

  View details for PubMedCentralID PMC3443284

• E-cadherin is under constitutive actomyosin-generated tension that is increased at cell-cell contacts upon externally applied stretch PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Borghi, N., Sorokina, M., Shcherbakova, O. G., Weis, W. I., Pruitt, B. L., Nelson, W. J., Dunn, A. R. 2012; 109 (31): 12568-12573

  Abstract

  Classical cadherins are transmembrane proteins at the core of intercellular adhesion complexes in cohesive metazoan tissues. The extracellular domain of classical cadherins forms intercellular bonds with cadherins on neighboring cells, whereas the cytoplasmic domain recruits catenins, which in turn associate with additional cytoskeleton binding and regulatory proteins. Cadherin/catenin complexes are hypothesized to play a role in the transduction of mechanical forces that shape cells and tissues during development, regeneration, and disease. Whether mechanical forces are transduced directly through cadherins is unknown. To address this question, we used a Förster resonance energy transfer (FRET)-based molecular tension sensor to test the origin and magnitude of tensile forces transmitted through the cytoplasmic domain of E-cadherin in epithelial cells. We show that the actomyosin cytoskeleton exerts pN-tensile force on E-cadherin, and that this tension requires the catenin-binding domain of E-cadherin and αE-catenin. Surprisingly, the actomyosin cytoskeleton constitutively exerts tension on E-cadherin at the plasma membrane regardless of whether or not E-cadherin is recruited to cell-cell contacts, although tension is further increased at cell-cell contacts when adhering cells are stretched. Our findings thus point to a constitutive role of E-cadherin in transducing mechanical forces between the actomyosin cytoskeleton and the plasma membrane, not only at cell-cell junctions but throughout the cell surface.

  View details for DOI 10.1073/pnas.1204390109

  View details for Web of Science ID 000307538200062

  View details for PubMedID 22802638

  View details for PubMedCentralID PMC3411997

• alpha E-catenin is an autoinhibited molecule that coactivates vinculin PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Choi, H., Pokutta, S., Cadwell, G. W., Bobkov, A. A., Bankston, L. A., Liddington, R. C., Weis, W. I. 2012; 109 (22): 8576-8581

  Abstract

  αE-catenin, an essential component of the adherens junction, interacts with the classical cadherin-β-catenin complex and with F-actin, but its precise role is unknown. αE-catenin also binds to the F-actin-binding protein vinculin, which also appears to be important in junction assembly. Vinculin and αE-catenin are homologs that contain a series of helical bundle domains, D1-D5. We mapped the vinculin-binding site to a sequence in D3a comprising the central two helices of a four-helix bundle. The crystal structure of this peptide motif bound to vinculin D1 shows that the two helices adopt a parallel, colinear arrangement suggesting that the αE-catenin D3a bundle must unfold in order to bind vinculin. We show that αE-catenin D3 binds strongly to vinculin, whereas larger fragments and full-length αE-catenin bind approximately 1,000-fold more weakly. Thus, intramolecular interactions within αE-catenin inhibit binding to vinculin. The actin-binding activity of vinculin is inhibited by an intramolecular interaction between the head (D1-D4) and the actin-binding D5 tail. In the absence of F-actin, there is no detectable binding of αE-catenin D3 to full-length vinculin; however, αE-catenin D3 promotes binding of vinculin to F-actin whereas full-length αE-catenin does not. These findings support the combinatorial or "coincidence" model of activation in which binding of high-affinity proteins to the vinculin head and tail is required to shift the conformational equilibrium of vinculin from a closed, autoinhibited state to an open, stable F-actin-binding state. The data also imply that αE-catenin must be activated in order to bind to vinculin.

  View details for DOI 10.1073/pnas.1203906109

  View details for Web of Science ID 000304881700048

  View details for PubMedID 22586082

  View details for PubMedCentralID PMC3365184

• Structure of the delta-opioid receptor bound to naltrindole NATURE Granier, S., Manglik, A., Kruse, A. C., Kobilka, T. S., Thian, F. S., Weis, W. I., Kobilka, B. K. 2012; 485 (7398): 400-U171

  Abstract

  The opioid receptor family comprises three members, the µ-, δ- and κ-opioid receptors, which respond to classical opioid alkaloids such as morphine and heroin as well as to endogenous peptide ligands like endorphins. They belong to the G-protein-coupled receptor (GPCR) superfamily, and are excellent therapeutic targets for pain control. The δ-opioid receptor (δ-OR) has a role in analgesia, as well as in other neurological functions that remain poorly understood. The structures of the µ-OR and κ-OR have recently been solved. Here we report the crystal structure of the mouse δ-OR, bound to the subtype-selective antagonist naltrindole. Together with the structures of the µ-OR and κ-OR, the δ-OR structure provides insights into conserved elements of opioid ligand recognition while also revealing structural features associated with ligand-subtype selectivity. The binding pocket of opioid receptors can be divided into two distinct regions. Whereas the lower part of this pocket is highly conserved among opioid receptors, the upper part contains divergent residues that confer subtype selectivity. This provides a structural explanation and validation for the 'message-address' model of opioid receptor pharmacology, in which distinct 'message' (efficacy) and 'address' (selectivity) determinants are contained within a single ligand. Comparison of the address region of the δ-OR with other GPCRs reveals that this structural organization may be a more general phenomenon, extending to other GPCR families as well.

  View details for DOI 10.1038/nature11111

  View details for Web of Science ID 000304099100049

  View details for PubMedID 22596164

  View details for PubMedCentralID PMC3523198

• Crystal structure of the mu-opioid receptor bound to a morphinan antagonist NATURE Manglik, A., Kruse, A. C., Kobilka, T. S., Thian, F. S., Mathiesen, J. M., Sunahara, R. K., Pardo, L., Weis, W. I., Kobilka, B. K., Granier, S. 2012; 485 (7398): 321-U170

  Abstract

  Opium is one of the world's oldest drugs, and its derivatives morphine and codeine are among the most used clinical drugs to relieve severe pain. These prototypical opioids produce analgesia as well as many undesirable side effects (sedation, apnoea and dependence) by binding to and activating the G-protein-coupled µ-opioid receptor (µ-OR) in the central nervous system. Here we describe the 2.8 Å crystal structure of the mouse µ-OR in complex with an irreversible morphinan antagonist. Compared to the buried binding pocket observed in most G-protein-coupled receptors published so far, the morphinan ligand binds deeply within a large solvent-exposed pocket. Of particular interest, the µ-OR crystallizes as a two-fold symmetrical dimer through a four-helix bundle motif formed by transmembrane segments 5 and 6. These high-resolution insights into opioid receptor structure will enable the application of structure-based approaches to develop better drugs for the management of pain and addiction.

  View details for DOI 10.1038/nature10954

  View details for Web of Science ID 000304099100032

  View details for PubMedID 22437502

  View details for PubMedCentralID PMC3523197

• Structure and dynamics of the M3 muscarinic acetylcholine receptor NATURE Kruse, A. C., Hu, J., Pan, A. C., Arlow, D. H., Rosenbaum, D. M., Rosemond, E., Green, H. F., Liu, T., Chae, P. S., Dror, R. O., Shaw, D. E., Weis, W. I., Wess, J., Kobilka, B. K. 2012; 482 (7386): 552-556

  Abstract

  Acetylcholine, the first neurotransmitter to be identified, exerts many of its physiological actions via activation of a family of G-protein-coupled receptors (GPCRs) known as muscarinic acetylcholine receptors (mAChRs). Although the five mAChR subtypes (M1-M5) share a high degree of sequence homology, they show pronounced differences in G-protein coupling preference and the physiological responses they mediate. Unfortunately, despite decades of effort, no therapeutic agents endowed with clear mAChR subtype selectivity have been developed to exploit these differences. We describe here the structure of the G(q/11)-coupled M3 mAChR ('M3 receptor', from rat) bound to the bronchodilator drug tiotropium and identify the binding mode for this clinically important drug. This structure, together with that of the G(i/o)-coupled M2 receptor, offers possibilities for the design of mAChR subtype-selective ligands. Importantly, the M3 receptor structure allows a structural comparison between two members of a mammalian GPCR subfamily displaying different G-protein coupling selectivities. Furthermore, molecular dynamics simulations suggest that tiotropium binds transiently to an allosteric site en route to the binding pocket of both receptors. These simulations offer a structural view of an allosteric binding mode for an orthosteric GPCR ligand and provide additional opportunities for the design of ligands with different affinities or binding kinetics for different mAChR subtypes. Our findings not only offer insights into the structure and function of one of the most important GPCR families, but may also facilitate the design of improved therapeutics targeting these critical receptors.

  View details for DOI 10.1038/nature10867

  View details for Web of Science ID 000300770500056

  View details for PubMedID 22358844

  View details for PubMedCentralID PMC3529910

• Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist NATURE Haga, K., Kruse, A. C., Asada, H., Yurugi-Kobayashi, T., Shiroishi, M., Zhang, C., Weis, W. I., Okada, T., Kobilka, B. K., Haga, T., Kobayashi, T. 2012; 482 (7386): 547-U147

  Abstract

  The parasympathetic branch of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G-protein-coupled receptors that mediate the response to acetylcholine released from parasympathetic nerves. Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases. The M2 muscarinic acetylcholine receptor (M2 receptor) is essential for the physiological control of cardiovascular function through activation of G-protein-coupled inwardly rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we report the structure of the antagonist-bound human M2 receptor, the first human acetylcholine receptor to be characterized structurally, to our knowledge. The antagonist 3-quinuclidinyl-benzilate binds in the middle of a long aqueous channel extending approximately two-thirds through the membrane. The orthosteric binding pocket is formed by amino acids that are identical in all five muscarinic receptor subtypes, and shares structural homology with other functionally unrelated acetylcholine binding proteins from different species. A layer of tyrosine residues forms an aromatic cap restricting dissociation of the bound ligand. A binding site for allosteric ligands has been mapped to residues at the entrance to the binding pocket near this aromatic cap. The structure of the M2 receptor provides insights into the challenges of developing subtype-selective ligands for muscarinic receptors and their propensity for allosteric regulation.

  View details for DOI 10.1038/nature10753

  View details for Web of Science ID 000300770500055

  View details for PubMedID 22278061

  View details for PubMedCentralID PMC3345277

• Structural Basis of Wnt Signaling Inhibition by Dickkopf Binding to LRP5/6 DEVELOPMENTAL CELL Ahn, V. E., Chu, M. L., Choi, H., Tran, D., Abo, A., Weis, W. I. 2011; 21 (5): 862-873

  Abstract

  LDL receptor-related proteins 5 and 6 (LRP5/6) are coreceptors for Wnt growth factors, and also bind Dkk proteins, secreted inhibitors of Wnt signaling. The LRP5/6 ectodomain contains four β-propeller/EGF-like domain repeats. The first two repeats, LRP6(1-2), bind to several Wnt variants, whereas LRP6(3-4) binds other Wnts. We present the crystal structure of the Dkk1 C-terminal domain bound to LRP6(3-4), and show that the Dkk1 N-terminal domain binds to LRP6(1-2), demonstrating that a single Dkk1 molecule can bind to both portions of the LRP6 ectodomain and thereby inhibit different Wnts. Small-angle X-ray scattering analysis of LRP6(1-4) bound to a noninhibitory antibody fragment or to full-length Dkk1 shows that in both cases the ectodomain adopts a curved conformation that places the first three repeats at a similar height relative to the membrane. Thus, Wnts bound to either portion of the LRP6 ectodomain likely bear a similar spatial relationship to Frizzled coreceptors.

  View details for DOI 10.1016/j.devce1.2011.09.003

  View details for Web of Science ID 000297233700009

  View details for PubMedID 22000856

  View details for PubMedCentralID PMC3215855

• The beta-Catenin Binding Protein ICAT Modulates Androgen Receptor Activity MOLECULAR ENDOCRINOLOGY Zhuo, M., Zhu, C., Sun, J., Weis, W. I., Sun, Z. 2011; 25 (10): 1677-1688

  Abstract

  Androgens have important roles in the development of the prostate gland and in prostate cancer. Since the finding that β-catenin is a cofactor of the androgen receptor (AR) and can augment AR signaling, several proteins have been found to affect AR signaling through their interaction with β-catenin. Here, we investigated inhibitor of β-catenin and T-cell factor (ICAT), a β-catenin binding protein that inhibits the canonical Wnt/β-catenin signaling pathway, in AR signaling. We demonstrated that expression of ICAT in two AR positive prostate cancer cell lines, LNCaP and LAPC4, augments ligand-dependent AR-mediated transcription. In contrast, short hairpin RNA knockdown of ICAT and β-catenin specifically blocks enhanced AR-mediated transcription by ICAT. Using both stable expression of ICAT and short hairpin RNA knockdown of ICAT expression approaches, we further showed that ICAT enhances expression of endogenous PSA and KLK2, two androgen response genes, and ligand-induced cell growth. In addition, we identified that ICAT and AR can form a ternary complex with β-catenin using in vitro glutathione S-transferase protein pulldown assays. Moreover, we detected the endogenous protein complex containing ICAT, AR, and β-catenin in prostate cancer cells using immunoprecipitation assays. Recruitment of endogenous ICAT onto the promoter region of the human PSA gene, an AR downstream target promoter, was also identified in LNCaP cells. Finally, using in vitro protein binding assays, we examined the effect of full-length and truncated ICAT on the AR-β-catenin interaction and observed that addition of full-length ICAT retained the interaction between β-catenin and AR proteins. Intriguingly, the truncated ICAT comprising the N-terminal helical domain showed a more pronounced effect on β-catenin binding to AR proteins. Our findings suggest a novel molecular mechanism underlying the cross talk between androgen and Wnt signaling pathways.

  View details for DOI 10.1210/me.2011-1023

  View details for Web of Science ID 000295322700001

  View details for PubMedID 21885566

  View details for PubMedCentralID PMC3182419

• Crystal structure of the beta(2) adrenergic receptor-Gs protein complex NATURE Rasmussen, S. G., DeVree, B. T., Zou, Y., Kruse, A. C., Chung, K. Y., Kobilka, T. S., Thian, F. S., Chae, P. S., Pardon, E., Calinski, D., Mathiesen, J. M., Shah, S. T., Lyons, J. A., Caffrey, M., Gellman, S. H., Steyaert, J., Skiniotis, G., Weis, W. I., Sunahara, R. K., Kobilka, B. K. 2011; 477 (7366): 549-U311

  Abstract

  G protein-coupled receptors (GPCRs) are responsible for the majority of cellular responses to hormones and neurotransmitters as well as the senses of sight, olfaction and taste. The paradigm of GPCR signalling is the activation of a heterotrimeric GTP binding protein (G protein) by an agonist-occupied receptor. The β(2) adrenergic receptor (β(2)AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric β(2)AR and nucleotide-free Gs heterotrimer. The principal interactions between the β(2)AR and Gs involve the amino- and carboxy-terminal α-helices of Gs, with conformational changes propagating to the nucleotide-binding pocket. The largest conformational changes in the β(2)AR include a 14 Å outward movement at the cytoplasmic end of transmembrane segment 6 (TM6) and an α-helical extension of the cytoplasmic end of TM5. The most surprising observation is a major displacement of the α-helical domain of Gαs relative to the Ras-like GTPase domain. This crystal structure represents the first high-resolution view of transmembrane signalling by a GPCR.

  View details for DOI 10.1038/nature10361

  View details for Web of Science ID 000295320900031

  View details for PubMedID 21772288

  View details for PubMedCentralID PMC3184188

• Protein Evolution in Cell and Tissue Development: Going Beyond Sequence and Transcriptional Analysis DEVELOPMENTAL CELL Dickinson, D. J., Weis, W. I., Nelson, W. J. 2011; 21 (1): 32-34

  Abstract

  Studies of animal evolution often focus on sequence and transcriptional analysis, based on an assumption that the evolution of development is driven by changes in gene expression. We argue that biochemical and cell biological approaches are also required, because sequence-conserved proteins can have different biochemical, cellular, and developmental properties.

  View details for DOI 10.1016/j.devcel.2011.06.004

  View details for Web of Science ID 000293310200011

  View details for PubMedID 21763606

  View details for PubMedCentralID PMC3145331

• Crystal Structure of a Rigid Four-Spectrin-Repeat Fragment of the Human Desmoplakin Plakin Domain JOURNAL OF MOLECULAR BIOLOGY Choi, H., Weis, W. I. 2011; 409 (5): 800-812

  Abstract

  The plakin protein family serves to connect cell-cell and cell-matrix adhesion molecules to the intermediate filament cytoskeleton. Desmoplakin (DP) is an integral part of desmosomes, where it links desmosomal cadherins to the intermediate filaments. The 1056-amino-acid N-terminal region of DP contains a plakin domain common to members of the plakin family. Plakin domains contain multiple copies of spectrin repeats (SRs). We determined the crystal structure of a fragment of DP, residues 175-630, consisting of four SRs and an inserted SH3 domain. The four repeats form an elongated, rigid structure. The SH3 domain is present in a loop between two helices of an SR and interacts extensively with the preceding SR in a manner that appears to limit inter-repeat flexibility. The intimate intramolecular association of the SH3 domain with the preceding SR is also observed in plectin, another plakin protein, but not in α-spectrin, suggesting that the SH3 domain of plakins contributes to the stability and rigidity of this subfamily of SR-containing proteins.

  View details for DOI 10.1016/j.jmb.2011.04.046

  View details for Web of Science ID 000291913300009

  View details for PubMedID 21536047

  View details for PubMedCentralID PMC3107870

• A Polarized Epithelium Organized by beta- and alpha-Catenin Predates Cadherin and Metazoan Origins SCIENCE Dickinson, D. J., Nelson, W. J., Weis, W. I. 2011; 331 (6022): 1336-1339

  Abstract

  A fundamental characteristic of metazoans is the formation of a simple, polarized epithelium. In higher animals, the structural integrity and functional polarization of simple epithelia require a cell-cell adhesion complex that contains a classical cadherin, the Wnt-signaling protein β-catenin and the actin-binding protein α-catenin. We show that the non-metazoan Dictyostelium discoideum forms a polarized epithelium that is essential for multicellular development. Although D. discoideum lacks a cadherin homolog, we identify an α-catenin ortholog that binds a β-catenin-related protein. Both proteins are essential for formation of the epithelium, polarized protein secretion, and proper multicellular morphogenesis. Thus, the organizational principles of metazoan multicellularity may be more ancient than previously recognized, and the role of the catenins in cell polarity predates the evolution of Wnt signaling and classical cadherins.

  View details for DOI 10.1126/science.1199633

  View details for Web of Science ID 000288215200050

  View details for PubMedID 21393547

  View details for PubMedCentralID PMC3152298

• Structural Basis for Langerin Recognition of Diverse Pathogen and Mammalian Glycans through a Single Binding Site JOURNAL OF MOLECULAR BIOLOGY Feinberg, H., Taylor, M. E., Razi, N., McBride, R., Knirel, Y. A., Graham, S. A., Drickamer, K., Weis, W. I. 2011; 405 (4): 1027-1039

  Abstract

  Langerin mediates the carbohydrate-dependent uptake of pathogens by Langerhans cells in the first step of antigen presentation to the adaptive immune system. Langerin binds to an unusually diverse number of endogenous and pathogenic cell surface carbohydrates, including mannose-containing O-specific polysaccharides derived from bacterial lipopolysaccharides identified here by probing a microarray of bacterial polysaccharides. Crystal structures of the carbohydrate-recognition domain from human langerin bound to a series of oligomannose compounds, the blood group B antigen, and a fragment of β-glucan reveal binding to mannose, fucose, and glucose residues by Ca(2+) coordination of vicinal hydroxyl groups with similar stereochemistry. Oligomannose compounds bind through a single mannose residue, with no other mannose residues contacting the protein directly. There is no evidence for a second Ca(2+)-independent binding site. Likewise, a β-glucan fragment, Glcβ1-3Glcβ1-3Glc, binds to langerin through the interaction of a single glucose residue with the Ca(2+) site. The fucose moiety of the blood group B trisaccharide Galα1-3(Fucα1-2)Gal also binds to the Ca(2+) site, and selective binding to this glycan compared to other fucose-containing oligosaccharides results from additional favorable interactions of the nonreducing terminal galactose, as well as of the fucose residue. Surprisingly, the equatorial 3-OH group and the axial 4-OH group of the galactose residue in 6SO(4)-Galβ1-4GlcNAc also coordinate Ca(2+), a heretofore unobserved mode of galactose binding in a C-type carbohydrate-recognition domain bearing the Glu-Pro-Asn signature motif characteristic of mannose binding sites. Salt bridges between the sulfate group and two lysine residues appear to compensate for the nonoptimal binding of galactose at this site.

  View details for DOI 10.1016/j.jmb.2010.11.039

  View details for Web of Science ID 000286962300010

  View details for PubMedID 21112338

  View details for PubMedCentralID PMC3065333

• Biochemical and Structural Characterization of beta-Catenin Interactions with Nonphosphorylated and CK2-Phosphorylated Lef-1 JOURNAL OF MOLECULAR BIOLOGY Sun, J., Weis, W. I. 2011; 405 (2): 519-530

  Abstract

  In the Wnt/β-catenin signaling pathway, β-catenin activates target genes through its interactions with the T-cell factor/lymphoid enhancer-binding factor (TCF/Lef) family of transcription factors. The crystal structures of complexes between the β-catenin armadillo domain and the Lef-1 N-terminal domain show that the overall conformation and many of the interactions are similar to other published structures of TCFs bound to β-catenin. However, a second salt bridge in other TCF-β-catenin structures is absent in the structure of β-catenin-Lef-1 complex, indicating that this feature is not obligatory for β-catenin binding. Casein kinase II (CK2) has been shown to act as a positive regulator of Wnt signaling, and Lef-1 is a substrate of CK2. In vitro phosphorylation of purified Lef-1 was used to examine the effect of CK2 on the interaction of Lef-1 with β-catenin. Mass spectrometry data show that CK2 phosphorylation of Lef-1 N-terminal domain results in a single phosphorylation site at Ser40. Isothermal titration calorimetry revealed that β-catenin binds to nonphosphorylated or CK2-phosphorylated Lef-1 with the same affinity, which is consistent with the absence of phospho-Ser40 interactions in the crystal structure of phosphorylated Lef-1 N-terminal domain bound to β-catenin. These data indicate that the effect of CK2 on the Wnt/β-catenin pathway does not appear to be at the level of the Lef-1-β-catenin interaction.

  View details for DOI 10.1016/j.jmb.2010.11.010

  View details for Web of Science ID 000286850300016

  View details for PubMedID 21075118

  View details for PubMedCentralID PMC3017643

• Structure of a nanobody-stabilized active state of the beta(2) adrenoceptor NATURE Rasmussen, S. G., Choi, H., Fung, J. J., Pardon, E., Casarosa, P., Chae, P. S., DeVree, B. T., Rosenbaum, D. M., Thian, F. S., Kobilka, T. S., Schnapp, A., Konetzki, I., Sunahara, R. K., Gellman, S. H., Pautsch, A., Steyaert, J., Weis, W. I., Kobilka, B. K. 2011; 469 (7329): 175-180

  Abstract

  G protein coupled receptors (GPCRs) exhibit a spectrum of functional behaviours in response to natural and synthetic ligands. Recent crystal structures provide insights into inactive states of several GPCRs. Efforts to obtain an agonist-bound active-state GPCR structure have proven difficult due to the inherent instability of this state in the absence of a G protein. We generated a camelid antibody fragment (nanobody) to the human β(2) adrenergic receptor (β(2)AR) that exhibits G protein-like behaviour, and obtained an agonist-bound, active-state crystal structure of the receptor-nanobody complex. Comparison with the inactive β(2)AR structure reveals subtle changes in the binding pocket; however, these small changes are associated with an 11 Å outward movement of the cytoplasmic end of transmembrane segment 6, and rearrangements of transmembrane segments 5 and 7 that are remarkably similar to those observed in opsin, an active form of rhodopsin. This structure provides insights into the process of agonist binding and activation.

  View details for DOI 10.1038/nature09648

  View details for Web of Science ID 000286143400030

  View details for PubMedID 21228869

  View details for PubMedCentralID PMC3058308

• Structure and function of an irreversible agonist-beta(2) adrenoceptor complex NATURE Rosenbaum, D. M., Zhang, C., Lyons, J. A., Holl, R., Aragao, D., Arlow, D. H., Rasmussen, S. G., Choi, H., DeVree, B. T., Sunahara, R. K., Chae, P. S., Gellman, S. H., Dror, R. O., Shaw, D. E., Weis, W. I., Caffrey, M., Gmeiner, P., Kobilka, B. K. 2011; 469 (7329): 236-240

  Abstract

  G-protein-coupled receptors (GPCRs) are eukaryotic integral membrane proteins that modulate biological function by initiating cellular signalling in response to chemically diverse agonists. Despite recent progress in the structural biology of GPCRs, the molecular basis for agonist binding and allosteric modulation of these proteins is poorly understood. Structural knowledge of agonist-bound states is essential for deciphering the mechanism of receptor activation, and for structure-guided design and optimization of ligands. However, the crystallization of agonist-bound GPCRs has been hampered by modest affinities and rapid off-rates of available agonists. Using the inactive structure of the human β(2) adrenergic receptor (β(2)AR) as a guide, we designed a β(2)AR agonist that can be covalently tethered to a specific site on the receptor through a disulphide bond. The covalent β(2)AR-agonist complex forms efficiently, and is capable of activating a heterotrimeric G protein. We crystallized a covalent agonist-bound β(2)AR-T4L fusion protein in lipid bilayers through the use of the lipidic mesophase method, and determined its structure at 3.5 Å resolution. A comparison to the inactive structure and an antibody-stabilized active structure (companion paper) shows how binding events at both the extracellular and intracellular surfaces are required to stabilize an active conformation of the receptor. The structures are in agreement with long-timescale (up to 30 μs) molecular dynamics simulations showing that an agonist-bound active conformation spontaneously relaxes to an inactive-like conformation in the absence of a G protein or stabilizing antibody.

  View details for DOI 10.1038/nature09665

  View details for Web of Science ID 000286143400043

  View details for PubMedID 21228876

  View details for PubMedCentralID PMC3074335

• In vitro and in vivo reconstitution of the cadherin-catenin-actin complex from Caenorhabditis elegans PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kwiatkowski, A. V., Maiden, S. L., Pokutta, S., Choi, H., Benjamin, J. M., Lynch, A. M., Nelson, W. J., Weis, W. I., Hardin, J. 2010; 107 (33): 14591-14596

  Abstract

  The ternary complex of cadherin, beta-catenin, and alpha-catenin regulates actin-dependent cell-cell adhesion. alpha-Catenin can bind beta-catenin and F-actin, but in mammals alpha-catenin either binds beta-catenin as a monomer or F-actin as a homodimer. It is not known if this conformational regulation of alpha-catenin is evolutionarily conserved. The Caenorhabditis elegans alpha-catenin homolog HMP-1 is essential for actin-dependent epidermal enclosure and embryo elongation. Here we show that HMP-1 is a monomer with a functional C-terminal F-actin binding domain. However, neither full-length HMP-1 nor a ternary complex of HMP-1-HMP-2(beta-catenin)-HMR-1(cadherin) bind F-actin in vitro, suggesting that HMP-1 is auto-inhibited. Truncation of either the F-actin or HMP-2 binding domain of HMP-1 disrupts C. elegans development, indicating that HMP-1 must be able to bind F-actin and HMP-2 to function in vivo. Our study defines evolutionarily conserved properties of alpha-catenin and suggests that multiple mechanisms regulate alpha-catenin binding to F-actin.

  View details for DOI 10.1073/pnas.1007349107

  View details for PubMedID 20689042

• Direct Ubiquitination of beta-Catenin by Siah-1 and Regulation by the Exchange Factor TBL1 JOURNAL OF BIOLOGICAL CHEMISTRY Dimitrova, Y. N., Li, J., Lee, Y., Rios-Esteves, J., Friedman, D. B., Choi, H., Weis, W. I., Wang, C., Chazin, W. J. 2010; 285 (18): 13507-13516

  Abstract

  Beta-catenin is a key component of the Wnt signaling pathway that functions as a transcriptional co-activator of Wnt target genes. Upon UV-induced DNA damage, beta-catenin is recruited for polyubiquitination and subsequent proteasomal degradation by a unique, p53-induced SCF-like complex (SCF(TBL1)), comprised of Siah-1, Siah-1-interacting protein (SIP), Skp1, transducin beta-like 1 (TBL1), and adenomatous polyposis coli (APC). Given the complexity of the various factors involved and the novelty of ubiquitination of the non-phosphorylated beta-catenin substrate, we have investigated Siah-1-mediated ubiquitination of beta-catenin in vitro and in cells. Overexpression and purification protocols were developed for each of the SCF(TBL1) proteins, enabling a systematic analysis of beta-catenin ubiquitination using an in vitro ubiquitination assay. This study revealed that Siah-1 alone was able to polyubiquitinate beta-catenin. In addition, TBL1 was shown to play a role in protecting beta-catenin from Siah-1 ubiquitination in vitro and from Siah-1-targeted proteasomal degradation in cells. Siah-1 and TBL1 were found to bind to the same armadillo repeat domain of beta-catenin, suggesting that polyubiquitination of beta-catenin is regulated by competition between Siah-1 and TBL1 during Wnt signaling.

  View details for DOI 10.1074/jbc.M109.049411

  View details for Web of Science ID 000276987700023

  View details for PubMedID 20181957

  View details for PubMedCentralID PMC2859511

• Trimeric Structure of Langerin JOURNAL OF BIOLOGICAL CHEMISTRY Feinberg, H., Powlesland, A. S., Taylor, M. E., Weis, W. I. 2010; 285 (17): 13285-13293

  Abstract

  Langerin, an endocytic receptor of Langerhans cells, binds pathogens such as human immunodeficiency virus by recognition of surface glycoconjugates and mediates their internalization into Birbeck granules. Langerin has an extracellular region consisting of a C-type carbohydrate-recognition domain (CRD) and a neck region that stabilizes formation of trimers. As in many other C-type lectins, oligomerization is required for high affinity binding to glycan ligands and is also likely to be important for determining specificity. To facilitate structural analysis of the human langerin trimer, a truncated form of the extracellular region, consisting of part of the neck and the CRD, has been characterized. Like the full-length protein, truncated langerin exists as a stable trimer in solution. Glycan array screening with the trimeric fragment shows that high mannose oligosaccharides are the best ligands for langerin. Structural analysis of the trimeric fragment of langerin confirms that the neck region forms a coiled-coil of alpha-helices. Multiple interactions between the neck region and the CRDs make the trimer a rigid unit with the three CRDs in fixed positions and the primary sugar-binding sites separated by a distance of 42 A. The fixed orientation of the sugar-binding sites in the trimer is likely to place constraints on the ligands that can be bound by langerin.

  View details for DOI 10.1074/jbc.M109.086058

  View details for Web of Science ID 000276787800083

  View details for PubMedID 20181944

  View details for PubMedCentralID PMC2857130

• alpha E-catenin regulates actin dynamics independently of cadherin-mediated cell-cell adhesion JOURNAL OF CELL BIOLOGY Benjamin, J. M., Kwiatkowski, A. V., Yang, C., Korobova, F., Pokutta, S., Svitkina, T., Weis, W. I., Nelson, W. J. 2010; 189 (2): 339-352

  Abstract

  alphaE-catenin binds the cell-cell adhesion complex of E-cadherin and beta-catenin (beta-cat) and regulates filamentous actin (F-actin) dynamics. In vitro, binding of alphaE-catenin to the E-cadherin-beta-cat complex lowers alphaE-catenin affinity for F-actin, and alphaE-catenin alone can bind F-actin and inhibit Arp2/3 complex-mediated actin polymerization. In cells, to test whether alphaE-catenin regulates actin dynamics independently of the cadherin complex, the cytosolic alphaE-catenin pool was sequestered to mitochondria without affecting overall levels of alphaE-catenin or the cadherin-catenin complex. Sequestering cytosolic alphaE-catenin to mitochondria alters lamellipodia architecture and increases membrane dynamics and cell migration without affecting cell-cell adhesion. In contrast, sequestration of cytosolic alphaE-catenin to the plasma membrane reduces membrane dynamics. These results demonstrate that the cytosolic pool of alphaE-catenin regulates actin dynamics independently of cell-cell adhesion.

  View details for DOI 10.1083/jcb.200910041

  View details for PubMedID 20404114

• Structural Correlates of Antibodies Associated with Acute Reversal of Amyloid beta-related Behavioral Deficits in a Mouse Model of Alzheimer Disease JOURNAL OF BIOLOGICAL CHEMISTRY Basi, G. S., Feinberg, H., Oshidari, F., Anderson, J., Barbour, R., Baker, J., Comery, T. A., Diep, L., Gill, D., Johnson-Wood, K., Goel, A., Grantcharova, K., Lee, M., Li, J., Partridge, A., Griswold-Prenner, I., Piot, N., Walker, D., Widom, A., Pangalos, M. N., Seubert, P., Jacobsen, J. S., Schenk, D., Weis, W. I. 2010; 285 (5): 3417-3427

  Abstract

  Immunotherapy targeting of amyloid beta (Abeta) peptide in transgenic mouse models of Alzheimer disease (AD) has been widely demonstrated to resolve amyloid deposition as well as associated neuronal, glial, and inflammatory pathologies. These successes have provided the basis for ongoing clinical trials of immunotherapy for treatment of AD in humans. Acute as well as chronic Abeta-targeted immunotherapy has also been demonstrated to reverse Abeta-related behavioral deficits assessing memory in AD transgenic mouse models. We observe that three antibodies targeting the same linear epitope of Abeta, Abeta(3-7), differ in their ability to reverse contextual fear deficits in Tg2576 mice in an acute testing paradigm. Reversal of contextual fear deficit by the antibodies does not correlate with in vitro recognition of Abeta in a consistent or correlative manner. To better define differences in antigen recognition at the atomic level, we determined crystal structures of Fab fragments in complex with Abeta. The conformation of the Abeta peptide recognized by all three antibodies was highly related and is also remarkably similar to that observed in independently reported Abeta:antibody crystal structures. Sequence and structural differences between the antibodies, particularly in CDR3 of the heavy chain variable region, are proposed to account for differing in vivo properties of the antibodies under study. These findings provide a structural basis for immunotherapeutic strategies targeting Abeta species postulated to underlie cognitive deficits in AD.

  View details for DOI 10.1074/jbc.M109.045187

  View details for Web of Science ID 000273829000056

  View details for PubMedID 19923222

  View details for PubMedCentralID PMC2823416

• Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor NATURE Bokoch, M. P., Zou, Y., Rasmussen, S. G., Liu, C. W., Nygaard, R., Rosenbaum, D. M., Fung, J. J., Choi, H., Thian, F. S., Kobilka, T. S., Puglisi, J. D., Weis, W. I., Pardo, L., Prosser, R. S., Mueller, L., Kobilka, B. K. 2010; 463 (7277): 108-U121

  Abstract

  G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs have revealed structural conservation extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the native ligand-binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the beta(2) adrenergic receptor: a salt bridge linking extracellular loops 2 and 3. Small-molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G-protein activation (agonist, neutral antagonist and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive-state crystal structures.

  View details for DOI 10.1038/nature08650

  View details for PubMedID 20054398

• Segmented Helical Structure of the Neck Region of the Glycan-Binding Receptor DC-SIGNR JOURNAL OF MOLECULAR BIOLOGY Feinberg, H., Tso, C. K., Taylor, M. E., Drickamer, K., Weis, W. I. 2009; 394 (4): 613-620

  Abstract

  Carbohydrate-recognition domains (CRDs) in the glycan-binding receptors DC-SIGN (dendritic-cell-specific intercellular adhesion molecule 1-grabbing nonintegrin; CD209) and DC-SIGNR (DC-SIGN-related receptor, also known as L-SIGN and variously designated CD209L and CD299) are projected from the membrane surface by extended neck domains containing multiple repeats of a largely conserved 23-amino-acid sequence motif. Crystals of a fragment of the neck domain of DC-SIGNR containing multiple repeats in which each molecule extends through multiple unit cells, such that the observed crystallographic asymmetric unit represents one repeat averaged over six repeats of the protein, have been obtained. The repeats are largely alpha-helical. Based on the structure and arrangement of the repeats in the crystal, the neck region can be described as a series of four-helix bundles connected by short, non-helical linkers. Combining the structure of the isolated neck domain with a previously determined overlapping structure of the distal end of the neck region with the CRDs attached provides a model of the almost-complete extracellular portion of the receptor. The results are consistent with previous characterization of the extended structure for the isolated neck region and the extracellular domain. The organization of the neck suggests how CRDs may be disposed differently in DC-SIGN compared with DC-SIGNR and in variant forms of DC-SIGNR assembled from polypeptides with different numbers of repeats in the neck domain.

  View details for DOI 10.1016/j.jmb.2009.10.006

  View details for Web of Science ID 000272602900004

  View details for PubMedID 19835887

  View details for PubMedCentralID PMC2971551

• Interactions of Plakoglobin and beta-Catenin with Desmosomal Cadherins BASIS OF SELECTIVE EXCLUSION OF alpha-AND beta-CATENIN FROM DESMOSOMES JOURNAL OF BIOLOGICAL CHEMISTRY Choi, H., Gross, J. C., Pokutta, S., Weis, W. I. 2009; 284 (46): 31776-31788

  Abstract

  Plakoglobin and beta-catenin are homologous armadillo repeat proteins found in adherens junctions, where they interact with the cytoplasmic domain of classical cadherins and with alpha-catenin. Plakoglobin, but normally not beta-catenin, is also a structural constituent of desmosomes, where it binds to the cytoplasmic domains of the desmosomal cadherins, desmogleins and desmocollins. Here, we report structural, biophysical, and biochemical studies aimed at understanding the molecular basis of selective exclusion of beta-catenin and alpha-catenin from desmosomes. The crystal structure of the plakoglobin armadillo domain bound to phosphorylated E-cadherin shows virtually identical interactions to those observed between beta-catenin and E-cadherin. Trypsin sensitivity experiments indicate that the plakoglobin arm domain by itself is more flexible than that of beta-catenin. Binding of plakoglobin and beta-catenin to the intracellular regions of E-cadherin, desmoglein1, and desmocollin1 was measured by isothermal titration calorimetry. Plakoglobin and beta-catenin bind strongly and with similar thermodynamic parameters to E-cadherin. In contrast, beta-catenin binds to desmoglein-1 more weakly than does plakoglobin. beta-Catenin and plakoglobin bind with similar weak affinities to desmocollin-1. Full affinity binding of desmoglein-1 requires sequences C-terminal to the region homologous to the catenin-binding domain of classical cadherins. Although pulldown assays suggest that the presence of N- and C-terminal beta-catenin "tails" that flank the armadillo repeat region reduces the affinity for desmosomal cadherins, calorimetric measurements show no significant effects of the tails on binding to the cadherins. Using purified proteins, we show that desmosomal cadherins and alpha-catenin compete directly for binding to plakoglobin, consistent with the absence of alpha-catenin in desmosomes.

  View details for DOI 10.1074/jbc.M109.047928

  View details for Web of Science ID 000271572700032

  View details for PubMedID 19759396

  View details for PubMedCentralID PMC2797248

• Structure and Biochemistry of Cadherins and Catenins COLD SPRING HARBOR PERSPECTIVES IN BIOLOGY Shapiro, L., Weis, W. I. 2009; 1 (3)

  Abstract

  Classical cadherins mediate specific adhesion at intercellular adherens junctions. Interactions between cadherin ectodomains from apposed cells mediate cell-cell contact, whereas the intracellular region functionally links cadherins to the underlying cytoskeleton. Structural, biophysical, and biochemical studies have provided important insights into the mechanism and specificity of cell-cell adhesion by classical cadherins and their interplay with the cytoskeleton. Adhesive binding arises through exchange of beta strands between the first extracellular cadherin domains (EC1) of partner cadherins from adjacent cells. This "strand-swap" binding mode is common to classical and desmosomal cadherins, but sequence alignments suggest that other cadherins will bind differently. The intracellular region of classical cadherins binds to p120 and beta-catenin, and beta-catenin binds to the F-actin binding protein alpha-catenin. Rather than stably bridging beta-catenin to actin, it appears that alpha-catenin actively regulates the actin cytoskeleton at cadherin-based cell-cell contacts.

  View details for DOI 10.1101/cshperspect.a003053

  View details for Web of Science ID 000279879100008

  View details for PubMedID 20066110

• The First Propeller Domain of LRP6 Regulates Sensitivity to DKK1 MOLECULAR BIOLOGY OF THE CELL Binnerts, M. E., Tomasevic, N., Bright, J. M., Leung, J., Ahn, V. E., Kim, K., Zhan, X., Liu, S., Yonkovich, S., Williams, J., Zhou, M., Gros, D., Dixon, M., Korver, W., Weis, W. I., Abo, A. 2009; 20 (15): 3552-3560

  Abstract

  The Wnt coreceptor LRP6 is required for canonical Wnt signaling. To understand the molecular regulation of LRP6 function, we generated a series of monoclonal antibodies against the extra cellular domain (ECD) of LRP6 and selected a high-affinity mAb (mAb135) that recognizes cell surface expression of endogenous LRP6. mAb135 enhanced Wnt dependent TCF reporter activation and antagonized DKK1 dependent inhibition of Wnt3A signaling, suggesting a role in modulation of LRP6 function. Detailed analysis of LRP6 domain mutants identified Ser 243 in the first propeller domain of LRP6 as a critical residue for mAb135 binding, implicating this domain in regulating the sensitivity of LRP6 to DKK1. In agreement with this notion, mAb135 directly disrupted the interaction of DKK1 with recombinant ECD LRP6 and a truncated form of the LRP6 ECD containing only repeats 1 and 2. Finally, we found that mAb135 completely protected LRP6 from DKK1 dependent internalization. Together, these results identify the first propeller domain as a novel regulatory domain for DKK1 binding to LRP6 and show that mAb against the first propeller domain of LRP6 can be used to modulate this interaction.

  View details for DOI 10.1091/mbc.E08-12-1252

  View details for Web of Science ID 000268545300011

  View details for PubMedID 19477926

• CRYSTAL STRUCTURES OF THE beta(2)-ADRENERGIC RECEPTOR 40th Erice Course on From Molecules to Medicine - Structure of Biological Macromolecules and Its Relevance in Combating New Diseases and Bioterrorism Weis, W. I., Rosenbaum, D. M., Rasmussen, S. G., Choi, H., Thian, F. S., Kobilka, T. S., Yao, X., Day, P. W., Parnot, C., Fung, J. J., Ratnala, V. R., Kobilka, B. K., Cherezov, V., Hanson, M. A., Kuhn, P., Stevens, R. C., Edwards, P. C., Schertler, G. F., Burghammer, M., Sanishvili, R., Fischetti, R. F., Masood, A., Rohrer, D. K. SPRINGER. 2009: 217–230

  View details for Web of Science ID 000266237200014

• Direct Inhibition of GSK3 beta by the Phosphorylated Cytoplasmic Domain of LRP6 in Wnt/beta-Catenin Signaling PLOS ONE Piao, S., Lee, S., Kim, H., Yum, S., Stamos, J. L., Xu, Y., Lee, S., Lee, J., Oh, S., Han, J., Park, B., Weis, W. I., Ha, N. 2008; 3 (12)

  Abstract

  Wnt/beta-catenin signaling plays a central role in development and is also involved in a diverse array of diseases. Binding of Wnts to the coreceptors Frizzled and LRP6/5 leads to phosphorylation of PPPSPxS motifs in the LRP6/5 intracellular region and the inhibition of GSK3beta bound to the scaffold protein Axin. However, it remains unknown how GSK3beta is specifically inhibited upon Wnt stimulation. Here, we show that overexpression of the intracellular region of LRP6 containing a Ser/Thr rich cluster and a PPPSPxS motif impairs the activity of GSK3beta in cells. Synthetic peptides containing the PPPSPxS motif strongly inhibit GSK3beta in vitro only when they are phosphorylated. Microinjection of these peptides into Xenopus embryos confirms that the phosphorylated PPPSPxS motif potentiates Wnt-induced second body axis formation. In addition, we show that the Ser/Thr rich cluster of LRP6 plays an important role in LRP6 binding to GSK3beta. These observations demonstrate that phosphorylated LRP6/5 both recruits and directly inhibits GSK3beta using two distinct portions of its cytoplasmic sequence, and suggest a novel mechanism of activation in this signaling pathway.

  View details for DOI 10.1371/journal.pone.0004046

  View details for Web of Science ID 000265463000032

  View details for PubMedID 19107203

  View details for PubMedCentralID PMC2603313

• Structural insights into G-protein-coupled receptor activation CURRENT OPINION IN STRUCTURAL BIOLOGY Weis, W. I., Kobilka, B. K. 2008; 18 (6): 734-740

  Abstract

  G-protein-coupled receptors (GPCRs) are the largest family of eukaryotic plasma membrane receptors, and are responsible for the majority of cellular responses to external signals. GPCRs share a common architecture comprising seven transmembrane (TM) helices. Binding of an activating ligand enables the receptor to catalyze the exchange of GTP for GDP in a heterotrimeric G protein. GPCRs are in a conformational equilibrium between inactive and activating states. Crystallographic and spectroscopic studies of the visual pigment rhodopsin and two beta-adrenergic receptors have defined some of the conformational changes associated with activation.

  View details for DOI 10.1016/j.sbi.2008.09.010

  View details for Web of Science ID 000262064300014

  View details for PubMedID 18957321

• Improved structures of full-length p97, an AAA ATPase: Implications for mechanisms of nucleotide-dependent conformational change STRUCTURE Davies, J. M., Brunger, A. T., Weis, W. I. 2008; 16 (5): 715-726

  Abstract

  The ATPases associated with various cellular activities (AAA) protein p97 has been implicated in a variety of cellular processes, including endoplasmic reticulum-associated degradation and homotypic membrane fusion. p97 belongs to a subgroup of AAA proteins that contains two nucleotide binding domains, D1 and D2. We determined the crystal structure of D2 at 3.0 A resolution. This model enabled rerefinement of full-length p97 in different nucleotide states against previously reported low-resolution diffraction data to significantly improved R values and Ramachandran statistics. Although the overall fold remained similar, there are significant improvements, especially around the D2 nucleotide binding site. The rerefinement illustrates the importance of knowledge of high-resolution structures of fragments covering most of the whole molecule. The structures suggest that nucleotide hydrolysis is transformed into larger conformational changes by pushing of one D2 domain by its neighbor in the hexamer, and transmission of nucleotide-state information through the D1-D2 linker to displace the N-terminal, effector binding domain.

  View details for DOI 10.1016/j.str.2008.02.010

  View details for Web of Science ID 000255728700011

  View details for PubMedID 18462676

• Munc18a controls SNARE assembly through its interaction with the syntaxin N-peptid EMBO JOURNAL Burkhardt, P., Hattendorf, D. A., Weis, W. I., Fasshauer, D. 2008; 27 (7): 923-933

  Abstract

  Sec1/Munc18-like (SM) proteins functionally interact with SNARE proteins in vesicular fusion. Despite their high sequence conservation, structurally disparate binding modes for SM proteins with syntaxins have been observed. Several SM proteins appear to bind only to a short peptide present at the N terminus of syntaxin, designated the N-peptide, while Munc18a binds to a 'closed' conformation formed by the remaining portion of syntaxin 1a. Here, we show that the syntaxin 16 N-peptide binds to the SM protein Vps45, but the remainder of syntaxin 16 strongly enhances the affinity of the interaction. Likewise, the N-peptide of syntaxin 1a serves as a second binding site in the Munc18a/syntaxin 1a complex. When the syntaxin 1a N-peptide is bound to Munc18a, SNARE complex formation is blocked. Removal of the N-peptide enables binding of syntaxin 1a to its partner SNARE SNAP-25, while still bound to Munc18a. This suggests that Munc18a controls the accessibility of syntaxin 1a to its partners, a role that might be common to all SM proteins.

  View details for DOI 10.1038/emboj.2008.37

  View details for Web of Science ID 000255291300001

  View details for PubMedID 18337752

• Biochemical and structural analysis of alpha-catenin in cell-cell contacts BIOCHEMICAL SOCIETY TRANSACTIONS Pokutta, S., Drees, F., Yamada, S., Nelson, W. J., Weis, W. I. 2008; 36: 141-147

  Abstract

  Cadherins are transmembrane adhesion molecules that mediate homotypic cell-cell contact. In adherens junctions, the cytoplasmic domain of cadherins is functionally linked to the actin cytoskeleton through a series of proteins known as catenins. E-cadherin binds to beta-catenin, which in turn binds to alpha-catenin to form a ternary complex. alpha-Catenin also binds to actin, and it was assumed previously that alpha-catenin links the cadherin-catenin complex to actin. However, biochemical, structural and live-cell imaging studies of the cadherin-catenin complex and its interaction with actin show that binding of beta-catenin to alpha-catenin prevents the latter from binding to actin. Biochemical and structural data indicate that alpha-catenin acts as an allosteric protein whose conformation and activity changes depending on whether or not it is bound to beta-catenin. Initial contacts between cells occur on dynamic lamellipodia formed by polymerization of branched actin networks, a process controlled by the Arp2/3 (actin-related protein 2/3) complex. alpha-Catenin can suppress the activity of Arp2/3 by competing for actin filaments. These findings lead to a model for adherens junction formation in which clustering of the cadherin-beta-catenin complex recruits high levels of alpha-catenin that can suppress the Arp2/3 complex, leading to cessation of lamellipodial movement and formation of a stable contact. Thus alpha-catenin appears to play a central role in cell-cell contact formation.

  View details for DOI 10.1042/BST0360141

  View details for Web of Science ID 000255371000001

  View details for PubMedID 18363554

  View details for PubMedCentralID PMC3369830

• GPCR engineering yields high-resolution structural insights into beta(2)-adrenergic receptor function SCIENCE Rosenbaum, D. M., Cherezov, V., Hanson, M. A., Rasmussen, S. G., Thian, F. S., Kobilka, T. S., Choi, H., Yao, X., Weis, W. I., Stevens, R. C., Kobilka, B. K. 2007; 318 (5854): 1266-1273

  Abstract

  The beta2-adrenergic receptor (beta2AR) is a well-studied prototype for heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) that respond to diffusible hormones and neurotransmitters. To overcome the structural flexibility of the beta2AR and to facilitate its crystallization, we engineered a beta2AR fusion protein in which T4 lysozyme (T4L) replaces most of the third intracellular loop of the GPCR ("beta2AR-T4L") and showed that this protein retains near-native pharmacologic properties. Analysis of adrenergic receptor ligand-binding mutants within the context of the reported high-resolution structure of beta2AR-T4L provides insights into inverse-agonist binding and the structural changes required to accommodate catecholamine agonists. Amino acids known to regulate receptor function are linked through packing interactions and a network of hydrogen bonds, suggesting a conformational pathway from the ligand-binding pocket to regions that interact with G proteins.

  View details for DOI 10.1126/science.1150609

  View details for Web of Science ID 000251086600034

  View details for PubMedID 17962519

• High-resolution crystal structure of an engineered human beta(2)-adrenergic G protein-coupled receptor SCIENCE Cherezov, V., Rosenbaum, D. M., Hanson, M. A., Rasmussen, S. G., Thian, F. S., Kobilka, T. S., Choi, H., Kuhn, P., Weis, W. I., Kobilka, B. K., Stevens, R. C. 2007; 318 (5854): 1258-1265

  Abstract

  Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors constitute the largest family of eukaryotic signal transduction proteins that communicate across the membrane. We report the crystal structure of a human beta2-adrenergic receptor-T4 lysozyme fusion protein bound to the partial inverse agonist carazolol at 2.4 angstrom resolution. The structure provides a high-resolution view of a human G protein-coupled receptor bound to a diffusible ligand. Ligand-binding site accessibility is enabled by the second extracellular loop, which is held out of the binding cavity by a pair of closely spaced disulfide bridges and a short helical segment within the loop. Cholesterol, a necessary component for crystallization, mediates an intriguing parallel association of receptor molecules in the crystal lattice. Although the location of carazolol in the beta2-adrenergic receptor is very similar to that of retinal in rhodopsin, structural differences in the ligand-binding site and other regions highlight the challenges in using rhodopsin as a template model for this large receptor family.

  View details for DOI 10.1126/science.1150577

  View details for Web of Science ID 000251086600033

  View details for PubMedID 17962520

  View details for PubMedCentralID PMC2583103

• Crystal structure of the human beta(2) adrenergic G-protein-coupled receptor NATURE Rasmussen, S. G., Choi, H., Rosenbaum, D. M., Kobilka, T. S., Thian, F. S., Edwards, P. C., Burghammer, M., Ratnala, V. R., Sanishvili, R., Fischetti, R. F., Schertler, G. F., Weis, W. I., Kobilka, B. K. 2007; 450 (7168): 383-U4

  Abstract

  Structural analysis of G-protein-coupled receptors (GPCRs) for hormones and neurotransmitters has been hindered by their low natural abundance, inherent structural flexibility, and instability in detergent solutions. Here we report a structure of the human beta2 adrenoceptor (beta2AR), which was crystallized in a lipid environment when bound to an inverse agonist and in complex with a Fab that binds to the third intracellular loop. Diffraction data were obtained by high-brilliance microcrystallography and the structure determined at 3.4 A/3.7 A resolution. The cytoplasmic ends of the beta2AR transmembrane segments and the connecting loops are well resolved, whereas the extracellular regions of the beta2AR are not seen. The beta2AR structure differs from rhodopsin in having weaker interactions between the cytoplasmic ends of transmembrane (TM)3 and TM6, involving the conserved E/DRY sequences. These differences may be responsible for the relatively high basal activity and structural instability of the beta2AR, and contribute to the challenges in obtaining diffraction-quality crystals of non-rhodopsin GPCRs.

  View details for DOI 10.1038/nature06325

  View details for Web of Science ID 000250918600046

  View details for PubMedID 17952055

• A monoclonal antibody for G protein-coupled receptor crystallography NATURE METHODS Day, P. W., Rasmussen, S. G., Parnot, C., Fung, J. J., Masood, A., Kobilka, T. S., Yao, X., Choi, H., Weis, W. I., Rohrer, D. K., Kobilka, B. K. 2007; 4 (11): 927-929

  Abstract

  G protein-coupled receptors (GPCRs) constitute the largest family of signaling proteins in mammals, mediating responses to hormones, neurotransmitters, and senses of sight, smell and taste. Mechanistic insight into GPCR signal transduction is limited by a paucity of high-resolution structural information. We describe the generation of a monoclonal antibody that recognizes the third intracellular loop (IL3) of the native human beta(2) adrenergic (beta(2)AR) receptor; this antibody was critical for acquiring diffraction-quality crystals.

  View details for DOI 10.1038/NMETH1112

  View details for Web of Science ID 000250575700017

  View details for PubMedID 17952087

• Catenins: playing both sides of the synapse CURRENT OPINION IN CELL BIOLOGY Kwiatkowski, A. V., Weis, W. I., Nelson, W. J. 2007; 19 (5): 551-556

  Abstract

  Synapses of the central nervous system (CNS) are specialized cell-cell junctions that mediate intercellular signal transmission from one neuron to another. The directional nature of signal relay requires synaptic contacts to be morphologically asymmetric with distinct protein components, while changes in synaptic communication during neural network formation require synapses to be plastic. Synapse morphology and plasticity require a dynamic actin cytoskeleton. Classical cadherins, which are junctional proteins associated with the actin cytoskeleton, localize to synapses and regulate synaptic adhesion, stability and remodeling. The major intracellular components of cadherin junctions are the catenin proteins, and increasing evidence suggests that cadherin-catenin complexes modulate an array of synaptic processes. Here we review the role of catenins in regulating the development of pre- and postsynaptic compartments and function in synaptic plasticity, with particular focus on their role in regulating the actin cytoskeleton.

  View details for DOI 10.1016/j.ceb.2007.08.005

  View details for Web of Science ID 000251164200009

  View details for PubMedID 17936606

  View details for PubMedCentralID PMC2674286

• Scavenger receptor C-type lectin binds to the leukocyte cell surface glycan Lewis(x) by a novel mechanism JOURNAL OF BIOLOGICAL CHEMISTRY Feinberg, H., Taylor, M. E., Weis, W. I. 2007; 282 (23): 17250-17258

  Abstract

  The scavenger receptor C-type lectin (SRCL) is unique in the family of class A scavenger receptors, because in addition to binding sites for oxidized lipoproteins it also contains a C-type carbohydrate-recognition domain (CRD) that interacts with specific glycans. Both human and mouse SRCL are highly specific for the Lewis(x) trisaccharide, which is commonly found on the surfaces of leukocytes and some tumor cells. Structural analysis of the CRD of mouse SRCL in complex with Lewis(x) and mutagenesis show the basis for this specificity. The interaction between mouse SRCL and Lewis(x) is analogous to the way that selectins and DC-SIGN bind to related fucosylated glycans, but the mechanism of the interaction is novel, because it is based on a primary galactose-binding site similar to the binding site in the asialoglycoprotein receptor. Crystals of the human receptor lacking bound calcium ions reveal an alternative conformation in which a glycan ligand would be released during receptor-mediated endocytosis.

  View details for DOI 10.1074/jbc.M701624200

  View details for Web of Science ID 000246946500058

  View details for PubMedID 17420244

  View details for PubMedCentralID PMC2289868

• Structure of the yeast polarity protein Sro7 reveals a SNARE regulatory mechanism NATURE Hattendorf, D. A., Andreeva, A., Gangar, A., Brennwald, P. J., Weis, W. I. 2007; 446 (7135): 567-571

  Abstract

  Polarized exocytosis requires coordination between the actin cytoskeleton and the exocytic machinery responsible for fusion of secretory vesicles at specific sites on the plasma membrane. Fusion requires formation of a complex between a vesicle-bound R-SNARE and plasma membrane Qa, Qb and Qc SNARE proteins. Proteins in the lethal giant larvae protein family, including lethal giant larvae and tomosyn in metazoans and Sro7 in yeast, interact with Q-SNAREs and are emerging as key regulators of polarized exocytosis. The crystal structure of Sro7 reveals two seven-bladed WD40 beta-propellers followed by a 60-residue-long 'tail', which is bound to the surface of the amino-terminal propeller. Deletion of the Sro7 tail enables binding to the Qbc SNARE region of Sec9 and this interaction inhibits SNARE complex assembly. The N-terminal domain of Sec9 provides a second, high-affinity Sro7 interaction that is unaffected by the tail. The results suggest that Sro7 acts as an allosteric regulator of exocytosis through interactions with factors that control the tail. Sequence alignments indicate that lethal giant larvae and tomosyn have a two-beta-propeller fold similar to that of Sro7, but only tomosyn appears to retain the regulatory tail.

  View details for DOI 10.1038/nature05635

  View details for Web of Science ID 000245242900055

  View details for PubMedID 17392788

• Multiple modes of binding enhance the affinity of DC-SIGN for high mannose N-linked glycans found on viral glycoproteins JOURNAL OF BIOLOGICAL CHEMISTRY Feinberg, H., Castelli, R., Drickamer, K., Seeberger, P. H., Weis, W. I. 2007; 282 (6): 4202-4209

  Abstract

  The dendritic cell surface receptor DC-SIGN and the closely related endothelial cell receptor DC-SIGNR specifically recognize high mannose N-linked carbohydrates on viral pathogens. Previous studies have shown that these receptors bind the outer trimannose branch Manalpha1-3[Manalpha1-6]Manalpha present in high mannose structures. Although the trimannoside binds to DC-SIGN or DC-SIGNR more strongly than mannose, additional affinity enhancements are observed in the presence of one or more Manalpha1-2Manalpha moieties on the nonreducing termini of oligomannose structures. The molecular basis of this enhancement has been investigated by determining crystal structures of DC-SIGN bound to a synthetic six-mannose fragment of a high mannose N-linked oligosaccharide, Manalpha1-2Manalpha1-3[Manalpha1-2Manalpha1-6]Manalpha1-6Man and to the disaccharide Manalpha1-2Man. The structures reveal mixtures of two binding modes in each case. Each mode features typical C-type lectin binding at the principal Ca2+-binding site by one mannose residue. In addition, other sugar residues form contacts unique to each binding mode. These results suggest that the affinity enhancement displayed toward oligosaccharides decorated with the Manalpha1-2Manalpha structure is due in part to multiple binding modes at the primary Ca2+ site, which provide both additional contacts and a statistical (entropic) enhancement of binding.

  View details for DOI 10.1074/jbc.M609689200

  View details for Web of Science ID 000244481900083

  View details for PubMedID 17150970

  View details for PubMedCentralID PMC2277367

• Structure and mechanism of Cadherins and catenins in cell-cell contacts ANNUAL REVIEW OF CELL AND DEVELOPMENTAL BIOLOGY Pokutta, S., Weis, W. I. 2007; 23: 237-261

  Abstract

  Cadherins are Ca(2+)-dependent cell adhesion molecules found in several kinds of cell-cell contact, including adherens junctions and desmosomes. In the presence of Ca(2+), cells expressing the same type of cadherin form stable contacts with one another, a phenomenon designated homophilic, or homotypic, adhesion. Most cadherins are single-pass transmembrane proteins whose extracellular regions mediate specific cell-cell interactions. The intracellular faces of these contacts are associated with the actin cytoskeleton in adherens junctions or the intermediate-filament system in desmosomes. The close coordination of the transmembrane adhesion molecules with the cytoskeleton is believed to be essential in coordinating morphogenetic movements of tissues during development and in conferring the appropriate mechanical properties to cell-cell contacts. Structural, biochemical, and biophysical analysis of the molecules that comprise these contacts has provided unique mechanistic insights into the specificity of homophilic adhesion, the functional connection to the underlying cytoskeleton, and the dynamics of junction formation.

  View details for DOI 10.1146/annurev.cellbio.22.010305.104241

  View details for Web of Science ID 000250896200010

  View details for PubMedID 17539752

• Re-solving the cadherin-catenin-actin conundrum JOURNAL OF BIOLOGICAL CHEMISTRY Weis, W. I., Nelson, W. J. 2006; 281 (47): 35593-35597

  View details for DOI 10.1074/jbc.R600027200

  View details for Web of Science ID 000242100500002

  View details for PubMedID 17005550

  View details for PubMedCentralID PMC3368706

• Crystal structure of the S-cerevisiae exocyst component Exo70p JOURNAL OF MOLECULAR BIOLOGY Hamburger, Z. A., Hamburger, A. E., West, A. P., Weis, W. I. 2006; 356 (1): 9-21

  Abstract

  The exocyst is an evolutionarily conserved multiprotein complex required for the targeting and docking of post-Golgi vesicles to the plasma membrane. Through its interactions with a variety of proteins, including small GTPases, the exocyst is thought to integrate signals from the cell and signal that vesicles arriving at the plasma membrane are ready for fusion. Here we describe the three-dimensional crystal structure of one of the components of the exocyst, Exo70p, from Saccharomyces cerevisiae at 3.5A resolution. Exo70p binds the small GTPase Rho3p in a GTP-dependent manner with an equilibrium dissociation constant of approximately 70 microM. Exo70p is an extended rod approximately 155 angstroms in length composed principally of alpha helices, and is a novel fold. The structure provides a first view of the Exo70 protein family and provides a framework to study the molecular function of this exocyst component.

  View details for DOI 10.1016/j.jmb.2005.09.099

  View details for Web of Science ID 000234938600002

  View details for PubMedID 16359701

• Thermodynamics of beta-catenin-ligand interactions - The roles of the N- and C-terminal tails in modulating binding affinity JOURNAL OF BIOLOGICAL CHEMISTRY Choi, H. J., Huber, A. H., Weis, W. I. 2006; 281 (2): 1027-1038

  Abstract

  beta-Catenin is a structural component of adherens junctions, where it binds to the cytoplasmic domain of cadherin cell adhesion molecules. beta-Catenin is also a transcriptional coactivator in the Wnt signaling pathway, where it binds to Tcf/Lef family transcription factors. In the absence of a Wnt signal, nonjunctional beta-catenin is present in a multiprotein complex containing the proteins axin and adenomatous polyposis coli (APC), both of which bind directly to beta-catenin. The thermodynamics of beta-catenin binding to E-cadherin, Lef-1, APC, axin, and the transcriptional inhibitor ICAT have been determined by isothermal titration calorimetry. Most of the interactions showed large, unfavorable entropy changes, consistent with these ligands being natively unstructured in the absence of beta-catenin. Phosphorylation of serine residues present in a sequence motif common to cadherins and APC increased the affinity for beta-catenin 300-700-fold, and surface plasmon resonance measurements revealed that phosphorylation of E-cadherin both enhanced its on rate and decreased its off rate. The effects of the N- and C-terminal "tails" that flank the beta-catenin armadillo repeat domain on ligand binding have also been investigated using constructs lacking one or both tails. Contrary to earlier studies that employed less direct binding assays, the tails did not affect the affinity of beta-catenin for tight ligands such as E-cadherin, Lef-1, and phosphorylated APC. However, the beta-catenin C-terminal tail was found to decrease the affinity for the weaker ligands APC and axin, suggesting that this region may have a regulatory role in beta-catenin degradation.

  View details for DOI 10.1074/jbc.M511338200

  View details for Web of Science ID 000234447200043

  View details for PubMedID 16293619

• Deconstructing the cadherin-catenin-actin complex CELL Yamada, S., Pokutta, S., Drees, F., Weis, W. I., Nelson, W. J. 2005; 123 (5): 889-901

  Abstract

  Spatial and functional organization of cells in tissues is determined by cell-cell adhesion, thought to be initiated through trans-interactions between extracellular domains of the cadherin family of adhesion proteins, and strengthened by linkage to the actin cytoskeleton. Prevailing dogma is that cadherins are linked to the actin cytoskeleton through beta-catenin and alpha-catenin, although the quaternary complex has never been demonstrated. We test this hypothesis and find that alpha-catenin does not interact with actin filaments and the E-cadherin-beta-catenin complex simultaneously, even in the presence of the actin binding proteins vinculin and alpha-actinin, either in solution or on isolated cadherin-containing membranes. Direct analysis in polarized cells shows that mobilities of E-cadherin, beta-catenin, and alpha-catenin are similar, regardless of the dynamic state of actin assembly, whereas actin and several actin binding proteins have higher mobilities. These results suggest that the linkage between the cadherin-catenin complex and actin filaments is more dynamic than previously appreciated.

  View details for DOI 10.1016/j.cell.2005.09.020

  View details for Web of Science ID 000233814100021

  View details for PubMedID 16325582

  View details for PubMedCentralID PMC3368712

• alpha-catenin is a molecular switch that binds E-cadherin-beta-catenin and regulates actin-filament assembly CELL Drees, F., Pokutta, S., Yamada, S., Nelson, W. J., Weis, W. I. 2005; 123 (5): 903-915

  Abstract

  Epithelial cell-cell junctions, organized by adhesion proteins and the underlying actin cytoskeleton, are considered to be stable structures maintaining the structural integrity of tissues. Contrary to the idea that alpha-catenin links the adhesion protein E-cadherin through beta-catenin to the actin cytoskeleton, in the accompanying paper we report that alpha-catenin does not bind simultaneously to both E-cadherin-beta-catenin and actin filaments. Here we demonstrate that alpha-catenin exists as a monomer or a homodimer with different binding properties. Monomeric alpha-catenin binds more strongly to E-cadherin-beta-catenin, whereas the dimer preferentially binds actin filaments. Different molecular conformations are associated with these different binding states, indicating that alpha-catenin is an allosteric protein. Significantly, alpha-catenin directly regulates actin-filament organization by suppressing Arp2/3-mediated actin polymerization, likely by competing with the Arp2/3 complex for binding to actin filaments. These results indicate a new role for alpha-catenin in local regulation of actin assembly and organization at sites of cadherin-mediated cell-cell adhesion.

  View details for DOI 10.1016/j.cell.2005.09.021

  View details for Web of Science ID 000233814100022

  View details for PubMedID 16325583

  View details for PubMedCentralID PMC3369825

• Structural basis of DC-SIGN ligand specificity Annual Meeting of the Society-for-Glycobiology Feinberg, H., Guo, Y., Conroy, E., Mitchell, D., Alvarez, R., Blixt, O., Taylor, M., Drickamer, K., Weis, W. OXFORD UNIV PRESS INC. 2005: 1195–95

  View details for Web of Science ID 000232920300048

• beta-catenin directly displaces Groucho/TLE repressors from Tcf/Lef in Wnt-mediated transcription activation NATURE STRUCTURAL & MOLECULAR BIOLOGY Daniels, D. L., Weis, W. I. 2005; 12 (4): 364-371

  Abstract

  Wnt growth factors mediate cell fate determination during embryogenesis and in the renewal of tissues in the adult. Wnts act by stabilizing cellular levels of the transcriptional coactivator beta-catenin, which forms complexes with sequence-specific DNA-binding Tcf/Lef transcription factors. In the absence of nuclear beta-catenin, Tcf/Lefs act as transcriptional repressors by binding to Groucho/TLE proteins. The molecular basis of the switch from transcriptional repression to activation during Wnt signaling has not been clear, in particular whether factors other than beta-catenin are required to disrupt the interaction between Groucho/TLE and Tcf/Lef. Using highly purified proteins, we demonstrate that beta-catenin displaces Groucho/TLE from Tcf/Lef by binding to a previously unidentified second, low-affinity binding site on Lef-1 that includes sequences just N-terminal to the DNA-binding domain, and that overlaps the Groucho/TLE-binding site.

  View details for DOI 10.1038/nsmb912

  View details for Web of Science ID 000228126200018

  View details for PubMedID 15768032

• Structure of the armadillo repeat domain of plakophilin 1 JOURNAL OF MOLECULAR BIOLOGY Choi, H. J., Weis, W. I. 2005; 346 (1): 367-376

  Abstract

  The p120ctn subfamily of armadillo domain proteins has roles in modulating intercellular adhesion by cadherin-containing junctions. We have determined the crystal structure of the arm repeat domain from plakophilin-1 (PKP1), a member of the p120ctn subfamily that is found in desmosomes. The structure reveals that the domain has nine instead of the expected ten arm repeats. A sequence predicted to be an arm repeat is instead a large insert which serves as a wedge that produces a significant bend in the overall domain structure. Structure-based sequence alignments indicate that the nine repeats and large insert are common to this subfamily of armadillo proteins. A prominent basic patch on the surface of the protein may serve as a binding site for partners of these proteins.

  View details for DOI 10.1016/j.jmb.2004.11.048

  View details for Web of Science ID 000226674900030

  View details for PubMedID 15663951

• Conformational changes of p97 during nucleotide hydrolysis determined by small-angle X-ray scattering STRUCTURE Davies, J. M., Tsuruta, H., May, A. P., Weis, W. I. 2005; 13 (2): 183-195

  Abstract

  Valosin-containing protein (VCP)/p97 is an AAA family ATPase that has been implicated in the removal of misfolded proteins from the endoplasmic reticulum and in membrane fusion. p97 forms a homohexamer whose protomers consist of an N-terminal (N) domain responsible for binding to effector proteins, followed by two AAA ATPase domains, D1 and D2. Small-angle X-ray scattering (SAXS) measurements of p97 in the presence of AMP-PNP (ATP state), ADP-AlF(x) (hydrolysis transition state), ADP, or no nucleotide reveal major changes in the positions of the N domains with respect to the hexameric ring during the ATP hydrolysis cycle. Nucleotide binding and hydrolysis experiments indicate that D2 inhibits nucleotide exchange by D1. The data suggest that the conversion of the chemical energy of ATP hydrolysis into mechanical work on substrates involves transmission of conformational changes generated by D2 through D1 to move N.

  View details for DOI 10.1016/j.str.2004.11.014

  View details for Web of Science ID 000227076400005

  View details for PubMedID 15698563

• Extended neck regions stabilize tetramers of the receptors DC-SIGN and DC-SIGNR JOURNAL OF BIOLOGICAL CHEMISTRY Feinberg, H., Guo, Y., Mitchell, D. A., Drickamer, K., Weis, W. I. 2005; 280 (2): 1327-1335

  Abstract

  The human cell surface receptors DC-SIGN (dendritic cell-specific intercellular adhesion molecule-grabbing nonintegrin) and DC-SIGNR (DC-SIGN-related) bind to oligosaccharide ligands found on human tissues as well as on pathogens including viruses, bacteria, and parasites. The extracellular portion of each receptor contains a membrane-distal carbohydrate-recognition domain (CRD) and forms tetramers stabilized by an extended neck region consisting of 23 amino acid repeats. Cross-linking analysis of full-length receptors expressed in fibroblasts confirms the tetrameric state of the intact receptors. Hydrodynamic studies on truncated receptors demonstrate that the portion of the neck of each protein adjacent to the CRD is sufficient to mediate the formation of dimers, whereas regions near the N terminus are needed to stabilize the tetramers. Some of the intervening repeats are missing from polymorphic forms of DC-SIGNR. Two different crystal forms of truncated DC-SIGNR comprising two neck repeats and the CRD reveal that the CRDs are flexibly linked to the neck, which contains alpha-helical segments interspersed with non-helical regions. Differential scanning calorimetry measurements indicate that the neck and CRDs are independently folded domains. Based on the crystal structures and hydrodynamic data, models for the full extracellular domains of the receptors have been generated. The observed flexibility of the CRDs in the tetramer, combined with previous data on the specificity of these receptors, suggests an important role for oligomerization in the recognition of endogenous glycans, in particular those present on the surfaces of enveloped viruses recognized by these proteins.

  View details for DOI 10.1074/jbc.M409925200

  View details for Web of Science ID 000226195200058

  View details for PubMedID 15509576

• Mechanism of phosphorylation-dependent binding of APC to beta-catenin and its role in beta-catenin degradation MOLECULAR CELL Ha, N. C., Tonozuka, T., Stamos, J. L., Choi, H. J., Weis, W. I. 2004; 15 (4): 511-521

  Abstract

  The transcriptional coactivator beta-catenin mediates Wnt growth factor signaling. In the absence of a Wnt signal, casein kinase 1 (CK1) and glycogen synthase kinase-3beta (GSK-3beta) phosphorylate cytosolic beta-catenin, thereby flagging it for recognition and destruction by the ubiquitin/proteosome machinery. Phosphorylation occurs in a multiprotein complex that includes the kinases, beta-catenin, axin, and the Adenomatous Polyposis Coli (APC) protein. The role of APC in this process is poorly understood. CK1epsilon and GSK-3beta phosphorylate APC, which increases its affinity for beta-catenin. Crystal structures of phosphorylated and nonphosphorylated APC bound to beta-catenin reveal a phosphorylation-dependent binding motif generated by mutual priming of CK1 and GSK-3beta substrate sequences. Axin is shown to act as a scaffold for substrate phosphorylation by these kinases. Phosphorylated APC and axin bind to the same surface of, and compete directly for, beta-catenin. The structural and biochemical data suggest a novel model for how APC functions in beta-catenin degradation.

  View details for Web of Science ID 000223565200006

  View details for PubMedID 15327768

• Structural basis for distinct ligand-binding and targeting properties of the receptors DC-SIGN and DC-SIGNR NATURE STRUCTURAL & MOLECULAR BIOLOGY Guo, Y., Feinberg, H., Conroy, E., Mitchell, D. A., Alvarez, R., Blixt, O., Taylor, M. E., Weis, W. I., Drickamer, K. 2004; 11 (7): 591-598

  Abstract

  Both the dendritic cell receptor DC-SIGN and the closely related endothelial cell receptor DC-SIGNR bind human immunodeficiency virus and enhance infection. However, biochemical and structural comparison of these receptors now reveals that they have very different physiological functions. By screening an extensive glycan array, we demonstrated that DC-SIGN and DC-SIGNR have distinct ligand-binding properties. Our structural and mutagenesis data explain how both receptors bind high-mannose oligosaccharides on enveloped viruses and why only DC-SIGN binds blood group antigens, including those present on microorganisms. DC-SIGN mediates endocytosis, trafficking as a recycling receptor and releasing ligand at endosomal pH, whereas DC-SIGNR does not release ligand at low pH or mediate endocytosis. Thus, whereas DC-SIGN has dual ligand-binding properties and functions both in adhesion and in endocytosis of pathogens, DC-SIGNR binds a restricted set of ligands and has only the properties of an adhesion receptor.

  View details for DOI 10.1038/nsmb784

  View details for Web of Science ID 000222274100007

  View details for PubMedID 15195147

• In vitro methods for investigating desmoplakin-intermediate filament interactions and their role in adhesive strength INTERMEDIATE FILAMENT CYTOSKELETON Hudson, T. Y., Fontao, L., Godsel, L. M., Choi, H. J., Huen, A. C., Borradori, L., Weis, W. I., Green, K. J. 2004; 78: 757-786

  View details for Web of Science ID 000227909200026

  View details for PubMedID 15646638

• Structural aspects of adherens junctions and desmosomes. Handbook of experimental pharmacology Choi, H., Weis, W. I. 2004: 23-52

  Abstract

  The cadherin-containing intercellular junctions, adherens junctions and desmosomes share an overall logical organization in which the extracellular regions of the cadherins on opposing cells interact, while their cytoplasmic domains are linked to the cytoskeleton through protein assemblies. In adherens junctions, beta-catenin binds to the cytoplasmic domain of cadherins and to alpha-catenin, which links the cadherin/beta-catenin complex to the actin cytoskeleton. In desmosomes, the beta-catenin homolog plakoglobin binds to desmosomal cadherins. The desmosomal cadherin/plakoglobin complex is linked to the intermediate filament system by the protein desmoplakin. In the past decade, components of these systems have been purified to homogeneity and studied biochemically and structurally, providing the beginnings of a mechanistic description of junction architecture and dynamics.

  View details for DOI 10.1007/978-3-540-68170-0_2

  View details for PubMedID 20455089

• Crystal structure of the CUB1-EGF-CUB2 region of mannose-binding protein associated serine protease-2 EMBO JOURNAL Feinberg, H., Uitdehaag, J. C., Davies, J. M., Wallis, R., Drickamer, K., Weis, W. I. 2003; 22 (10): 2348-2359

  Abstract

  Serum mannose-binding proteins (MBPs) are C-type lectins that recognize cell surface carbohydrate structures on pathogens, and trigger killing of these targets by activating the complement pathway. MBPs circulate as a complex with MBP-associated serine proteases (MASPs), which become activated upon engagement of a target cell surface. The minimal functional unit for complement activation is a MASP homodimer bound to two MBP trimeric subunits. MASPs have a modular structure consisting of an N-terminal CUB domain, a Ca(2+)-binding EGF-like domain, a second CUB domain, two complement control protein modules and a C-terminal serine protease domain. The CUB1-EGF-CUB2 region mediates homodimerization and binding to MBP. The crystal structure of the MASP-2 CUB1-EGF-CUB2 dimer reveals an elongated structure with a prominent concave surface that is proposed to be the MBP-binding site. A model of the full six-domain structure and its interaction with MBPs suggests mechanisms by which binding to a target cell transmits conformational changes from MBP to MASP that allow activation of its protease activity.

  View details for Web of Science ID 000182957100005

  View details for PubMedID 12743029

• A structural analysis of full length VCP, a AAA protein. 47th Annual Meeting of the Biophysical-Society DeLaBarre, B., Rouiller, I., May, A. P., Weis, W. I., Wilson-Kubalek, E. M., Brunger, A. T. CELL PRESS. 2003: 356A–356A

  View details for Web of Science ID 000183123801742

• Conformational changes of the multifunction p97 AAA ATPase during its ATPase cycle NATURE STRUCTURAL BIOLOGY Rouiller, I., DeLaBarre, B., May, A. P., Weis, W. I., Brunger, A. T., Milligan, R. A., Wilson-Kubalek, E. M. 2002; 9 (12): 950-957

  Abstract

  p97 (also called VCP), a member of the AAA ATPase family, is involved in several cellular processes, including membrane fusion and extraction of proteins from the endoplasmic reticulum for cytoplasmic degradation. We have studied the conformational changes that p97 undergoes during the ATPase cycle by cryo-EM and single-particle analysis. Three-dimensional maps show that the two AAA domains, D1 and D2, as well as the N-domains, experience conformational changes during ATP binding, ATP hydrolysis, P(i) release and ADP release. The N-domain is flexible in most nucleotide states except after ATP hydrolysis. The rings formed by D1 and D2 rotate with respect to each other, and the size of their axial openings fluctuates. Taken together, our results depict the movements that this and possibly other AAA ATPases can undergo during an ATPase cycle.

  View details for DOI 10.1038/nsb872

  View details for Web of Science ID 000179409400016

  View details for PubMedID 12434150

• ICAT inhibits beta-catenin binding to Tcf/Lef-family transcription factors and the general coactivator p300 using independent structural modules MOLECULAR CELL Daniels, D. L., Weis, W. I. 2002; 10 (3): 573-584

  Abstract

  In the canonical Wnt signaling pathway, beta-catenin activates target genes through its interactions with Tcf/Lef-family transcription factors and additional transcriptional coactivators. The crystal structure of ICAT, an inhibitor of beta-catenin-mediated transcription, bound to the armadillo repeat domain of beta-catenin, has been determined. ICAT contains an N-terminal helilical domain that binds to repeats 11 and 12 of beta-catenin, and an extended C-terminal region that binds to repeats 5-10 in a manner similar to that of Tcfs and other beta-catenin ligands. Full-length ICAT dissociates complexes of beta-catenin, Lef-1, and the transcriptional coactivator p300, whereas the helical domain alone selectively blocks binding to p300. The C-terminal armadillo repeats of beta-catenin may be an attractive target for compounds designed to disrupt aberrant beta-catenin-mediated transcription associated with various cancers.

  View details for Web of Science ID 000178330900015

  View details for PubMedID 12408825

• Structures of two intermediate filament-binding fragments of desmoplakin reveal a unique repeat motif structure NATURE STRUCTURAL BIOLOGY Choi, H. J., Park-Snyder, S., Pascoe, L. T., Green, K. J., Weis, W. I. 2002; 9 (8): 612-620

  Abstract

  Desmosomes are intercellular junctions in which cadherin cell adhesion molecules are linked to the intermediate filament (IF) system. Desmoplakin is a member of the plakin family of IF-binding proteins. The C-terminal domain of desmoplakin (DPCT) mediates binding to IFs in desmosomes. The DPCT sequence contains three regions, termed A, B and C, consisting of 4.5 copies of a 38-amino acid repeat motif. We demonstrate that these regions form discrete subdomains that bind to IFs and report the crystal structures of domains B and C. In contrast to the elongated structures formed by other kinds of repeat motifs, the plakin repeats form a globular structure with a unique fold. A conserved basic groove found on the domain may represent an IF-binding site.

  View details for DOI 10.1038/nsb818

  View details for Web of Science ID 000177214200015

  View details for PubMedID 12101406

• Three-dimensional structure of the amino-terminal domain of syntaxin 6, a SNAP-25 C homolog PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Misura, K. M., Bock, J. B., Gonzalez, L. C., Scheller, R. H., Weis, W. I. 2002; 99 (14): 9184-9189

  Abstract

  Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are required for intracellular membrane fusion, and are differentially localized throughout the cell. SNAREs on vesicle and target membranes contain "SNARE motifs" which interact to form a four-helix bundle that contributes to the fusion of two membranes. SNARE motif sequences fall into four classes, homologous to the neuronal proteins syntaxin 1a, VAMP 2, and the N- and C-terminal SNARE motifs of SNAP-25 (S25N and S25C), and it is thought that one member from each class interacts to form a SNARE complex. Many SNAREs also feature N-terminal domains believed to function in regulating SNARE complex assembly or other aspects of vesicle transport. Syntaxin 6 is a SNARE found primarily in endosomal transport vesicles and whose SNARE motif shows significant homology to both syntaxin 1a and S25C. The crystal structure of the syntaxin 6 N-terminal domain reveals strong structural similarity with the N-terminal domains of syntaxin family members syntaxin 1a, Sso1p, and Vam3p, despite a very low level of sequence similarity. The syntaxin 6 SNARE motif can substitute for S25C in in vitro binding experiments, supporting the classification of syntaxin 6 as an S25C family member. Secondary structure prediction of SNARE proteins shows that the N-terminal domains of many syntaxin, S25N, and S25C family members are likely to be similar to one another, but are distinct from those of VAMP family members, indicating that syntaxin, S25N, and S25C SNAREs may have shared a common ancestor.

  View details for DOI 10.1073/pnas.132274599

  View details for Web of Science ID 000176775400022

  View details for PubMedID 12082176

  View details for PubMedCentralID PMC123115

• Biochemical and structural definition of the 1-afadin- and actin-binding sites of alpha-catenin JOURNAL OF BIOLOGICAL CHEMISTRY Pokutta, S., Drees, F., Takai, Y., NELSON, W. J., Weis, W. I. 2002; 277 (21): 18868-18874

  Abstract

  alpha-Catenin is an integral component of adherens junctions, where it links cadherins to the actin cytoskeleton. alpha-Catenin is also required for the colocalization of the nectin/afadin/ponsin adhesion system to adherens junctions, and it specifically associates with the nectin-binding protein afadin. A proteolytic fragment of alpha-catenin, residues 385-651, contains the afadin-binding site. The three-dimensional structure of this fragment comprises two side-by-side four-helix bundles, both of which are required for afadin binding. The alpha-catenin fragment 385-651 binds afadin more strongly than the full-length protein, suggesting that the full-length protein harbors a cryptic binding site for afadin. Comparison of the alpha-catenin 385-651 structure with the recently solved structure of the alpha-catenin M-fragment (Yang, J., Dokurno, P., Tonks, N. K., and Barford, D. (2001) EMBO J. 20, 3645-3656) reveals a surprising flexibility in the orientation of the two four-helix bundles. alpha-Catenin and the actin-binding protein vinculin share sequence and most likely structural similarity within their actin-binding domains. Despite this homology, actin binding requires additional sequences adjacent to this region.

  View details for DOI 10.1074/jbc.M201463200

  View details for Web of Science ID 000175975800081

  View details for PubMedID 11907041

  View details for PubMedCentralID PMC3368618

• Orientation of bound ligands in mannose-binding proteins - Implications for multivalent ligand recognition JOURNAL OF BIOLOGICAL CHEMISTRY Ng, K. K., Kolatkar, A. R., Park-Snyder, S., Feinberg, H., Clark, D. A., Drickamer, K., Weis, W. I. 2002; 277 (18): 16088-16095

  Abstract

  Mannose-binding proteins (MBPs) are C-type animal lectins that recognize high mannose oligosaccharides on pathogenic cell surfaces. MBPs bind to their carbohydrate ligands by forming a series of Ca(2+) coordination and hydrogen bonds with two hydroxyl groups equivalent to the 3- and 4-OH of mannose. In this work, the determinants of the orientation of sugars bound to rat serum and liver MBPs (MBP-A and MBP-C) have been systematically investigated. The crystal structures of MBP-A soaked with monosaccharides and disaccharides and also the structure of the MBP-A trimer cross-linked by a high mannose asparaginyl oligosaccharide reveal that monosaccharides or alpha1-6-linked mannose bind to MBP-A in one orientation, whereas alpha1-2- or alpha1-3-linked mannose binds in an orientation rotated 180 degrees around a local symmetry axis relating the 3- and 4-OH groups. In contrast, a similar set of ligands all bind to MBP-C in a single orientation. The mutation of MBP-A His(189) to its MBP-C equivalent, valine, causes Man alpha 1-3Man to bind in a mixture of orientations. These data combined with modeling indicate that the residue at this position influences the orientation of bound ligands in MBP. We propose that the control of binding orientation can influence the recognition of multivalent ligands. A lateral association of trimers in the cross-linked crystals may reflect interactions within higher oligomers of MBP-A that are stabilized by multivalent ligands.

  View details for DOI 10.1074/jbc.M200493200

  View details for Web of Science ID 000175510400116

  View details for PubMedID 11850428

• The cytoplasmic face of cell contact sites CURRENT OPINION IN STRUCTURAL BIOLOGY Pokutta, S., Weis, W. I. 2002; 12 (2): 255-262

  Abstract

  The cytoplasmic face of cell contact sites comprises large macromolecular assemblies that link transmembrane cell adhesion molecules to the cytoskeleton. These assemblies are dynamic structures that are the targets of regulatory signals that control cell adhesiveness. Recent studies of the biochemistry and structure of the cadherin-catenin complex, vinculin and proteins of the ezrin/radixin/moesin family have begun to reveal the architecture of these assemblies and the mechanisms that are involved in their regulation.

  View details for Web of Science ID 000175043000017

  View details for PubMedID 11959505

• Structural basis for selective recognition of oligosaccharides by DC-SIGN and DC-SIGNR SCIENCE Feinberg, H., Mitchell, D. A., Drickamer, K., Weis, W. I. 2001; 294 (5549): 2163-2166

  Abstract

  Dendritic cell specific intracellular adhesion molecule-3 (ICAM-3) grabbing nonintegrin (DC-SIGN), a C-type lectin present on the surface of dendritic cells, mediates the initial interaction of dendritic cells with T cells by binding to ICAM-3. DC-SIGN and DC-SIGNR, a related receptor found on the endothelium of liver sinusoids, placental capillaries, and lymph nodes, bind to oligosaccharides that are present on the envelope of human immunodeficiency virus (HIV), an interaction that strongly promotes viral infection of T cells. Crystal structures of carbohydrate-recognition domains of DC-SIGN and of DC-SIGNR bound to oligosaccharide, in combination with binding studies, reveal that these receptors selectively recognize endogenous high-mannose oligosaccharides and may represent a new avenue for developing HIV prophylactics.

  View details for Web of Science ID 000172647700050

  View details for PubMedID 11739956

• Molecular mechanisms of beta-catenin recognition by adenomatous polyposis coli revealed by the structure of an APC-beta-catenin complex EMBO JOURNAL Spink, K. E., Fridman, S. G., Weis, W. I. 2001; 20 (22): 6203-6212

  Abstract

  The adenomatous polyposis coli (APC) tumor suppressor protein plays a critical role in regulating cellular levels of the oncogene product beta-catenin. APC binds to beta-catenin through a series of homologous 15 and 20 amino acid repeats. We have determined the crystal structure of a 15 amino acid beta-catenin binding repeat from APC bound to the armadillo repeat region of beta-catenin. Although it lacks significant sequence homology, the N-terminal half of the repeat binds in a manner similar to portions of E-cadherin and XTcf3, but the remaining interactions are unique to APC. We discuss the implications of this new structure for the design of therapeutics, and present evidence from structural, biochemical and sequence data, which suggest that the 20 amino acid repeats can adopt two modes of binding to beta-catenin.

  View details for Web of Science ID 000172403000004

  View details for PubMedID 11707392

• Crystal structure and biophysical properties of a complex between the N-terminal SNARE region of SNAP25 and syntaxin 1a. JOURNAL OF BIOLOGICAL CHEMISTRY Misura, K. M., Gonzalez, L. C., May, A. P., Scheller, R. H., Weis, W. I. 2001; 276 (44): 41301-41309

  Abstract

  SNARE proteins are required for intracellular membrane fusion. In the neuron, the plasma membrane SNAREs syntaxin 1a and SNAP25 bind to VAMP2 found on neurotransmitter-containing vesicles. These three proteins contain "SNARE regions" that mediate their association into stable tetrameric coiled-coil structures. Syntaxin 1a contributes one such region, designated H3, and SNAP25 contributes two SNARE regions to the fusogenic complex with VAMP2. Syntaxin 1a H3 (syn1aH3) and SNAP25 can form a stable assembly, which can then be bound by VAMP2 to form the full SNARE complex. Here we show that syn1aH3 can also form a stable but kinetically trapped complex with the N-terminal SNARE region of SNAP25 (S25N). The crystal structure of this complex reveals an extended parallel four-helix bundle similar to that of the core SNARE and the syn1aH3-SNAP25 complexes. The inherent ability of syn1aH3 and S25N to associate stably in vitro implies that the intracellular fusion machinery must prevent formation of, or remove, any non-productive complexes. Comparison with the syn1aH3-SNAP25 complex suggests that the linkage of the N- and C-terminal SNAP25 SNARE regions is kinetically advantageous in preventing formation of the non-productive syn1aH3-S25N complex. We also demonstrate that the syn1aH3-S25N complex can be disassembled by alpha-SNAP and N-ethylmaleimide-sensitive factor.

  View details for Web of Science ID 000171925600123

  View details for PubMedID 11533035

• beta-catenin: molecular plasticity and drug design TRENDS IN BIOCHEMICAL SCIENCES Daniels, D. L., Spink, K. E., Weis, W. I. 2001; 26 (11): 672-678

  Abstract

  The protein beta-catenin is an essential component of intercellular junctions and the Wnt growth factor signaling pathway. In many cancers, mutation of Wnt pathway components leads to activation of oncogenes by the beta-catenin-Tcf transcription factor complex. This complex is therefore an attractive target for anti-cancer drugs, but any such compound must selectively interfere with the beta-catenin-Tcf complex without disrupting other essential interactions of beta-catenin. Recent structural and biochemical studies have probed the molecular basis of ligand interaction by beta-catenin, and highlighted the possibilities and challenges of designing inhibitors of the beta-catenin-Tcf complex.

  View details for Web of Science ID 000172266900008

  View details for PubMedID 11701326

• A novel SNARE N-terminal domain revealed by the crystal structure of Sec22b JOURNAL OF BIOLOGICAL CHEMISTRY Gonzalez, L. C., Weis, W. I., Scheller, R. H. 2001; 276 (26): 24203-24211

  Abstract

  Intra-cellular membrane fusion is facilitated by the association of SNAREs from opposite membranes into stable alpha-helical bundles. Many SNAREs, in addition to their alpha-helical regions, contain N-terminal domains that likely have essential regulatory functions. To better understand this regulation, we have determined the 2.4-A crystal structure of the 130-amino acid N-terminal domain of mouse Sec22b (mSec22b), a SNARE involved in endoplasmic reticulum/Golgi membrane trafficking. The domain consists of a mixed alpha-helical/beta-sheet fold that resembles a circular permutation of the actin/poly-proline binding protein, profilin, and the GAF/PAS family of regulatory modules. The structure is distinct from the previously characterized N-terminal domain of syntaxin 1A, and, unlike syntaxin 1A, the N-terminal domain of mSec22b has no effect on the rate of SNARE assembly in vitro. An analysis of surface conserved residues reveals a potential protein interaction site. Key residues in this site are distinct in two mammalian Sec22 variants that lack SNARE domains. Finally, sequence analysis indicates that a similar domain is likely present in the endosomal/lysosomal SNARE VAMP7.

  View details for Web of Science ID 000169531100129

  View details for PubMedID 11309394

• Unraveling the mechanism of the vesicle transport ATPase NSF, the N-ethylmaleimide-sensitive factor JOURNAL OF BIOLOGICAL CHEMISTRY May, A. P., Whiteheart, S. W., Weis, W. I. 2001; 276 (25): 21991-21994

  View details for Web of Science ID 000169412700001

  View details for PubMedID 11301340

• The structure of the beta-catenin/E-cadherin complex and the molecular basis of diverse ligand recognition by beta-catenin CELL Huber, A. H., Weis, W. I. 2001; 105 (3): 391-402

  Abstract

  As a component of adherens junctions and the Wnt signaling pathway, beta-catenin binds cadherins, Tcf family transcription factors, and the tumor suppressor APC. We have determined the crystal structures of both unphosphorylated and phosphorylated E-cadherin cytoplasmic domain complexed with the arm repeat region of beta-catenin. The interaction spans all 12 arm repeats, and features quasi-independent binding regions that include helices which interact with both ends of the arm repeat domain and an extended stretch of 14 residues which closely resembles a portion of XTcf-3. Phosphorylation of E-cadherin results in interactions with a hydrophobic patch of beta-catenin that mimics the binding of an amphipathic XTcf-3 helix. APC contains sequences homologous to the phosphorylated region of cadherin, and is likely to bind similarly.

  View details for Web of Science ID 000168515900008

  View details for PubMedID 11348595

• Self-association of the H3 region of syntaxin 1A - Implications for intermediates in snare complex assembly JOURNAL OF BIOLOGICAL CHEMISTRY Misura, K. M., Scheller, R. H., Weis, W. I. 2001; 276 (16): 13273-13282

  Abstract

  Intracellular membrane fusion requires SNARE proteins found on the vesicle and target membranes. SNAREs associate by formation of a parallel four-helix bundle, and it has been suggested that formation of this complex promotes membrane fusion. The membrane proximal region of the cytoplasmic domain of the SNARE syntaxin 1A, designated H3, contributes one of the four helices to the SNARE complex. In the crystal structure of syntaxin 1A H3, four molecules associate as a homotetramer composed of two pairs of parallel helices that are anti-parallel to each other. The H3 oligomer observed in the crystals is also found in solution, as assessed by gel filtration and chemical cross-linking studies. The crystal structure reveals that the highly conserved Phe-216 packs against conserved Gln-226 residues present on the anti-parallel pair of helices. Modeling indicates that Phe-216 prevents parallel tetramer formation. Mutation of Phe-216 to Leu appears to allow formation of parallel tetramers, whereas mutation to Ala destabilizes the protein. These results indicate that Phe-216 has a role in preventing formation of stable parallel helical bundles, thus favoring the interaction of the H3 region of syntaxin 1a with other proteins involved in membrane fusion.

  View details for Web of Science ID 000168198600111

  View details for PubMedID 11118447

• The cadherin cytoplasmic domain is unstructured in the absence of beta-catenin - A possible-mechanism for regulating cadherin turnover JOURNAL OF BIOLOGICAL CHEMISTRY Huber, A. H., STEWART, D. B., Laurents, D. V., NELSON, W. J., Weis, W. I. 2001; 276 (15): 12301-12309

  Abstract

  Cadherins are single pass transmembrane proteins that mediate Ca(2+)-dependent homophilic cell-cell adhesion by linking the cytoskeletons of adjacent cells. In adherens junctions, the cytoplasmic domain of cadherins bind to beta-catenin, which in turn binds to the actin-associated protein alpha-catenin. The physical properties of the E-cadherin cytoplasmic domain and its interactions with beta-catenin have been investigated. Proteolytic sensitivity, tryptophan fluorescence, circular dichroism, and (1)H NMR measurements indicate that murine E-cadherin cytoplasmic domain is unstructured. Upon binding to beta-catenin, the domain becomes resistant to proteolysis, suggesting that it structures upon binding. Cadherin-beta-catenin complex stability is modestly dependent on ionic strength, indicating that, contrary to previous proposals, the interaction is not dominated by electrostatics. Comparison of 18 cadherin sequences indicates that their cytoplasmic domains are unlikely to be structured in isolation. This analysis also reveals the presence of PEST sequences, motifs associated with ubiquitin/proteosome degradation, that overlap the previously identified beta-catenin-binding site. It is proposed that binding of cadherins to beta-catenin prevents recognition of degradation signals that are exposed in the unstructured cadherin cytoplasmic domain, favoring a cell surface population of catenin-bound cadherins capable of participating in cell adhesion.

  View details for Web of Science ID 000168081800115

  View details for PubMedID 11121423

• Protein-protein interactions in intracellular membrane fusion CURRENT OPINION IN STRUCTURAL BIOLOGY Misura, K. M., May, A. P., Weis, W. I. 2000; 10 (6): 662-671

  Abstract

  The fusion of intracellular vesicles with their target membranes is an essential feature of the compartmental structure of eukaryotic cells. This process requires proteins that dictate the targeting of a vesicle to the correct cellular location, mediate bilayer fusion and, in some systems, regulate the precise time at which fusion occurs. Recent biophysical and structural studies of these proteins have begun to provide a foundation for understanding their functions at a molecular level.

  View details for Web of Science ID 000166509100007

  View details for PubMedID 11114503

• Mechanism of pH-dependent N-acetylgalactosamine binding by a functional mimic of the hepatocyte asialoglycoprotein receptor JOURNAL OF BIOLOGICAL CHEMISTRY Feinberg, H., Torgersen, D., Drickamer, K., Weis, W. I. 2000; 275 (45): 35176-35184

  Abstract

  Efficient release of ligands from the Ca(2+)-dependent carbohydrate-recognition domain (CRD) of the hepatic asialoglycoprotein receptor at endosomal pH requires a small set of conserved amino acids that includes a critical histidine residue. When these residues are incorporated at corresponding positions in an homologous galactose-binding derivative of serum mannose-binding protein, the pH dependence of ligand binding becomes more like that of the receptor. The modified CRD displays 40-fold preferential binding to N-acetylgalactosamine compared with galactose, making it a good functional mimic of the asialoglycoprotein receptor. In the crystal structure of the modified CRD bound to N-acetylgalactosamine, the histidine (His(202)) contacts the 2-acetamido methyl group and also participates in a network of interactions involving Asp(212), Arg(216), and Tyr(218) that positions a water molecule in a hydrogen bond with the sugar amide group. These interactions appear to produce the preference for N-acetylgalactosamine over galactose and are also likely to influence the pK(a) of His(202). Protonation of His(202) would disrupt its interaction with an asparagine that serves as a ligand for Ca(2+) and sugar. The structure of the modified CRD without sugar displays several different conformations that may represent structures of intermediates in the release of Ca(2+) and sugar ligands caused by protonation of His(202).

  View details for Web of Science ID 000165422800048

  View details for PubMedID 10931846

• Structure of a C-type carbohydrate recognition domain from the macrophage mannose receptor JOURNAL OF BIOLOGICAL CHEMISTRY Feinberg, H., Park-Snyder, S., Kolatkar, A. R., Heise, C. T., Taylor, M. E., Weis, W. I. 2000; 275 (28): 21539-21548

  Abstract

  The mannose receptor of macrophages and liver endothelium mediates clearance of pathogenic organisms and potentially harmful glycoconjugates. The extracellular portion of the receptor includes eight C-type carbohydrate recognition domains (CRDs), of which one, CRD-4, shows detectable binding to monosaccharide ligands. We have determined the crystal structure of CRD-4. Although the basic C-type lectin fold is preserved, a loop extends away from the core of the domain to form a domain-swapped dimer in the crystal. Of the two Ca(2+) sites, only the principal site known to mediate carbohydrate binding in other C-type lectins is occupied. This site is altered in a way that makes sugar binding impossible in the mode observed in other C-type lectins. The structure is likely to represent an endosomal form of the domain formed when Ca(2+) is lost from the auxiliary calcium site. The structure suggests a mechanism for endosomal ligand release in which the auxiliary calcium site serves as a pH sensor. Acid pH-induced removal of this Ca(2+) results in conformational rearrangements of the receptor, rendering it unable to bind carbohydrate ligands.

  View details for Web of Science ID 000088230600078

  View details for PubMedID 10779515

• Structural basis of the Axin-adenomatous polyposis coli interaction EMBO JOURNAL Spink, K. E., Polakis, P., Weis, W. I. 2000; 19 (10): 2270-2279

  Abstract

  Axin and the adenomatous polyposis coli (APC) tumor suppressor protein are components of the Wnt/Wingless growth factor signaling pathway. In the absence of Wnt signal, Axin and APC regulate cytoplasmic levels of the proto-oncogene beta-catenin through the formation of a large complex containing these three proteins, glycogen synthase kinase 3beta (GSK3beta) and several other proteins. Both Axin and APC are known to be critical for beta-catenin regulation, and truncations in APC that eliminate the Axin-binding site result in human cancers. A protease-resistant domain of Axin that contains the APC-binding site is a member of the regulators of G-protein signaling (RGS) superfamily. The crystal structures of this domain alone and in complex with an Axin-binding sequence from APC reveal that the Axin-APC interaction occurs at a conserved groove on a face of the protein that is distinct from the G-protein interface of classical RGS proteins. The molecular interactions observed in the Axin-APC complex provide a rationale for the evolutionary conservation seen in both proteins.

  View details for Web of Science ID 000087140800014

  View details for PubMedID 10811618

• Three-dimensional structure of the neuronal-Sec1-syntaxin 1a complex NATURE Misura, K. M., Scheller, R. H., Weis, W. I. 2000; 404 (6776): 355-362

  Abstract

  Syntaxin 1a and neuronal Sec1 (nSec1) form an evolutionarily conserved heterodimer that is essential for vesicle trafficking and membrane fusion. The crystal structure of the nSec1-syntaxin 1a complex, determined at 2.6 A resolution, reveals that major conformational rearrangements occur in syntaxin relative to both the core SNARE complex and isolated syntaxin. We identify regions of the two proteins that seem to determine the binding specificity of particular Sec1 proteins for syntaxin isoforms, which is likely to be important for the fidelity of membrane trafficking. The structure also indicates mechanisms that might couple the action of upstream effector proteins to conformational changes in syntaxin 1a and nSec1 that lead to core complex formation and membrane fusion.

  View details for Web of Science ID 000086119000040

  View details for PubMedID 10746715

• Structure of the dimerization and beta-catenin-binding region of alpha-catenin MOLECULAR CELL Pokutta, S., Weis, W. I. 2000; 5 (3): 533-543

  Abstract

  In adherens junctions, alpha-catenin links the cadherin-beta-catenin complex to the actin-based cytoskeleton. alpha-catenin is a homodimer in solution, but forms a 1:1 heterodimer with beta-catenin. The crystal structure of the alpha-catenin dimerization domain, residues 82-279, shows that alpha-catenin dimerizes through formation of a four-helix bundle in which two antiparallel helices are contributed by each protomer. A slightly larger fragment, comprising residues 57-264, binds to beta-catenin. A chimera consisting of the alpha-catenin-binding region of beta-catenin linked to the amino terminus of alpha-catenin 57-264 behaves as a monomer in solution, as expected, since beta-catenin binding disrupts the alpha-catenin dimer. The crystal structure of this chimera reveals the interaction between alpha- and beta-catenin, and provides a basis for understanding adherens junction assembly.

  View details for Web of Science ID 000086457600012

  View details for PubMedID 10882138

• Crystal structure of the amino-terminal domain of N-ethylmaleimide-sensitive fusion protein NATURE CELL BIOLOGY May, A. P., Misura, K. M., Whiteheart, S. W., Weis, W. I. 1999; 1 (3): 175-182

  Abstract

  The cytosolic ATPase N-ethylmaleimide-sensitive fusion protein (NSF) disassembles complexes of membrane-bound proteins known as SNAREs, an activity essential for vesicular trafficking. The amino-terminal domain of NSF (NSF-N) is required for the interaction of NSF with the SNARE complex through the adaptor protein alpha-SNAP. The crystal structure of NSF-N reveals two subdomains linked by a single stretch of polypeptide. A polar interface between the two subdomains indicates that they can move with respect to one another during the catalytic cycle of NSF. Structure-based sequence alignments indicate that in addition to NSF orthologues, the p97 family of ATPases contain an amino-terminal domain of similar structure.

  View details for Web of Science ID 000083102500017

  View details for PubMedID 10559905

• Ca2+-dependent structural changes in C-type mannose-binding proteins BIOCHEMISTRY Ng, K. K., Park-Snyder, S., Weis, W. I. 1998; 37 (51): 17965-17976

  Abstract

  C-type animal lectins are a diverse family of proteins which mediate cell-surface carbohydrate-recognition events through a conserved carbohydrate-recognition domain (CRD). Most members of this family possess a carbohydrate-binding activity that depends strictly on the binding of Ca2+ at two sites, designated 1 and 2, in the CRD. The structural transitions associated with Ca2+ binding in C-type lectins have been investigated by determining high-resolution crystal structures of rat serum mannose-binding protein (MBP) bound to one Ho3+ in place of Ca2+, and the apo form of rat liver MBP. The removal of Ca2+ does not affect the core structure of the CRD, but dramatic conformational changes occur in the loops. The most significant structural change in the absence of Ca2+ is the isomerization of a cis-peptide bond preceding a conserved proline residue in Ca2+ site 2. This bond adopts the cis conformation in all Ca2+-bound structures, whereas both cis and trans conformations are observed in the absence of Ca2+. The pattern of structural changes in the three loops that interact with Ca2+ is dictated in large part by the conformation of the prolyl peptide bond. The highly conserved nature of Ca2+ site 2 suggests that the transitions observed in MBPs are general features of Ca2+ binding in C-type lectins.

  View details for Web of Science ID 000077848400038

  View details for PubMedID 9922165

• Coupling of prolyl peptide bond isomerization and Ca2+ binding in a C-type mannose-binding protein BIOCHEMISTRY Ng, K. K., Weis, W. I. 1998; 37 (51): 17977-17989

  Abstract

  A proline residue flanked by two polar residues is a highly conserved sequence motif in the Ca2+- and carbohydrate-binding site of C-type animal lectins. Crystal structures of several C-type lectins have shown that the two flanking residues are only observed to act as Ca2+ ligands when the peptide bond preceding the proline residue is in the cis conformation. In contrast, structures of the apo- and one-ion forms of mannose-binding proteins (MBPs) reveal that, when the Ca2+-binding site is empty, the peptide bond preceding the proline can adopt either the cis or trans conformation, and distinct structures in adjacent regions are associated with the two proline isomers. In this work, measurements of Ca2+-induced changes in intrinsic tryptophan fluorescence, and fluorescence energy transfer from tryptophan to Tb3+, reveal a slow conformational change in rat liver MBP (MBP-C) accompanying the binding of either Ca2+ or Tb3+. The Ca2+-induced increase in intrinsic tryptophan fluorescence shows biphasic kinetics: a burst phase with a rate constant greater than 1 s(-1) is followed by a slow phase with a single-exponential rate constant ranging from 0.01 to 0.05 s(-1) (36 degrees C) that depends on the concentration of Ca2+. Likewise, addition of EGTA to Ca2+-bound or Tb3+-bound MBP-C causes a decrease in intrinsic tryptophan fluorescence with biphasic kinetics consisting of a burst phase with a rate constant greater than 1 s(-1), followed by a slow phase with a single-exponential rate constant of 0.065 s(-1). In contrast, Tb3+ fluorescence produced by resonant energy transfer from MBP-C decreases in a single kinetic phase with a rate constant greater than 1 s(-1), implying that the slow change in tryptophan fluorescence monitors a conformational change that is not limited in rate by ion dissociation. The rate constants of the slow phases accompanying Ca2+ binding and release are strongly affected by temperature and are weakly accelerated by the prolyl isomerase cyclophilin. These data strongly suggest that the binding of either Ca2+ or Tb3+ to MBP-C is coupled to a conformational change that involves the cis-trans isomerization of a peptide bond. Fitting of the data to kinetic models indicates that, in the absence of Ca2+, the proline in approximately 80% of the molecules is in the trans conformation. The slow kinetics associated with cis-trans proline isomerization may be exploited by endocytic receptors to facilitate sorting of carbohydrate-bearing ligands from the receptor in the endosome.

  View details for Web of Science ID 000077848400039

  View details for PubMedID 9922166

• Crystal structure of the hexamerization domain of N-ethylmaleimide-sensitive fusion protein CELL Lenzen, C. U., Steinmann, D., Whiteheart, S. W., Weis, W. I. 1998; 94 (4): 525-536

  Abstract

  N-ethylmaleimide-sensitive fusion protein (NSF) is a cytosolic ATPase required for many intracellular vesicle fusion reactions. NSF consists of an amino-terminal region that interacts with other components of the vesicle trafficking machinery, followed by two homologous ATP-binding cassettes, designated D1 and D2, that possess essential ATPase and hexamerization activities, respectively. The crystal structure of D2 bound to Mg2+-AMPPNP has been determined at 1.75 A resolution. The structure consists of a nucleotide-binding and a helical domain, and it is unexpectedly similar to the first two domains of the clamp-loading subunit delta' of E. coli DNA polymerase III. The structure suggests several regions responsible for coupling of ATP hydrolysis to structural changes in full-length NSF.

  View details for Web of Science ID 000075541100014

  View details for PubMedID 9727495

• Mechanism of N-acetylgalactosamine binding to a C-type animal lectin carbohydrate-recognition domain JOURNAL OF BIOLOGICAL CHEMISTRY Kolatkar, A. R., Leung, A. K., Isecke, R., Brossmer, R., Drickamer, K., Weis, W. I. 1998; 273 (31): 19502-19508

  Abstract

  The mammalian hepatic asialoglycoprotein receptor, a member of the C-type animal lectin family, displays preferential binding to N-acetylgalactosamine compared with galactose. The structural basis for selective binding to N-acetylgalactosamine has been investigated. Regions of the carbohydrate-recognition domain of the receptor believed to be important in preferential binding to N-acetylgalactosamine have been inserted into the homologous carbohydrate-recognition domain of a mannose-binding protein mutant that was previously altered to bind galactose. Introduction of a single histidine residue corresponding to residue 256 of the hepatic asialoglycoprotein receptor was found to cause a 14-fold increase in the relative affinity for N-acetylgalactosamine compared with galactose. The relative ability of various acyl derivatives of galactosamine to compete for binding to this modified carbohydrate-recognition domain suggest that it is a good model for the natural N-acetylgalactosamine binding site of the asialoglycoprotein receptor. Crystallographic analysis of this mutant carbohydrate-recognition domain in complex with N-acetylgalactosamine reveals a direct interaction between the inserted histidine residue and the methyl group of the N-acetyl substituent of the sugar. Evidence for the role of the side chain at position 208 of the receptor in positioning this key histidine residue was obtained from structural analysis and mutagenesis experiments. The corresponding serine residue in the modified carbohydrate-recognition domain of mannose-binding protein forms a hydrogen bond to the imidazole side chain. When this serine residue is changed to valine, loss in selectivity for N-acetylgalactosamine is observed. The structure of this mutant reveals that the beta-branched valine side chain interacts directly with the histidine side chain, resulting in an altered imidazole ring orientation.

  View details for Web of Science ID 000075125200024

  View details for PubMedID 9677372

• The C-type lectin superfamily in the immune system IMMUNOLOGICAL REVIEWS Weis, W. I., Taylor, M. E., Drickamer, K. 1998; 163: 19-34

  Abstract

  Protein-carbohydrate interactions serve multiple functions in the immune system. Many animal lectins (sugar-binding proteins) mediate both pathogen recognition and cell-cell interactions using structurally related Ca(2+)-dependent carbohydrate-recognition domains (C-type CRDs). Pathogen recognition by soluble collections such as serum mannose-binding protein and pulmonary surfactant proteins, and also the macrophage cell-surface mannose receptor, is effected by binding of terminal monosaccharide residues characteristic of bacterial and fungal cell surfaces. The broad selectivity of the monosaccharide-binding site and the geometrical arrangement of multiple CRDs in the intact lectins explains the ability of the proteins to mediate discrimination between self and non-self. In contrast, the much narrower binding specificity of selectin cell adhesion molecules results from an extended binding site within a single CRD. Other proteins, particularly receptors on the surface of natural killer cells, contain C-type lectin-like domains (CTLDs) that are evolutionarily divergent from the C-type lectins and which would be predicted to function through different mechanisms.

  View details for Web of Science ID 000074973900003

  View details for PubMedID 9700499

• Cell-surface carbohydrate recognition by animal and viral lectins CURRENT OPINION IN STRUCTURAL BIOLOGY Weis, W. I. 1997; 7 (5): 624-630

  Abstract

  Many animal and viral lectins are specific for monosaccharides found in particular glycosidic linkages, or for larger oligosaccharide structures. Recent crystal structures of complexes between these proteins and receptor fragments have provided insights into the recognition of linkage isomers and oligosaccharide conformation.

  View details for Web of Science ID A1997YB57900004

  View details for PubMedID 9345619

• Three-dimensional structure of the armadillo repeat region of beta-catenin CELL Huber, A. H., NELSON, W. J., Weis, W. I. 1997; 90 (5): 871-882

  Abstract

  Beta-catenin is essential for cadherin-based cell adhesion and Wnt/Wingless growth factor signaling. In these roles, it binds to cadherins, Tcf-family transcription factors, and the tumor suppressor gene product Adenomatous Polyposis Coli (APC). A core region of beta-catenin, composed of 12 copies of a 42 amino acid sequence motif known as an armadillo repeat, mediates these interactions. The three-dimensional structure of a protease-resistant fragment of beta-catenin containing the armadillo repeat region has been determined. The 12 repeats form a superhelix of helices that features a long, positively charged groove. Although unrelated in sequence, the beta-catenin binding regions of cadherins, Tcfs, and APC are acidic and are proposed to interact with this groove.

  View details for Web of Science ID A1997XV56300008

  View details for PubMedID 9298899

• Structure of a selectin-like mutant of mannose-binding protein complexed with sialylated and sulfated Lewis(x) oligosaccharides BIOCHEMISTRY Ng, K. K., Weis, W. I. 1997; 36 (5): 979-988

  Abstract

  Rat serum mannose-binding protein in which residues 211-213 have been changed to the Lys-Lys-Lys sequence found in E-selectin binds HL-60 cells and the oligosaccharide 3'-NeuAc-Le(x). To understand how this mutant, designated K3, mimics the carbohydrate-binding properties of E-selectin, structures of K3 alone and in complexes with 3'-NeuAc-Le(x), 3'-sulfo-Le(x) and 4'-sulfo-Le(x) have been determined at 1.95-2.1 A resolution by X-ray crystallography. The region of K3 that interacts with bound oligosaccharides superimposes closely with the corresponding region of unliganded E-selectin. In each of the oligosaccharide-protein complexes, the 2- and 3-OH of Fuc coordinate Ca2+ and form a network of cooperative hydrogen bonds with amino acid side chains that also coordinate the Ca2+. Lys211 of the K3 mutant, which corresponds to Lys111 of E-selectin, interacts with each of the three bound ligands: the N zeta atom donates a hydrogen bond to the 4-OH of Gal in 3'-NeuAc-Le(x), forms a water-mediated hydrogen bond with the 4-OH of Gal in 3'-sulfo-Le(x), and forms a salt bridge with the sulfate group of 4'-sulfo-Le(x). Lys213 packs against an otherwise exposed aromatic residue and forms a water-mediated hydrogen bond with Lys211 which may help to position that residue for interactions with bound oligosaccharides. These structures are consistent with previous mutagenesis and chemical modification studies which demonstrate the importance of the Ca2+ ligands as well as Lys111 and Lys113 for carbohydrate binding in the selectins, and they provide a structural basis for understanding the selective recognition of negatively charged Le(x) derivatives by the selectins.

  View details for Web of Science ID A1997WG07200001

  View details for PubMedID 9033386

• Structural basis of galactose recognition by C-type animal lectins JOURNAL OF BIOLOGICAL CHEMISTRY Kolatkar, A. R., Weis, W. I. 1996; 271 (12): 6679-6685

  Abstract

  The asialoglycoprotein receptors and many other C-type (Ca2+-dependent) animal lectins specifically recognize galactose- or N-acetylgalactosamine-terminated oligosaccharides. Analogous binding specificity can be engineered into the homologous rat mannose-binding protein A by changing three amino acids and inserting a glycine-rich loop (Iobst, S. T., and Drickamer, K. (1994) J. Biol. Chem. 269, 15512-15519). Crystal structures of this mutant complexed with beta-methyl galactoside and N-acetylgalactosamine (GalNAc) reveal that as with wild-type mannose-binding proteins, the 3- and 4-OH groups of the sugar directly coordinate Ca2+ and form hydrogen bonds with amino acids that also serve as Ca2+ ligands. The different stereochemistry of the 3- and 4-OH groups in mannose and galactose, combined with a fixed Ca2+ coordination geometry, leads to different pyranose ring locations in the two cases. The glycine-rich loop provides selectivity against mannose by holding a critical tryptophan in a position optimal for packing with the apolar face of galactose but incompatible with mannose binding. The 2-acetamido substituent of GalNAc is in the vicinity of amino acid positions identified by site-directed mutagenesis (Iobst, S. T., and Drickamer, K. (1996) J. Biol. Chem. 271, 6686-6693) as being important for the formation of a GalNAc-selective binding site.

  View details for Web of Science ID A1996UB15700021

  View details for PubMedID 8636086

• Structural analysis of monosaccharide recognition by rat liver mannose-binding protein JOURNAL OF BIOLOGICAL CHEMISTRY Ng, K. K., Drickamer, K., Weis, W. I. 1996; 271 (2): 663-674

  Abstract

  The structural basis of carbohydrate recognition by rat liver mannose-binding protein (MBP-C) has been explored by determining the three-dimensional structure of the C-type carbohydrate-recognition domain (CRD) of MBP-C using x-ray crystallography. The structure was solved by molecular replacement using rat serum mannose-binding protein (MBP-A) as a search model and was refined to maximum Bragg spacings of 1.7 A. Despite their almost identical folds, the dimeric structures formed by the two MBP CRDs differ dramatically. Complexes of MBP-C with methyl glycosides of mannose, N-acetylglucosamine, and fucose were prepared by soaking MBP-C crystals in solutions containing these sugars. Surprisingly, the pyranose ring of mannose is rotated 180 degrees relative to the orientation observed previously in MBP-A, but the local interactions between sugar and protein are preserved. For each of the bound sugars, vicinal, equatorial hydroxyl groups equivalent to the 3- and 4-OH groups of mannose directly coordinate Ca2+ and form hydrogen bonds with residues also serving as Ca2+ ligands. Few interactions are observed between other parts of the sugar and the protein. A complex formed between free galactose and MBP-C reveals a similar mode of binding, with the anomeric hydroxyl group serving as one of the Ca2+ ligands. A second binding site for mannose has also been observed in one of two copies in the asymmetric unit at a sugar concentration of 1.3 M. These structures explain how MBPs recognize a wide range of monosaccharides and suggest how fine specificity differences between MBP-A and MBP-C may be achieved.

  View details for Web of Science ID A1996TP88900014

  View details for PubMedID 8557671

• Direct observation of protein solvation and discrete disorder with experimental crystallographic phases SCIENCE Burling, F. T., Weis, W. I., Flaherty, K. M., Brunger, A. T. 1996; 271 (5245): 72-77

  Abstract

  A complete and accurate set of experimental crystallographic phases to a resolution of 1.8 angstroms was obtained for a 230-residue dimeric fragment of rat mannose-binding protein A with the use of multiwavelength anomalous dispersion (MAD) phasing. An accurate image of the crystal structure could thus be obtained without resort to phases calculated from a model. Partially reduced disulfide bonds, local disorder, and differences in the mobility of chemically equivalent molecules are apparent in the experimental electron density map. A solvation layer is visible that includes well-ordered sites of hydration around polar and charged protein atoms, as well as diffuse, partially disordered solvent shells around exposed hydrophobic groups. Because the experimental phases and the resulting electron density map are free from the influence of a model, they provide a stringent test of theoretical models of macromolecular solvation, motion, and conformational heterogeneity.

  View details for Web of Science ID A1996TP02200046

  View details for PubMedID 8539602

• Structural basis of lectin-carbohydrate recognition ANNUAL REVIEW OF BIOCHEMISTRY Weis, W. I., Drickamer, K. 1996; 65: 441-473

  Abstract

  Lectins are responsible for cell surface sugar recognition in bacteria, animals, and plants. Examples include bacterial toxins; animal receptors that mediate cell-cell interactions, uptake of glycoconjugates, and pathogen neutralization; and plant toxins and mitogens. The structural basis for selective sugar recognition by members of all of these groups has been investigated by x-ray crystallography. Mechanisms for sugar recognition have evolved independently in diverse protein structural frameworks, but share some key features. Relatively low affinity binding sites for monosaccharides are formed at shallow indentations on protein surfaces. Selectivity is achieved through a combination of hydrogen bonding to the sugar hydroxyl groups with van der Waals packing, often including packing of a hydrophobic sugar face against aromatic amino acid side chains. Higher selectivity of binding is achieved by extending binding sites through additional direct and water-mediated contacts between oligosaccharides and the protein surface. Dramatically increased affinity for oligosaccharides results from clustering of simple binding sites in oligomers of the lectin polypeptides. The geometry of such oligomers helps to establish the ability of the lectins to distinguish surface arrays of polysaccharides in some instances and to crosslink glycoconjugates in others.

  View details for Web of Science ID A1996UV92200015

  View details for PubMedID 8811186

• TRIMERIC STRUCTURE OF A C-TYPE MANNOSE-BINDING PROTEIN STRUCTURE Weis, W. I., Drickamer, K. 1994; 2 (12): 1227-1240

  Abstract

  Mannose-binding proteins (MBPs) are C-type (Ca(2+)-dependent) animal lectins found in serum. They recognize cell-surface oligosaccharide structures characteristic of pathogenic bacteria and fungi, and trigger the neutralization of these organisms. Like most lectins, MBPs display weak intrinsic affinity for monovalent sugar ligands, but bind avidly to multivalent ligands.We report physical studies in solution and the crystal structure determined at 1.8 A Bragg spacings of a trimeric fragment of MBP-A, containing the carbohydrate-recognition domain (CRD) and the neck domain that links the carboxy-terminal CRD to the collagen-like portion of the intact molecule. The neck consists of a parallel triple-stranded coiled coil of alpha-helices linked by four residues to the CRD. The isolated neck peptide does not form stable helices in aqueous solution. The previously characterized carbohydrate-binding sites lie at the distal end of the trimer and are separated from each other by 53 A.The carbohydrate-binding sites in MBP-A are too far apart for a single trimer to bind multivalently to a typical mammalian high-mannose oligosaccharide. Thus MBPs can recognize pathogens selectively by binding avidly only to the widely spaced, repetitive sugar arrays on pathogenic cell surfaces. Sequence alignments reveal that other C-type lectins are likely to have a similar oligomeric structure, but differences in their detailed organization will have an important role in determining their interactions with oligosaccharides.

  View details for Web of Science ID A1994QB49400012

  View details for PubMedID 7704532

• BINDING OF SUGAR LIGANDS TO CA2+-DEPENDENT ANIMAL LECTINS .1. ANALYSIS OF MANNOSE-BINDING BY SITE-DIRECTED MUTAGENESIS AND NMR JOURNAL OF BIOLOGICAL CHEMISTRY IOBST, S. T., Wormald, M. R., Weis, W. I., Dwek, R. A., Drickamer, K. 1994; 269 (22): 15505-15511

  Abstract

  The Ca(2+)-dependent carbohydrate-recognition domain (CRD) of rat serum mannose-binding protein has been subjected to site-directed mutagenesis to determine the importance of individual residues in ligation of mannose and related sugars. The effects of the mutations were assessed by direct binding assays, competition binding studies, partial proteolysis, and NMR analysis of sugar-CRD titrations. As suggested by the crystal structure of the mannose-binding CRD complexed with oligosaccharide ligand, asparagine and glutamic acid residues that interact with hydroxyl groups 3 and 4 of the sugar, as well as with one of the two bound Ca2+, are critical for ligand binding. In addition, the beta-carbon of His189 contributes substantially to the binding affinity, apparently through a van der Waals contact with C-4 of the sugar ligand. van der Waals contacts between the imidazole ring of His189 and the 2 hydroxyl group of mannose, and between Ile207 and C-6 of mannose, observed in the crystal structure, contribute less to stability of the ligand complex. The effects of changes at positions 189 and 207 on the ability of the CRD to distinguish between alpha-and beta-methyl L-fucosides suggest that fucose may bind in an alternative orientation compared to the arrangement originally proposed based on the mannose-CRD complex.

  View details for Web of Science ID A1994NP51300020

  View details for PubMedID 8195194

• RECOGNITION OF CELL-SURFACE CARBOHYDRATES BY C-TYPE ANIMAL LECTINS SmithKline-Beecham-Pharmaceuticals 7th US Research Symposium - Cellular Adhesion: Molecular Definition to Therapeutic Potential Weis, W. I. PLENUM PRESS DIV PLENUM PUBLISHING CORP. 1994: 55–75

  View details for Web of Science ID A1994BA81Y00004

• STRUCTURE OF A C-TYPE MANNOSE-BINDING PROTEIN COMPLEXED WITH AN OLIGOSACCHARIDE NATURE Weis, W. I., Drickamer, K., Hendrickson, W. A. 1992; 360 (6400): 127-134

  Abstract

  C-type (Ca(2+)-dependent) animal lectins such as mannose-binding proteins mediate many cell-surface carbohydrate-recognition events. The crystal structure at 1.7 A resolution of the carbohydrate-recognition domain of rat mannose-binding protein complexed with an oligomannose asparaginyl-oligosaccharide reveals that Ca2+ forms coordination bonds with the carbohydrate ligand. Carbohydrate specificity is determined by a network of coordination and hydrogen bonds that stabilizes the ternary complex of protein, Ca2+ and sugar. Two branches of the oligosaccharide crosslink neighbouring carbohydrate-recognition domains in the crystal, enabling multivalent binding to a single oligosaccharide chain to be visualized directly.

  View details for Web of Science ID A1992JX75200049

  View details for PubMedID 1436090

• MOLECULAR MECHANISMS OF COMPLEX CARBOHYDRATE-RECOGNITION AT THE CELL-SURFACE COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY Weis, W. I., Quesenberry, M. S., Taylor, M. E., Bezouska, K., Hendrickson, W. A., Drickamer, K. 1992; 57: 281-289

  View details for Web of Science ID A1992LV41600031

  View details for PubMedID 1339665

• STRUCTURE OF THE CALCIUM-DEPENDENT LECTIN DOMAIN FROM A RAT MANNOSE-BINDING PROTEIN DETERMINED BY MAD PHASING SCIENCE Weis, W. I., Kahn, R., Fourme, R., Drickamer, K., Hendrickson, W. A. 1991; 254 (5038): 1608-1615

  Abstract

  Calcium-dependent (C-type) animal lectins participate in many cell surface recognition events mediated by protein-carbohydrate interactions. The C-type lectin family includes cell adhesion molecules, endocytic receptors, and extracellular matrix proteins. Mammalian mannose-binding proteins are C-type lectins that function in antibody-independent host defense against pathogens. The crystal structure of the carbohydrate-recognition domain of a rat mannose-binding protein, determined as the holmium-substituted complex by multiwavelength anomalous dispersion (MAD) phasing, reveals an unusual fold consisting of two distinct regions, one of which contains extensive nonregular secondary structure stabilized by two holmium ions. The structure explains the conservation of 32 residues in all C-type carbohydrate-recognition domains, suggesting that the fold seen here is common to these domains. The strong anomalous scattering observed at the Ho LIII edge demonstrates that traditional heavy atom complexes will be generally amenable to the MAD phasing method.

  View details for Web of Science ID A1991GV07300035

  View details for PubMedID 1721241

• PHYSICAL CHARACTERIZATION AND CRYSTALLIZATION OF THE CARBOHYDRATE-RECOGNITION DOMAIN OF A MANNOSE-BINDING PROTEIN FROM RAT JOURNAL OF BIOLOGICAL CHEMISTRY Weis, W. I., Crichlow, G. V., Murthy, H. M., Hendrickson, W. A., Drickamer, K. 1991; 266 (31): 20678-20686

  Abstract

  A portion of rat mannose-binding protein A (MBP-A), a Ca(2+)-dependent animal lectin, has been overproduced in a bacterial expression system, biochemically characterized, and crystallized. A fragment corresponding to the COOH-terminal 115 residues of native MBP-A, produced by subtilisin digestion of the bacterially expressed protein, contains the carbohydrate-recognition domain (CRD). Gel filtration, chemical cross-linking, and crystallographic self-rotation function analyses indicate that the subtilisin fragment is a dimer, although the complete bacterially expressed fragment, containing the neck and CRD of MBP-A, is a trimer. Crystals of the minimal CRD, obtained only as a complex with a Man6GlcNAc2Asn glycopeptide, diffract to Bragg spacings of at least 1.7 A. Several trivalent lanthanide ions (Ln3+) can substitute for Ca2+, as assessed by their ability to support carbohydrate binding and to protect the CRD from proteolysis in a manner similar to that observed for Ca2+. These assays indicate that Ln2+ binds about 30 times more tightly than Ca2+ to the CRD, and that two Ca2+ or Ln3+ bind to each monomer, a result confirmed by determination of the Ho3+ positions in a Ho(3+)-containing crystal of the CRD. Crystals grown in the presence of Ln3+ belong to different space groups from those obtained with Ca2+ and are therefore not useable for traditional crystallographic phase determination methods, but are well-suited for high resolution structure determination by multiwavelength anomalous dispersion phasing.

  View details for Web of Science ID A1991GN00100017

  View details for PubMedID 1939118

• RIGID PROTEIN MOTION AS A MODEL FOR CRYSTALLOGRAPHIC TEMPERATURE FACTORS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kuriyan, J., Weis, W. I. 1991; 88 (7): 2773-2777

  Abstract

  The extent to which the librations of rigid molecules can model the crystallographic temperature factor profiles of proteins has been examined. For all proteins considered, including influenza virus hemagglutinin, glutathione reductase, myohemerythrin, myoglobin, and streptavidin, a simple 10-parameter model [V. Schomaker and K. N. Trueblood (1968) Acta Crystallogr. Sect. B 24, 63-76] is found to reproduce qualitatively the patterns of maxima and minima in the isotropic backbone meansquare displacements. Large deviations between the rigid molecule and individual atomic temperature factors are found to be correlated with a region in hemagglutinin for which the refined structural model is unsatisfactory and with errors in the structure in a partially incorrect model of myohemerythrin. For the high-resolution glutathione reductase structure, better results are obtained on treating each of the compact domains in the structure as independent rigid bodies. The method allows for the refinement of reliable temperature factors with the introduction of minimal parameters and may prove useful for the evaluation of models in the early stages of x-ray structure refinement. While these results by themselves do not establish the nature of the underlying displacements, the success of the rigid protein model in reproducing qualitative features of temperature factor profiles suggests that rigid body refinement results should be considered in any interpretation of crystallographic thermal parameters.

  View details for Web of Science ID A1991FE86400034

  View details for PubMedID 2011586

• REFINEMENT OF THE INFLUENZA-VIRUS HEMAGGLUTININ BY SIMULATED ANNEALING JOURNAL OF MOLECULAR BIOLOGY Weis, W. I., Brunger, A. T., Skehel, J. J., WILEY, D. C. 1990; 212 (4): 737-761

  Abstract

  We have applied the method of simulated annealing to the refinement of the 3 A resolution crystal structure of the influenza virus hemagglutinin glycoprotein, using the program X-PLOR. Two different methods were introduced into X-PLOR to treat the non-crystallographic symmetry present in this and in other crystal structures. In the first, only the unique protomer atoms are refined; by application of the non-crystallographic symmetry operators to the protomer atoms, the X-ray structure factor derivatives are effectively averaged, and a non-bonded energy term models the interactions of the protomer with its neighbors in the oligomer without explicit refinement of the other protomers in the crystallographic asymmetric unit. In the second method, the entire asymmetric unit is refined, but an effective energy term is added to the empirical energy that restrains symmetry-related atomic positions to their average values after least-squares superposition. Several other modifications and additions were made to previously published X-PLOR protocols, including weighting of the X-ray terms, maintenance of the temperature of the molecular dynamics simulation, treatment of charged groups, changes in the values of certain empirical energy parameters, and the use of N-linked carbohydrate empirical energy parameters. The hemagglutinin refinement proceeded in several stages. An initial round of simulated annealing of the monomer was followed by rigid-body refinement of the 3-fold non-crystallographic symmetry axis position and a second round of monomer refinement. A third round was performed on the trimer using non-crystallographic symmetry restraints in all regions except those in lattice contacts showing obvious derivations from 3-fold symmetry. The refinement was completed with several rounds of conventional positional and isotropic temperature factor refinement needed to correct bad model geometry introduced by high-temperature molecular dynamics in regions of weak electron density. This structure was then used as the basis for refinement of three crystallographically isomorphous hemagglutinin structures, including complexes with the influenza virus receptor, sialic acid. Model geometry comparable to well-refined high-resolution structures was obtained with relatively little manual intervention, demonstrating the ability of simulated annealing refinement to produce highly idealized structures at moderate resolution.

  View details for Web of Science ID A1990DB07400014

  View details for PubMedID 2329580

• THE STRUCTURE OF A MEMBRANE-FUSION MUTANT OF THE INFLUENZA-VIRUS HEMAGGLUTININ EMBO JOURNAL Weis, W. I., CUSACK, S. C., Brown, J. H., Daniels, R. S., Skehel, J. J., WILEY, D. C. 1990; 9 (1): 17-24

  Abstract

  The haemagglutinin glycoprotein (HA) of influenza virus specifically mediates fusion of the viral and host cell endosomal membranes at the acidic pH of endosomes. The HAs from mutant viruses with raised fusion pH optima contain amino acid substitutions in regions of the HA structure thought to be involved in the fusion process [Daniels et al. (1985b) Cell, 40, 431-439]. We have determined the neutral pH crystal structure of one such mutant, HA2 112 Asp----Gly. A water molecule appears to partially replace the aspartate side chain, and no changes are observed in the surrounding structure. It appears that four intra-chain hydrogen bonds that stabilize the location of the N-terminus of HA2 are lost in the mutant, resulting in a local destabilization that facilitates the extrusion of the N-terminus at higher pH.

  View details for Web of Science ID A1990CJ35900003

  View details for PubMedID 2295311

• MEMBRANE-FUSION BY INFLUENZA-VIRUSES AND THE MECHANISM OF ACTION OF AMANTADINE INTERNATIONAL WORKSHOP ON THE USE OF X-RAY CRYSTALLOGRAPHY IN THE DESIGN OF ANTIVIRAL AGENTS Wharton, S. A., Hay, A. J., Sugrue, R. J., Skehel, J. J., Weis, W. I., WILEY, D. C. ACADEMIC PRESS INC. 1990: 1–12

  View details for Web of Science ID A1990BQ49U00001

• IMMUNE RECOGNITION OF INFLUENZA-VIRUS HEMAGGLUTININ PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY OF LONDON SERIES B-BIOLOGICAL SCIENCES Skehel, J. J., BARNET, B. C., Burt, D. S., Daniels, R. S., Douglas, A. R., Graham, C. M., Hodgson, J., Knossow, M., Mills, K. H., Riska, P. F., Thomas, D. B., Weis, W., WILEY, D. C., WRIGLEY, N. G. 1989; 323 (1217): 479-?

  Abstract

  Haemagglutinin glycoproteins are the components of influenza virus membranes against which infectivity-neutralizing antibodies are directed. Sequence analysis of natural and laboratory-selected variant haemagglutinins indicates the regions of the molecule recognized by antibodies and by helper T cells; the identity of these regions and the relations between them are discussed.

  View details for Web of Science ID A1989AB41200003

  View details for PubMedID 2569205

• STUDIES ON THE STRUCTURE OF THE INFLUENZA-VIRUS HEMAGGLUTININ AT THE PH OF MEMBRANE-FUSION JOURNAL OF GENERAL VIROLOGY Ruigrok, R. W., Aitken, A., Calder, L. J., Martin, S. R., Skehel, J. J., Wharton, S. A., Weis, W., WILEY, D. C. 1988; 69: 2785-2795

  Abstract

  At the pH required to trigger the membrane fusion activity of the influenza virus haemagglutinin (HA) the soluble ectodomain of the molecule, BHA, which is released from virus by bromelain digestion, aggregates into rosettes. Analyses of soluble proteolytic fragments derived from the rosettes indicated that aggregation is mediated by association of the conserved hydrophobic amino-terminal region of BHA2, the smaller glycopolypeptide component of each BHA subunit. Further analyses of the structure of the soluble fragments and of HA in its low pH conformation by electron microscopy, spectroscopy and in crosslinking experiments showed that, although the membrane distal globular domains lose their trimer structure at the pH of fusion, the central fibrous stem of the molecule remains trimeric and assumes a more stable conformation. The increase in length of BHA2 at low pH observed microscopically appears to result from movement of the amino-terminal region to the membrane proximal end of the molecule and in virus incubated at low pH the amino terminus may insert into the virus membrane. The consequences of these possibilities for the mechanism of membrane fusion are discussed.

  View details for Web of Science ID A1988Q900800010

  View details for PubMedID 3183628

• STRUCTURE OF THE INFLUENZA-VIRUS HEMAGGLUTININ COMPLEXED WITH ITS RECEPTOR, SIALIC-ACID NATURE Weis, W., Brown, J. H., Cusack, S., Paulson, J. C., Skehel, J. J., WILEY, D. C. 1988; 333 (6172): 426-431

  Abstract

  The three-dimensional structures of influenza virus haemagglutinins complexed with cell receptor analogues show sialic acids bound to a pocket of conserved amino acids surrounded by antibody-binding sites. Sialic acid fills the conserved pocket, demonstrating that it is the influenza virus receptor. The proximity of the antibody-binding sites suggests that antibodies neutralize virus infectivity by preventing virus-to-cell binding. The structures suggest approaches to the design of anti-viral drugs that could block attachment of viruses to cells.

  View details for Web of Science ID A1988N634000051

  View details for PubMedID 3374584

• CONFORMATIONAL ASPECTS OF THE ACID-INDUCED FUSION MECHANISM OF INFLUENZA-VIRUS HEMAGGLUTININ - CIRCULAR-DICHROISM AND FLUORESCENCE STUDIES JOURNAL OF BIOLOGICAL CHEMISTRY Wharton, S. A., Ruigrok, R. W., Martin, S. R., Skehel, J. J., Bayley, P. M., Weis, W., WILEY, D. C. 1988; 263 (9): 4474-4480

  Abstract

  Circular dichroism and tryptophan fluorescence spectroscopy have been used to investigate the structures of the influenza virus membrane glycoprotein hemagglutinin, acid-treated hemagglutinin, and fragments of hemagglutinin derived by proteolysis. The conformational change in hemagglutinin which occurs at the pH of membrane fusion (pH 5-6) was associated with a significant change of the environment of tyrosine residues, a change in the environment of tryptophan residues, but no changes in secondary structure. Tryptic digestion of the hemagglutinin in its low pH conformation which releases one of the subunit polypeptides (HA1) caused minimal changes in tyrosine and tryptophan environments but a small secondary structural change in HA1. The secondary structure of the remainder of the molecule (HA2) was very similar to that predicted from the known x-ray crystallographic structure of the native molecule. However, fluorescence spectroscopy indicated a tertiary change in structure in the coiled coil of alpha-helices which form the fibrous central stem of the molecule. These results are consistent with a conformational change required for membrane fusion which involves a decrease of HA1/HA1, HA1/HA2 interactions and changes in tertiary structure not accompanied by changes in secondary structure.

  View details for Web of Science ID A1988M662000065

  View details for PubMedID 3346256