Axel Brunger

All Publications

• In-SituStructure and Topography of AMPA Receptor Scaffolding Complexes Visualized by CryoET. bioRxiv : the preprint server for biology Held, R. G., Liang, J., Esquivies, L., Khan, Y. A., Wang, C., Azubel, M., Brunger, A. T. 2024

  Abstract

  Most synapses in the brain transmit information by the presynaptic release of vesicular glutamate, driving postsynaptic depolarization through AMPA-type glutamate receptors (AMPARs). The nanometer-scale topography of synaptic AMPARs regulates response amplitude by controlling the number of receptors activated by synaptic vesicle fusion. The mechanisms controlling AMPAR topography and their interactions with postsynaptic scaffolding proteins are unclear, as is the spatial relationship between AMPARs and synaptic vesicles. Here, we used cryo-electron tomography to map the molecular topography of AMPARs and visualize their in-situ structure. Clustered AMPARs form structured complexes with postsynaptic scaffolding proteins resolved by sub-tomogram averaging. Sub-synaptic topography mapping reveals the presence of AMPAR nanoclusters with exclusion zones beneath synaptic vesicles. Our molecular-resolution maps visualize the predominant information transfer path in the nervous system.

  View details for DOI 10.1101/2024.10.19.619226

  View details for PubMedID 39464045

• Pre-fusion AAA+ remodeling of target-SNARE protein complexes enables synaptic transmission. bioRxiv : the preprint server for biology White, K. I., Khan, Y. A., Qiu, K., Balaji, A., Couoh-Cardel, S., Esquivies, L., Pfuetzner, R. A., Diao, J., Brunger, A. T. 2024

  Abstract

  Membrane fusion is driven by SNARE complex formation across cellular contexts, including vesicle fusion during synaptic transmission. Multiple proteins organize trans-SNARE complex assembly and priming, leading to fusion. One target membrane SNARE, syntaxin, forms nanodomains at the active zone, and another, SNAP-25, enters non-fusogenic complexes with it. Here, we show that the AAA+ protein NSF (N-ethylmaleimide sensitive factor) and SNAP (soluble NSF attachment protein) must act prior to fusion. We show that syntaxin clusters are conserved, that NSF colocalizes with them, and characterize SNARE populations within and near these clusters using cryo-EM. Supercomplexes of NSF, alpha-SNAP, and either a syntaxin tetramer or two binary complexes of syntaxin-SNAP-25 reveal atomic details of SNARE processing and show how sequential ATP hydrolysis drives disassembly. These results suggest a functional role for syntaxin clusters as reservoirs and a corresponding role for NSF in syntaxin liberation and SNARE protein quality control preceding fusion.

  View details for DOI 10.1101/2024.10.11.617886

  View details for PubMedID 39416070

• Sec18 side-loading is essential for universal SNARE recycling across cellular contexts. bioRxiv : the preprint server for biology Khan, Y. A., Ian White, K., Pfuetzner, R. A., Singal, B., Esquivies, L., Mckenzie, G., Liu, F., DeLong, K., Choi, U. B., Montabana, E., Mclaughlin, T., Wickner, W. T., Brunger, A. T. 2024

  Abstract

  SNARE proteins drive membrane fusion as their core domains zipper into a parallel four-helix bundle1,2. After fusion, these bundles are disassembled by the AAA+ protein Sec18/NSF and its adaptor Sec17/ α-SNAP3,4 to make them available for subsequent rounds of membrane fusion. SNARE domains are often flanked by C-terminal transmembrane or N-terminal domains5. Previous structures of the NSF-α-SNAP-SNARE complex revealed SNARE domain threaded through the D1 ATPase ring6, posing a topological constraint as SNARE transmembrane domains would prevent complete substrate threading as suggested for other AAA+ systems7. Here, in vivo mass-spectrometry reveals N-terminal SNARE domain interactions with Sec18, exacerbating this topological issue. Cryo-EM structures of a yeast SNARE complex, Sec18, and Sec17 in a non-hydrolyzing condition shows SNARE Sso1 threaded through the D1 and D2 ATPase rings of Sec18, with its folded, N-terminal Habc domain interacting with the D2 ring. This domain does not unfold during Sec18/NSF activity. Cryo-EM structures under hydrolyzing conditions revealed substrate-released and substrate-free states of Sec18 with a coordinated opening in the side of the ATPase rings. Thus, Sec18/NSF operates by substrate side-loading and unloading topologically constrained SNARE substrates.

  View details for DOI 10.1101/2024.08.30.610324

  View details for PubMedID 39257774

  View details for PubMedCentralID PMC11384006

• Observing isolated synaptic vesicle association and fusion ex vivo. Nature protocols Leitz, J., Wang, C., Esquivies, L., Peters, J. J., Gopal, N., Pfuetzner, R. A., Wang, A. L., Brunger, A. T. 2024

  Abstract

  Here, we present a protocol for isolating functionally intact glutamatergic synaptic vesicles from whole-mouse brain tissue and using them in a single-vesicle assay to examine their association and fusion with plasma membrane mimic vesicles. This is a Protocol Extension, building on our previous protocol, which used a purely synthetic system comprised of reconstituted proteins in liposomes. We also describe the generation of a peptide based on the vesicular glutamate transporter, which is essential in the isolation process of glutamatergic synaptic vesicles. This method uses easily accessible reagents to generate fusion-competent glutamatergic synaptic vesicles through immunoisolation. The generation of the vGlut peptide can be accomplished in 6 d, while the isolation of the synaptic vesicles by using the peptide can be accomplished in 2 d, with an additional day to fluorescently label the synaptic vesicles for use in a single-vesicle hybrid fusion assay. The single-vesicle fusion assay can be accomplished in 1 d and can unambiguously delineate synaptic vesicle association, dissociation, Ca2+-independent and Ca2+-dependent fusion modalities. This assay grants control of the synaptic vesicle environment while retaining the complexity of the synaptic vesicles themselves. This protocol can be adapted to studies of other types of synaptic vesicles or, more generally, different secretory or transport vesicles. The workflow described here requires expertise in biochemistry techniques, in particular, protein purification and fluorescence imaging. We assume that the laboratory has protein-purification equipment, including chromatography systems.

  View details for DOI 10.1038/s41596-024-01014-x

  View details for PubMedID 38956381

• Nanoscale architecture of synaptic vesicles and scaffolding complexes revealed by cryo-electron tomography. Proceedings of the National Academy of Sciences of the United States of America Held, R. G., Liang, J., Brunger, A. T. 2024; 121 (27): e2403136121

  Abstract

  The spatial distribution of proteins and their arrangement within the cellular ultrastructure regulates the opening of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in response to glutamate release at the synapse. Fluorescence microscopy imaging revealed that the postsynaptic density (PSD) and scaffolding proteins in the presynaptic active zone (AZ) align across the synapse to form a trans-synaptic "nanocolumn," but the relation to synaptic vesicle release sites is uncertain. Here, we employ focused-ion beam (FIB) milling and cryoelectron tomography to image synapses under near-native conditions. Improved image contrast, enabled by FIB milling, allows simultaneous visualization of supramolecular nanoclusters within the AZ and PSD and synaptic vesicles. Surprisingly, membrane-proximal synaptic vesicles, which fuse to release glutamate, are not preferentially aligned with AZ or PSD nanoclusters. These synaptic vesicles are linked to the membrane by peripheral protein densities, often consistent in size and shape with Munc13, as well as globular densities bridging the synaptic vesicle and plasma membrane, consistent with prefusion complexes of SNAREs, synaptotagmins, and complexin. Monte Carlo simulations of synaptic transmission events using biorealistic models guided by our tomograms predict that clustering AMPARs within PSD nanoclusters increases the variability of the postsynaptic response but not its average amplitude. Together, our data support a model in which synaptic strength is tuned at the level of single vesicles by the spatial relationship between scaffolding nanoclusters and single synaptic vesicle fusion sites.

  View details for DOI 10.1073/pnas.2403136121

  View details for PubMedID 38923992

• VAMP2 chaperones alpha-synuclein in synaptic vesicle co-condensates. Nature cell biology Wang, C., Zhang, K., Cai, B., Haller, J. E., Carnazza, K. E., Hu, J., Zhao, C., Tian, Z., Hu, X., Hall, D., Qiang, J., Hou, S., Liu, Z., Gu, J., Zhang, Y., Seroogy, K. B., Burre, J., Fang, Y., Liu, C., Brunger, A. T., Li, D., Diao, J. 2024

  Abstract

  alpha-Synuclein (alpha-Syn) aggregation is closely associated with Parkinson's disease neuropathology. Physiologically, alpha-Syn promotes synaptic vesicle (SV) clustering and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly. However, the underlying structural and molecular mechanisms are uncertain and it is not known whether this function affects the pathological aggregation of alpha-Syn. Here we show that the juxtamembrane region of vesicle-associated membrane protein 2 (VAMP2)-a component of the SNARE complex that resides on SVs-directly interacts with the carboxy-terminal region of alpha-Syn through charged residues to regulate alpha-Syn's function in clustering SVs and promoting SNARE complex assembly by inducing a multi-component condensed phase of SVs, alpha-Syn and other components. Moreover, VAMP2 binding protects alpha-Syn against forming aggregation-prone oligomers and fibrils in these condensates. Our results suggest a molecular mechanism that maintains alpha-Syn's function and prevents its pathological amyloid aggregation, the failure of which may lead to Parkinson's disease.

  View details for DOI 10.1038/s41556-024-01456-1

  View details for PubMedID 38951706

• Structure and topography of the synaptic V-ATPase-synaptophysin complex. Nature Wang, C., Jiang, W., Leitz, J., Yang, K., Esquivies, L., Wang, X., Shen, X., Held, R., Adams, D. J., Basta, T., Hampton, L., Jian, R., Jiang, L., Stowell, M. H., Baumeister, W., Guo, Q., Brunger, A. T. 2024

  Abstract

  Synaptic vesicles are organelles with a precisely defined protein and lipid composition1,2, yet the molecular mechanisms for the biogenesis of synaptic vesicles are mainly unknown. Here, we discovered a well-defined interface between the synaptic vesicle V-ATPase and synaptophysin by in situ cryo-electron tomography and single particle cryo-electron microscopy of functional synaptic vesicles isolated from mouse brains3. The synaptic vesicle V-ATPase is an ATP-dependent proton pump that establishes the protein gradient across the synaptic vesicle, which in turn drives the uptake of neurotransmitters4,5. Synaptophysin6 and its paralogs synaptoporin7 and synaptogyrin8 belong to a family of abundant synaptic vesicle proteins whose function is still unclear. We performed structural and functional studies of synaptophysin knockout mice, confirming the identity of synaptophysin as an interaction partner with the V-ATPase. Although there is little change in the conformation of the V-ATPase upon interaction with synaptophysin, the presence of synaptophysin in synaptic vesicles profoundly affects the copy number of V-ATPases. This effect on the topography of synaptic vesicles suggests that synaptophysin assists in their biogenesis. In support of this model, we observed that synaptophysin knockout mice exhibit severe seizure susceptibility, suggesting an imbalance of neurotransmitter release as a physiological consequence of the absence of synaptophysin.

  View details for DOI 10.1038/s41586-024-07610-x

  View details for PubMedID 38838737

• Beyond the MUN domain, Munc13 controls priming and depriming of synaptic vesicles. Cell reports Leitz, J., Wang, C., Esquivies, L., Pfuetzner, R. A., Peters, J. J., Couoh-Cardel, S., Wang, A. L., Brunger, A. T. 2024; 43 (5): 114026

  Abstract

  Synaptic vesicle docking and priming are dynamic processes. At the molecular level, SNAREs (soluble NSF attachment protein receptors), synaptotagmins, and other factors are critical for Ca2+-triggered vesicle exocytosis, while disassembly factors, including NSF (N-ethylmaleimide-sensitive factor) and α-SNAP (soluble NSF attachment protein), disassemble and recycle SNAREs and antagonize fusion under some conditions. Here, we introduce a hybrid fusion assay that uses synaptic vesicles isolated from mouse brains and synthetic plasma membrane mimics. We included Munc18, Munc13, complexin, NSF, α-SNAP, and an ATP-regeneration system and maintained them continuously-as in the neuron-to investigate how these opposing processes yield fusogenic synaptic vesicles. In this setting, synaptic vesicle association is reversible, and the ATP-regeneration system produces the most synchronous Ca2+-triggered fusion, suggesting that disassembly factors perform quality control at the early stages of synaptic vesicle association to establish a highly fusogenic state. We uncovered a functional role for Munc13 ancillary to the MUN domain that alleviates an α-SNAP-dependent inhibition of Ca2+-triggered fusion.

  View details for DOI 10.1016/j.celrep.2024.114026

  View details for PubMedID 38809756

• A new method for isolation and purification of fusion-competent inhibitory synaptic vesicles. Current research in physiology Gopal, N., Leitz, J., Wang, C., Esquivies, L., Pfuetzner, R. A., Brunger, A. T. 2024; 7: 100121

  Abstract

  Synaptic vesicles specific to inhibitory GABA-releasing neurons are critical for regulating neuronal excitability. To study the specific molecular composition, architecture, and function of inhibitory synaptic vesicles, we have developed a new method to isolate and purify GABA synaptic vesicles from mouse brains. GABA synaptic vesicles were immunoisolated from mouse brain tissue using an engineered fragment antigen-binding region (Fab) against the vesicular GABA transporter (vGAT) and purified. Western blot analysis confirmed that the GABA synaptic vesicles were specifically enriched for vGAT and largely depleted of contaminants from other synaptic vesicle types, such as vesicular glutamate transporter (vGLUT1), and other cellular organelles. This degree of purity was achieved despite the relatively low abundance of vGAT vesicles compared to the total synaptic vesicle pool in mammalian brains. Cryo-electron microscopy images of these isolated GABA synaptic vesicles revealed intact morphology with circular shape and protruding proteinaceous densities. The GABA synaptic vesicles are functional, as assessed by a hybrid (ex vivo/in vitro) vesicle fusion assay, and they undergo synchronized fusion with synthetic plasma membrane mimic vesicles in response to Ca2+-triggering, but, as a negative control, not to Mg2+-triggering. Our immunoisolation method could also be applied to other types of vesicles.

  View details for DOI 10.1016/j.crphys.2024.100121

  View details for PubMedID 38572021

  View details for PubMedCentralID PMC10990708

• Neutral lysophosphatidylcholine mediates alpha-synuclein-induced synaptic vesicle clustering. Proceedings of the National Academy of Sciences of the United States of America Lai, Y., Zhao, C., Tian, Z., Wang, C., Fan, J., Hu, X., Tu, J., Li, T., Leitz, J., Pfuetzner, R. A., Liu, Z., Zhang, S., Su, Z., Burre, J., Li, D., Sudhof, T. C., Zhu, Z., Liu, C., Brunger, A. T., Diao, J. 2023; 120 (44): e2310174120

  Abstract

  alpha-synuclein (alpha-Syn) is a presynaptic protein that is involved in Parkinson's and other neurodegenerative diseases and binds to negatively charged phospholipids. Previously, we reported that alpha-Syn clusters synthetic proteoliposomes that mimic synaptic vesicles. This vesicle-clustering activity depends on a specific interaction of alpha-Syn with anionic phospholipids. Here, we report that alpha-Syn surprisingly also interacts with the neutral phospholipid lysophosphatidylcholine (lysoPC). Even in the absence of anionic lipids, lysoPC facilitates alpha-Syn-induced vesicle clustering but has no effect on Ca2+-triggered fusion in a single vesicle-vesicle fusion assay. The A30P mutant of alpha-Syn that causes familial Parkinson disease has a reduced affinity to lysoPC and does not induce vesicle clustering. Taken together, the alpha-Syn-lysoPC interaction may play a role in alpha-Syn function.

  View details for DOI 10.1073/pnas.2310174120

  View details for PubMedID 37883437

• Sensory deficit screen identifies nsf mutation that differentially affects SNARE recycling and quality control. Cell reports Gao, Y., Khan, Y. A., Mo, W., White, K. I., Perkins, M., Pfuetzner, R. A., Trapani, J. G., Brunger, A. T., Nicolson, T. 2023; 42 (4): 112345

  Abstract

  The AAA+ NSF complex is responsible for SNARE complex disassembly both before and after membrane fusion. Loss of NSF function results in pronounced developmental and degenerative defects. In a genetic screen for sensory deficits in zebrafish, we identified a mutation in nsf, I209N, that impairs hearing and balance in a dosage-dependent manner without accompanying defects in motility, myelination, and innervation. Invitro experiments demonstrate that while the I209N NSF protein recognizes SNARE complexes, the effects on disassembly are dependent upon the type of SNARE complex and I209N concentration. Higher levels of I209N protein produce a modest decrease in binary (syntaxin-SNAP-25) SNARE complex disassembly and residual ternary (syntaxin-1A-SNAP-25-synaptobrevin-2) disassembly, whereas at lower concentrations binary disassembly activity is strongly reduced and ternary disassembly activity is absent. Our study suggests that the differential effect on disassembly of SNARE complexes leads to selective effects on NSF-mediated membrane trafficking and auditory/vestibular function.

  View details for DOI 10.1016/j.celrep.2023.112345

  View details for PubMedID 37027300

• Structure-based design of a SARS-CoV-2 Omicron-specific inhibitor. Proceedings of the National Academy of Sciences of the United States of America Yang, K., Wang, C., Kreutzberger, A. J., White, K. I., Pfuetzner, R. A., Esquivies, L., Kirchhausen, T., Brunger, A. T. 2023; 120 (13): e2300360120

  Abstract

  The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) introduced a relatively large number of mutations, including three mutations in the highly conserved heptad repeat 1 (HR1) region of the spike glycoprotein (S) critical for its membrane fusion activity. We show that one of these mutations, N969K induces a substantial displacement in the structure of the heptad repeat 2 (HR2) backbone in the HR1HR2 postfusion bundle. Due to this mutation, fusion-entry peptide inhibitors based on the Wuhan strain sequence are less efficacious. Here, we report an Omicron-specific peptide inhibitor designed based on the structure of the Omicron HR1HR2 postfusion bundle. Specifically, we inserted an additional residue in HR2 near the Omicron HR1 K969 residue to better accommodate the N969K mutation and relieve the distortion in the structure of the HR1HR2 postfusion bundle it introduced. The designed inhibitor recovers the loss of inhibition activity of the original longHR2_42 peptide with the Wuhan strain sequence against the Omicron variant in both a cell-cell fusion assay and a vesicular stomatitis virus (VSV)-SARS-CoV-2 chimera infection assay, suggesting that a similar approach could be used to combat future variants. From a mechanistic perspective, our work suggests the interactions in the extended region of HR2 may mediate the initial landing of HR2 onto HR1 during the transition of the S protein from the prehairpin intermediate to the postfusion state.

  View details for DOI 10.1073/pnas.2300360120

  View details for PubMedID 36940324

• Analysis of tripartite Synaptotagmin-1-SNARE-complexin-1 complexes in solution. FEBS open bio Jaczynska, K., Esquivies, L., Pfuetzner, R. A., Alten, B., Brewer, K. D., Zhou, Q., Kavalali, E. T., Brunger, A. T., Rizo, J. 2022

  Abstract

  Characterizing interactions of Synaptotagmin-1 with the SNARE complex is crucial to understand the mechanism of neurotransmitter release. X-ray crystallography revealed how the Synaptotagmin-1 C2 B domain binds to the SNARE complex through a so-called primary interface and to a complexin-1-SNARE complex through a so-called tripartite interface. Mutagenesis and electrophysiology supported the functional relevance of both interfaces, and extensive additional data validated the primary interface. However, ITC evidence suggesting that binding via the tripartite interface occurs in solution was called into question by subsequent NMR data. Here we describe joint efforts to address this apparent contradiction. Using the same ITC approach with the same C2 B domain mutant used previously (C2 BKA-Q ) but including ion exchange chromatography to purify it, which is crucial to remove polyacidic contaminants, we were unable to observe the substantial endothermic ITC signal that was previously attributed to binding of this mutant to the complexin-1-SNARE complex through the tripartite interface. We were also unable to detect substantial populations of the tripartite interface in NMR analyses of the ITC samples or in measurements of paramagnetic relaxation effects, despite the high sensitivity of this method to detect weak protein complexes. However, these experiments do not rule out the possibility of very low affinity (KD >1mM) binding though this interface. These results emphasize the need to develop methods to characterize the structure of synaptotagmin-1-SNARE complexes between two membranes, and to perform further structure-function analyses to establish the physiological relevance of the tripartite interface.

  View details for DOI 10.1002/2211-5463.13503

  View details for PubMedID 36305864

• The core complex of the Ca2+-triggered presynaptic fusion machinery. Journal of molecular biology Brunger, A. T., Leitz, J. 2022: 167853

  Abstract

  Synaptic neurotransmitter release is mediated by an orchestra of presynaptic proteins that precisely control and trigger fusion between synaptic vesicles and the neuron terminal at the active zone upon an action potential. Critical to this process are the neuronal SNAREs (Soluble N-ethylmaleimide sensitive factor Attachment protein REceptor), the Ca2+-sensor synaptotagmin, the activator/regulator complexin, and other factors. Here, we review the interactions between the SNARE complex and synaptotagmin, with focus on the so-called primary interface between synaptotagmin and the SNARE complex that has been validated in terms of its physiological relevance. We discuss several other but less validated interfaces as well, including the so-called tripartite interface, and we discuss the pros and cons for these possible alternative interfaces. We also present new molecular dynamics simulations of the tripartite interface and new data of an inhibitor of the primary interface in a reconstituted system of synaptic vesicle fusion.

  View details for DOI 10.1016/j.jmb.2022.167853

  View details for PubMedID 36243149

• Nanomolar inhibition of SARS-CoV-2 infection by an unmodified peptide targeting the prehairpin intermediate of the spike protein. Proceedings of the National Academy of Sciences of the United States of America Yang, K., Wang, C., Kreutzberger, A. J., Ojha, R., Kuivanen, S., Couoh-Cardel, S., Muratcioglu, S., Eisen, T. J., White, K. I., Held, R. G., Subramanian, S., Marcus, K., Pfuetzner, R. A., Esquivies, L., Doyle, C. A., Kuriyan, J., Vapalahti, O., Balistreri, G., Kirchhausen, T., Brunger, A. T. 2022; 119 (40): e2210990119

  Abstract

  Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge currently available coronavirus disease 2019 vaccines and monoclonal antibody therapies through epitope change on the receptor binding domain of the viral spike glycoprotein. Hence, there is a specific urgent need for alternative antivirals that target processes less likely to be affected by mutation, such as the membrane fusion step of viral entry into the host cell. One such antiviral class includes peptide inhibitors, which block formation of the so-called heptad repeat 1 and 2 (HR1HR2) six-helix bundle of the SARS-CoV-2 spike (S) protein and thus interfere with viral membrane fusion. We performed structural studies of the HR1HR2 bundle, revealing an extended, well-folded N-terminal region of HR2 that interacts with the HR1 triple helix. Based on this structure, we designed an extended HR2 peptide that achieves single-digit nanomolar inhibition of SARS-CoV-2 in cell-based and virus-based assays without the need for modifications such as lipidation or chemical stapling. The peptide also strongly inhibits all major SARS-CoV-2 variants to date. This extended peptide is ∼100-fold more potent than all previously published short, unmodified HR2 peptides, and it has a very long inhibition lifetime after washout in virus infection assays, suggesting that it targets a prehairpin intermediate of the SARS-CoV-2 S protein. Together, these results suggest that regions outside the HR2 helical region may offer new opportunities for potent peptide-derived therapeutics for SARS-CoV-2 and its variants, and even more distantly related viruses, and provide further support for the prehairpin intermediate of the S protein.

  View details for DOI 10.1073/pnas.2210990119

  View details for PubMedID 36122200

• Nanomolar inhibition of SARS-CoV-2 infection by an unmodified peptide targeting the prehairpin intermediate of the spike protein PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Yang, K., Wang, C., Kreutzberger, A. B., Ojha, R., Kuivanen, S., Couoh-Cardel, S., Muratcioglu, S., Eisen, T. J., White, K., Held, R. G., Subramanian, S., Marcus, K., Pfuetzner, R. A., Esquivies, L., Doyle, C. A., Kuriyan, J., Vapalahti, O., Balistreri, G., Kirchhausen, T., Brunger, A. T. 2022; 119 (40)

  View details for DOI 10.1073/pnas.2210990119/-/DCSupplemental

  View details for Web of Science ID 000986627700001

• Nanomolar inhibition of SARS-CoV-2 infection by an unmodified peptide targeting the pre-hairpin intermediate of the spike protein. bioRxiv : the preprint server for biology Yang, K., Wang, C., Kreutzberger, A. J., Ojha, R., Kuivanen, S., Couoh-Cardel, S., Muratcioglu, S., Eisen, T. J., White, K. I., Held, R. G., Subramanian, S., Marcus, K., Pfuetzner, R. A., Esquivies, L., Doyle, C. A., Kuriyan, J., Vapalahti, O., Balistreri, G., Kirchhausen, T., Brunger, A. T. 2022

  Abstract

  Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge currently available COVID-19 vaccines and monoclonal antibody therapies through epitope change on the receptor binding domain of the viral spike glycoprotein. Hence, there is a specific urgent need for alternative antivirals that target processes less likely to be affected by mutation, such as the membrane fusion step of viral entry into the host cell. One such antiviral class includes peptide inhibitors which block formation of the so-called HR1HR2 six-helix bundle of the SARS-CoV-2 spike (S) protein and thus interfere with viral membrane fusion. Here we performed structural studies of the HR1HR2 bundle, revealing an extended, well-folded N-terminal region of HR2 that interacts with the HR1 triple helix. Based on this structure, we designed an extended HR2 peptide that achieves single-digit nanomolar inhibition of SARS-CoV-2 in cell-based fusion, VSV-SARS-CoV-2 chimera, and authentic SARS-CoV-2 infection assays without the need for modifications such as lipidation or chemical stapling. The peptide also strongly inhibits all major SARS-CoV-2 variants to date. This extended peptide is ~100-fold more potent than all previously published short, unmodified HR2 peptides, and it has a very long inhibition lifetime after washout in virus infection assays, suggesting that it targets a pre-hairpin intermediate of the SARS-CoV-2 S protein. Together, these results suggest that regions outside the HR2 helical region may offer new opportunities for potent peptide-derived therapeutics for SARS-CoV-2 and its variants, and even more distantly related viruses, and provide further support for the pre-hairpin intermediate of the S protein.Significance Statement: SARS-CoV-2 infection requires fusion of viral and host membranes, mediated by the viral spike glycoprotein (S). Due to the importance of viral membrane fusion, S has been a popular target for developing vaccines and therapeutics. We discovered a simple peptide that inhibits infection by all major variants of SARS-CoV-2 with nanomolar efficacies. In marked contrast, widely used shorter peptides that lack a key N-terminal extension are about 100 x less potent than this peptide. Our results suggest that a simple peptide with a suitable sequence can be a potent and cost-effective therapeutic against COVID-19 and they provide new insights at the virus entry mechanism.

  View details for DOI 10.1101/2022.08.11.503553

  View details for PubMedID 35982670

• Discovery of a drug to treat airway mucus hypersecretion. Clinical and translational medicine Dickey, B. F., Lai, Y., Frick, M., Brunger, A. T. 2022; 12 (8): e972

  View details for DOI 10.1002/ctm2.972

  View details for PubMedID 35908252

• Structural conservation among variants of the SARS-CoV-2 spike postfusion bundle. Proceedings of the National Academy of Sciences of the United States of America Yang, K., Wang, C., White, K. I., Pfuetzner, R. A., Esquivies, L., Brunger, A. T. 2022; 119 (16): e2119467119

  Abstract

  Significance Emergence of viral pathogens necessitates new approaches to study viral fusion and entry into host cells. A key step in mediating fusion involves the formation of a six-helix bundle within the spike protein. Rapid structural characterization of this state has been difficult, hindering understanding of emerging variants. We developed a method to efficiently determine high-resolution bundle structures by molecular scaffolding and cryogenic electron microscopy. Using this method, we determined bundle structures of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. These structures reveal local effects of mutations on HR1HR2 interactions but global conservation of the bundle architecture among SARS-CoV-2 variants. We predict that inhibitors disrupting the postfusion bundle might be broadly efficacious against variants and even more distantly related lethal viruses.

  View details for DOI 10.1073/pnas.2119467119

  View details for PubMedID 35363556

• A feature-guided, focused 3D signal permutation method for subtomogram averaging. Journal of structural biology Jacob Peters, J., Leitz, J., Guo, Q., Beck, F., Baumeister, W., Brunger, A. T. 2022: 107851

  Abstract

  Advances in electron microscope instrumentation, cryo-electron tomography data collection, and subtomogram averaging have allowed for the in-situ visualization of molecules and their complexes in their native environment. Current data processing pipelines commonly extract subtomograms as a cubic subvolume with the key assumption that the selected object of interest is discrete from its surroundings. However, in instances when the object is in its native environment, surrounding densities may negatively affect the subsequent alignment and refinement processes, leading to loss of information due to misalignment. For example, the strong densities from surrounding membranes may dominate the alignment process for membrane proteins. Here, we developed methods for feature-guided subtomogram alignment and 3D signal permutation for subtomogram averaging. Our 3D signal permutation method randomizes and filters voxels outside a mask of any shape and blurs the boundary of the mask that encapsulates the object of interest. The randomization preserves global statistical properties such as mean density and standard deviation of voxel density values, effectively producing a featureless background surrounding the object of interest. This signal permutation process can be repeatedly applied with intervening alignments of the 3D signal-permuted subvolumes, recentering of the mask, and optional adjustments of the shape of the mask. We have implemented these methods in a new processing pipeline which starts from tomograms, contains feature-guided subtomogram extraction and alignment, 3D signal-permutation, and subtomogram visualization tools. As an example, feature-guided alignment and 3D signal permutation leads to improved subtomogram average maps for a dataset of synaptic protein complexes in their native environment.

  View details for DOI 10.1016/j.jsb.2022.107851

  View details for PubMedID 35346811

• Inhibition of calcium-triggered secretion by hydrocarbon-stapled peptides. Nature Lai, Y., Fois, G., Flores, J. R., Tuvim, M. J., Zhou, Q., Yang, K., Leitz, J., Peters, J., Zhang, Y., Pfuetzner, R. A., Esquivies, L., Jones, P., Frick, M., Dickey, B. F., Brunger, A. T. 2022

  Abstract

  Membrane fusion triggered by Ca2+ is orchestrated by a conserved set of proteins to mediate synaptic neurotransmitter release, mucin secretion and other regulated exocytic processes1-4. For neurotransmitter release, the Ca2+ sensitivity is introduced by interactions between the Ca2+ sensor synaptotagmin and the SNARE complex5, and sequence conservation and functional studies suggest that this mechanism is also conserved for mucin secretion6. Disruption of Ca2+-triggered membrane fusion by a pharmacological agent would have therapeutic value for mucus hypersecretion as it is the major cause of airway obstruction in the pathophysiology of respiratory viral infection, asthma, chronic obstructive pulmonary disease and cystic fibrosis7-11. Here we designed a hydrocarbon-stapled peptide that specifically disrupts Ca2+-triggered membrane fusion by interfering with the so-called primary interface between the neuronal SNARE complex and the Ca2+-binding C2B domain of synaptotagmin-1. In reconstituted systems with these neuronal synaptic proteins or with their airway homologues syntaxin-3, SNAP-23, VAMP8, synaptotagmin-2, along with Munc13-2 and Munc18-2, the stapled peptide strongly suppressed Ca2+-triggered fusion at physiological Ca2+ concentrations. Conjugation of cell-penetrating peptides to the stapled peptide resulted in efficient delivery into cultured human airway epithelial cells and mouse airway epithelium, where it markedly and specifically reduced stimulated mucin secretion in both systems, and substantially attenuated mucus occlusion of mouse airways. Taken together, peptides that disrupt Ca2+-triggered membrane fusion may enable the therapeutic modulation of mucin secretory pathways.

  View details for DOI 10.1038/s41586-022-04543-1

  View details for PubMedID 35322233

• The protein-folding problem: Not yet solved. Science (New York, N.Y.) Moore, P. B., Hendrickson, W. A., Henderson, R., Brunger, A. T. 1800; 375 (6580): 507

  View details for DOI 10.1126/science.abn9422

  View details for PubMedID 35113705

• Screening of Hydrocarbon-Stapled Peptides for Inhibition of Calcium-Triggered Exocytosis. Frontiers in pharmacology Lai, Y., Tuvim, M. J., Leitz, J., Peters, J., Pfuetzner, R. A., Esquivies, L., Zhou, Q., Czako, B., Cross, J. B., Jones, P., Dickey, B. F., Brunger, A. T. 2022; 13: 891041

  Abstract

  The so-called primary interface between the SNARE complex and synaptotagmin-1 (Syt1) is essential for Ca2+-triggered neurotransmitter release in neuronal synapses. The interacting residues of the primary interface are conserved across different species for synaptotagmins (Syt1, Syt2, Syt9), SNAP-25, and syntaxin-1A homologs involved in fast synchronous release. This Ca2+-independent interface forms prior to Ca2+-triggering and plays a role in synaptic vesicle priming. This primary interface is also conserved in the fusion machinery that is responsible for mucin granule membrane fusion. Ca2+-stimulated mucin secretion is mediated by the SNAREs syntaxin-3, SNAP-23, VAMP8, Syt2, and other proteins. Here, we designed and screened a series of hydrocarbon-stapled peptides consisting of SNAP-25 fragments that included some of the key residues involved in the primary interface as observed in high-resolution crystal structures. We selected a subset of four stapled peptides that were highly alpha-helical as assessed by circular dichroism and that inhibited both Ca2+-independent and Ca2+-triggered ensemble lipid-mixing with neuronal SNAREs and Syt1. In a single-vesicle content-mixing assay with reconstituted neuronal SNAREs and Syt1 or with reconstituted airway SNAREs and Syt2, the selected peptides also suppressed Ca2+-triggered fusion. Taken together, hydrocarbon-stapled peptides that interfere with the primary interface consequently inhibit Ca2+-triggered exocytosis. Our inhibitor screen suggests that these compounds may be useful to combat mucus hypersecretion, which is a major cause of airway obstruction in the pathophysiology of COPD, asthma, and cystic fibrosis.

  View details for DOI 10.3389/fphar.2022.891041

  View details for PubMedID 35814209

• Conformational dynamics of auto-inhibition in the ER calcium sensor STIM1. eLife van Dorp, S., Qiu, R., Choi, U. B., Wu, M. M., Yen, M., Kirmiz, M., Brunger, A. T., Lewis, R. S. 2021; 10

  Abstract

  The dimeric ER Ca2+ sensor STIM1 controls store-operated Ca2+ entry (SOCE) through the regulated binding of its CRAC activation domain (CAD) to Orai channels in the plasma membrane. In resting cells, the STIM1 CC1 domain interacts with CAD to suppress SOCE, but the structural basis of this interaction is unclear. Using single-molecule Forster resonance energy transfer (smFRET) and protein crosslinking approaches, we show that CC1 interacts dynamically with CAD in a domain-swapped configuration with an orientation predicted to sequester its Orai-binding region adjacent to the ER membrane. Following ER Ca2+ depletion and release from CAD, cysteine crosslinking indicates that the two CC1 domains become closely paired along their entire length in the active Orai-bound state. These findings provide a structural basis for the dual roles of CC1: sequestering CAD to suppress SOCE in resting cells and propelling it towards the plasma membrane to activate Orai and SOCE after store depletion.

  View details for DOI 10.7554/eLife.66194

  View details for PubMedID 34730514

• Conformational dynamics of auto-inhibition in the ER calcium sensor STIM1 ELIFE van Dorp, S., Qiu, R., Choi, U. B., Wu, M. M., Yen, M., Kirmiz, M., Brunger, A. T., Lewis, R. S. 2021; 10

  View details for DOI 10.7554/eLife.66194.sa2

  View details for Web of Science ID 000730064200001

• Prefused lysosomes cluster on autophagosomes regulated by VAMP8. Cell death & disease Chen, Q., Hao, M., Wang, L., Li, L., Chen, Y., Shao, X., Tian, Z., Pfuetzner, R. A., Zhong, Q., Brunger, A. T., Guan, J., Diao, J. 2021; 12 (10): 939

  Abstract

  Lysosome-autophagosome fusion is critical to autophagosome maturation. Although several proteins that regulate this fusion process have been identified, the prefusion architecture and its regulation remain unclear. Herein, we show that upon stimulation, multiple lysosomes form clusters around individual autophagosomes, setting the stage for membrane fusion. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein on lysosomes-vesicle-associated membrane protein 8 (VAMP8)-plays an important role in forming this prefusion state of lysosomal clusters. To study the potential role of phosphorylation on spontaneous fusion, we investigated the effect of phosphorylation of C-terminal residues of VAMP8. Using a phosphorylation mimic, we observed a decrease of fusion in an ensemble lipid mixing assay and an increase of unfused lysosomes associated with autophagosomes. These results suggest that phosphorylation not only reduces spontaneous fusion for minimizing autophagic flux under normal conditions, but also preassembles multiple lysosomes to increase the fusion probability for resuming autophagy upon stimulation. VAMP8 phosphorylation may thus play an important role in chemotherapy drug resistance by influencing autophagosome maturation.

  View details for DOI 10.1038/s41419-021-04243-0

  View details for PubMedID 34645799

• The AAA+superfamily: a review of the structural and mechanistic principles of these molecular machines. Critical reviews in biochemistry and molecular biology Khan, Y. A., White, K. I., Brunger, A. T. 2021: 1-32

  Abstract

  ATPases associated with diverse cellular activities (AAA+proteins) are a superfamily of proteins found throughout all domains of life. The hallmark of this family is a conserved AAA+domain responsible for a diverse range of cellular activities. Typically, AAA+proteins transduce chemical energy from the hydrolysis of ATP into mechanical energy through conformational change, which can drive a variety of biological processes. AAA+proteins operate in a variety of cellular contexts with diverse functions including disassembly of SNARE proteins, protein quality control, DNA replication, ribosome assembly, and viral replication. This breadth of function illustrates both the importance of AAA+proteins in health and disease and emphasizes the importance of understanding conserved mechanisms of chemo-mechanical energy transduction. This review is divided into three major portions. First, the core AAA+fold is presented. Next, the seven different clades of AAA+proteins and structural details and reclassification pertaining to proteins in each clade are described. Finally, two well-known AAA+proteins, NSF and its close relative p97, are reviewed in detail.

  View details for DOI 10.1080/10409238.2021.1979460

  View details for PubMedID 34632886

• Sec17/Sec18 can support membrane fusion without help from completion of SNARE zippering. eLife Song, H., Torng, T. L., Orr, A. S., Brunger, A. T., Wickner, W. T. 2021; 10

  Abstract

  Membrane fusion requires R-, Qa-, Qb-, and Qc-family SNAREs that zipper into RQaQbQc coiled coils, driven by the sequestration of apolar amino acids. Zippering has been thought to provide all the force driving fusion. Sec17/aSNAP can form an oligomeric assembly with SNAREs with the Sec17 C-terminus bound to Sec18/NSF, the central region bound to SNAREs, and a crucial apolar loop near the N-terminus poised to insert into membranes. We now report that Sec17 and Sec18 will drive robust fusion without requiring zippering completion. Zippering-driven fusion is blocked by deleting the C-terminal quarter of any Q-SNARE domain or by replacing the apolar amino acids of the Qa-SNARE which face the center of the 4-SNARE coiled coils with polar residues. These blocks, singly or combined, are bypassed by Sec17 and Sec18, and SNARE-dependent fusion is restored without help from completing zippering.

  View details for DOI 10.7554/eLife.67578

  View details for PubMedID 33944780

• Molecular Characterization of AMPA-Receptor-Containing Vesicles. Frontiers in molecular neuroscience Peters, J. J., Leitz, J., Oses-Prieto, J. A., Burlingame, A. L., Brunger, A. T. 2021; 14: 754631

  Abstract

  Regulated delivery of AMPA receptors (AMPARs) to the postsynaptic membrane is an essential step in synaptic strength modification, and in particular, long-term potentiation (LTP). While LTP has been extensively studied using electrophysiology and light microscopy, several questions regarding the molecular mechanisms of AMPAR delivery via trafficking vesicles remain outstanding, including the gross molecular make up of AMPAR trafficking organelles and identification and location of calcium sensors required for SNARE complex-dependent membrane fusion of such trafficking vesicles with the plasma membrane. Here, we isolated AMPA-containing vesicles (ACVs) from whole mouse brains via immunoisolation and characterized them using immunoelectron microscopy, immunoblotting, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). We identified several proteins on ACVs that were previously found to play a role in AMPAR trafficking, including synaptobrevin-2, Rabs, the SM protein Munc18-1, the calcium-sensor synaptotagmin-1, as well as several new candidates, including synaptophysin and synaptogyrin on ACV membranes. Additionally, we identified two populations of ACVs based on size and molecular composition: small-diameter, synaptobrevin-2- and GluA1-containing ACVs, and larger transferrin- receptor-, GluA1-, GluA2-, and GluA3-containing ACVs. The small-diameter population of ACVs may represent a fusion-capable population of vesicles due to the presence of synaptobrevin-2. Because the fusion of ACVs may be a requisite of LTP, this population could represent trafficking vesicles related to LTP.

  View details for DOI 10.3389/fnmol.2021.754631

  View details for PubMedID 34720876

  View details for PubMedCentralID PMC8554035

• Deorphanizing FAM19A proteins as pan-neurexin ligands with an unusual biosynthetic binding mechanism. The Journal of cell biology Khalaj, A. J., Sterky, F. H., Sclip, A., Schwenk, J., Brunger, A. T., Fakler, B., Sudhof, T. C. 2020; 219 (9)

  Abstract

  Neurexins are presynaptic adhesion molecules that organize synapses by binding to diverse trans-synaptic ligands, but how neurexins are regulated is incompletely understood. Here we identify FAM19A/TAFA proteins, "orphan" cytokines, as neurexin regulators that interact with all neurexins, except for neurexin-1gamma, via an unusual mechanism. Specifically, we show that FAM19A1-A4 bind to the cysteine-loop domain of neurexins by forming intermolecular disulfide bonds during transport through the secretory pathway. FAM19A-binding required both the cysteines of the cysteine-loop domain and an adjacent sequence of neurexins. Genetic deletion of neurexins suppressed FAM19A1 expression, demonstrating that FAM19As physiologically interact with neurexins. In hippocampal cultures, expression of exogenous FAM19A1 decreased neurexin O-glycosylation and suppressed its heparan sulfate modification, suggesting that FAM19As regulate the post-translational modification of neurexins. Given the selective expression of FAM19As in specific subtypes of neurons and their activity-dependent regulation, these results suggest that FAM19As serve as cell type-specific regulators of neurexin modifications.

  View details for DOI 10.1083/jcb.202004164

  View details for PubMedID 32706374

• Role of Aberrant Spontaneous Neurotransmission in SNAP25-Associated Encephalopathies. Neuron Alten, B. n., Zhou, Q. n., Shin, O. H., Esquivies, L. n., Lin, P. Y., White, K. I., Sun, R. n., Chung, W. K., Monteggia, L. M., Brunger, A. T., Kavalali, E. T. 2020

  Abstract

  SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) complex, composed of synaptobrevin, syntaxin, and SNAP25, forms the essential fusion machinery for neurotransmitter release. Recent studies have reported several mutations in the gene encoding SNAP25 as a causative factor for developmental and epileptic encephalopathies of infancy and childhood with diverse clinical manifestations. However, it remains unclear how SNAP25 mutations give rise to these disorders. Here, we show that although structurally clustered mutations in SNAP25 give rise to related synaptic transmission phenotypes, specific alterations in spontaneous neurotransmitter release are a key factor to account for disease heterogeneity. Importantly, we identified a single mutation that augments spontaneous release without altering evoked release, suggesting that aberrant spontaneous release is sufficient to cause disease in humans.

  View details for DOI 10.1016/j.neuron.2020.10.012

  View details for PubMedID 33147442

• NSF-MEDIATED DISASSEMBLY OF ON- AND OFF PATHWAY SNARE COMPLEXES AND INHIBITION BY COMPLEXIN Choi, U., Zhao, M., White, I., Brunger, A. WILEY. 2019: 19–20

  View details for Web of Science ID 000488134600011

• Synaptic vesicle fusion: today and beyond. Nature structural & molecular biology Brose, N., Brunger, A., Cafiso, D., Chapman, E. R., Diao, J., Hughson, F. M., Jackson, M. B., Jahn, R., Lindau, M., Ma, C., Rizo, J., Shin, Y., Sollner, T. H., Tamm, L., Yoon, T., Zhang, Y. 2019; 26 (8): 663–68

  View details for DOI 10.1038/s41594-019-0277-z

  View details for PubMedID 31384060

• The pre-synaptic fusion machinery. Current opinion in structural biology Brunger, A. T., Choi, U. B., Lai, Y., Leitz, J., White, K. I., Zhou, Q. 2019; 54: 179–88

  Abstract

  Here, we review recent insights into the neuronal presynaptic fusion machinery that releases neurotransmitter molecules into the synaptic cleft upon stimulation. The structure of the pre-fusion state of the SNARE/complexin-1/synaptotagmin-1 synaptic protein complex suggests a new model for the initiation of fast Ca2+-triggered membrane fusion. Functional studies have revealed roles of the essential factors Munc18 and Munc13, demonstrating that a part of their function involves the proper assembly of synaptic protein complexes. Near-atomic resolution structures of the NSF/alphaSNAP/SNARE complex provide first glimpses of the molecular machinery that disassembles the SNARE complex during the synaptic vesicle cycle. These structures show how this machinery captures the SNARE substrate and provide clues as to a possible processing mechanism.

  View details for PubMedID 30986753

• Nsf-Mediated Disassembly of On-and Off Pathway Snare Complexes and Inhibition by Complexin Choi, U. B., Zhao, M., White, K., Brunger, A. CELL PRESS. 2019: 528A

  View details for DOI 10.1016/j.bpj.2018.11.2844

  View details for Web of Science ID 000460779802654

• The pre-synaptic fusion machinery CURRENT OPINION IN STRUCTURAL BIOLOGY Brunger, A. T., Choi, U. B., Lai, Y., Leitz, J., White, K., Zhou, Q. 2019; 54: 179–88

  View details for DOI 10.1016/j.sbi.2019.03.007

  View details for Web of Science ID 000473554100023

• 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: 234–41

  View details for DOI 10.1107/S2059798318013177

  View details for Web of Science ID 000460206200012

• 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

• 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

• Structures of neurexophilin-neurexin complexes reveal a regulatory mechanism of alternative splicing. The EMBO journal Wilson, S. C., White, K. I., Zhou, Q. n., Pfuetzner, R. A., Choi, U. B., Südhof, T. C., Brunger, A. T. 2019: e101603

  Abstract

  Neurexins are presynaptic, cell-adhesion molecules that specify the functional properties of synapses via interactions with trans-synaptic ligands. Neurexins are extensively alternatively spliced at six canonical sites that regulate multifarious ligand interactions, but the structural mechanisms underlying alternative splicing-dependent neurexin regulation are largely unknown. Here, we determined high-resolution structures of the complex of neurexophilin-1 and the second laminin/neurexin/sex-hormone-binding globulin domain (LNS2) of neurexin-1 and examined how alternative splicing at splice site #2 (SS2) regulates the complex. Our data reveal a unique, extensive, neurexophilin-neurexin binding interface that extends the jelly-roll β-sandwich of LNS2 of neurexin-1 into neurexophilin-1. The SS2A insert of LNS2 augments this interface, increasing the binding affinity of LNS2 for neurexophilin-1. Taken together, our data reveal an unexpected architecture of neurexophilin-neurexin complexes that accounts for the modulation of binding by alternative splicing, which in turn regulates the competition of neurexophilin for neurexin binding with other ligands.

  View details for DOI 10.15252/embj.2019101603

  View details for PubMedID 31566781

• Structural principles of SNARE complex recognition by the AAA+ protein NSF. eLife White, K. I., Zhao, M., Choi, U. B., Pfuetzner, R. A., Brunger, A. T. 2018; 7

  Abstract

  The recycling of SNARE proteins following complex formation and membrane fusion is an essential process in eukaryotic trafficking. A highly conserved AAA+ protein, NSF (N-ethylmaleimide sensitive factor) and an adaptor protein, SNAP (soluble NSF attachment protein), disassembles the SNARE complex. We report electron-cryomicroscopy structures of the complex of NSF, alphaSNAP, and the full-length soluble neuronal SNARE complex (composed of syntaxin-1A, synaptobrevin-2, SNAP-25A) in the presence of ATP under non-hydrolyzing conditions at ~3.9 A resolution. These structures reveal electrostatic interactions by which two alphaSNAP molecules interface with a specific surface of the SNARE complex. This interaction positions the SNAREs such that the 15 N-terminal residues of SNAP-25A are loaded into the D1 ring pore of NSF via a spiral pattern of interactions between a conserved tyrosine NSF residue and SNAP-25A backbone atoms. This loading process likely precedes ATP hydrolysis. Subsequent ATP hydrolysis then drives complete disassembly.

  View details for PubMedID 30198481

• Structural principles of SNARE complex recognition by the AAA plus protein NSF ELIFE White, K., Zhao, M., Choi, U. B., Pfuetzner, R. A., Brunger, A. T. 2018; 7

  View details for DOI 10.7554/eLife.38888

  View details for Web of Science ID 000445720700001

• Ca2+-Triggered Synaptic Vesicle Fusion Initiated by Release of Inhibition TRENDS IN CELL BIOLOGY Brunger, A. T., Leitz, J., Zhou, Q., Choi, U. B., Lai, Y. 2018; 28 (8): 631-645

  View details for DOI 10.1016/j.tcb.2018.03.004

  View details for Web of Science ID 000439109600005

• NSF-mediated disassembly of on and off-pathway SNARE complexes and inhibition by complexin. eLife Choi, U. B., Zhao, M., White, K. I., Pfuetzner, R. A., Esquivies, L., Zhou, Q., Brunger, A. T. 2018; 7

  Abstract

  SNARE complex disassembly by the ATPase NSF is essential for neurotransmitter release and other membrane trafficking processes. We developed a single molecule FRET assay to monitor repeated rounds of NSF-mediated disassembly and reassembly of individual SNARE complexes. For ternary neuronal SNARE complexes, disassembly proceeds in a single step within 100 msec. We observed short- (< 0.32 sec) and long-lived ({greater than or equal to} 0.32 sec) disassembled states. The long-lived states represent fully disassembled SNARE complex, while the short-lived states correspond to failed disassembly or immediate re-assembly. Either high ionic strength or decreased alphaSNAP concentration reduces the disassembly rate while increasing the frequency of short-lived states. NSF is also capable of disassembling anti-parallel ternary SNARE complexes, suggesting a role in quality control. Finally, complexin-1 competes with alphaSNAP binding to the SNARE complex; addition of complexin-1 has an effect similar to that of decreasing the alphaSNAP concentration, suggesting a possible regulatory role in disassembly.

  View details for PubMedID 29985126

• NSF-mediated disassembly of on- and off-pathway SNARE complexes and inhibition by complexin ELIFE Choi, U. B., Zhao, M., White, K., Pfuetzner, R. A., Esquivies, L., Zhou, Q., Brunger, A. T. 2018; 7

  View details for DOI 10.7554/eLife.36497

  View details for Web of Science ID 000444081900001

• Ca2+-Triggered Synaptic Vesicle Fusion Initiated by Release of Inhibition. Trends in cell biology Brunger, A. T., Leitz, J. n., Zhou, Q. n., Choi, U. B., Lai, Y. n. 2018

  Abstract

  Recent structural and functional studies of the synaptic vesicle fusion machinery suggest an inhibited tripartite complex consisting of neuronal soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs), synaptotagmin, and complexin prior to Ca2+-triggered synaptic vesicle fusion. We speculate that Ca2+-triggered fusion commences with the release of inhibition by Ca2+ binding to synaptotagmin C2 domains. Subsequently, fusion is assisted by SNARE complex zippering and by active membrane remodeling properties of synaptotagmin. This additional, inhibitory role of synaptotagmin may be a general principle since other recent studies suggest that Ca2+ binding to extended synaptotagmin C2 domains enables lipid transport by releasing an inhibited state of the system, and that Munc13 may nominally be in an inhibited state, which is released upon Ca2+ binding to one of its C2 domains.

  View details for PubMedID 29706534

• Molecular Mechanisms of Fast Neurotransmitter Release ANNUAL REVIEW OF BIOPHYSICS, VOL 47 Brunger, A. T., Choi, U. B., Lai, Y., Leitz, J., Zhou, Q., Dill, K. A. 2018; 47: 469–97

  Abstract

  This review summarizes current knowledge of synaptic proteins that are central to synaptic vesicle fusion in presynaptic active zones, including SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors), synaptotagmin, complexin, Munc18 (mammalian uncoordinated-18), and Munc13 (mammalian uncoordinated-13), and highlights recent insights in the cooperation of these proteins for neurotransmitter release. Structural and functional studies of the synaptic fusion machinery suggest new molecular models of synaptic vesicle priming and Ca2+-triggered fusion. These studies will be a stepping-stone toward answering the question of how the synaptic vesicle fusion machinery achieves such high speed and sensitivity.

  View details for PubMedID 29792815

• The Conformational Flexibility of the Acyltransferase from the Disorazole Polyketide Synthase Is Revealed by an X-ray Free-Electron Laser Using a Room-Temperature Sample Delivery Method for Serial Crystallography BIOCHEMISTRY Mathews, I. I., Allison, K., Robbins, T., Lyubimov, A. Y., Uervirojnangkoorn, M., Brunger, A. T., Khosla, C., DeMirci, H., McPhillips, S. E., Hollenbeck, M., Soltis, M., Cohen, A. E. 2017; 56 (36): 4751–56

  Abstract

  The crystal structure of the trans-acyltransferase (AT) from the disorazole polyketide synthase (PKS) was determined at room temperature to a resolution of 2.5 Å using a new method for the direct delivery of the sample into an X-ray free-electron laser. A novel sample extractor efficiently delivered limited quantities of microcrystals directly from the native crystallization solution into the X-ray beam at room temperature. The AT structure revealed important catalytic features of this core PKS enzyme, including the occurrence of conformational changes around the active site. The implications of these conformational changes for polyketide synthase reaction dynamics are discussed.

  View details for DOI 10.1021/acs.biochem.7b00711

  View details for Web of Science ID 000410867600005

  View details for PubMedID 28832129

  View details for PubMedCentralID PMC5721673

• Morphologies of synaptic protein membrane fusion interfaces PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Gipson, P., Fukuda, Y., Danev, R., Lai, Y., Chen, D., Baumeister, W., Brunger, A. T. 2017; 114 (34): 9110–15

  Abstract

  Neurotransmitter release is orchestrated by synaptic proteins, such as SNAREs, synaptotagmin, and complexin, but the molecular mechanisms remain unclear. We visualized functionally active synaptic proteins reconstituted into proteoliposomes and their interactions in a native membrane environment by electron cryotomography with a Volta phase plate for improved resolvability. The images revealed individual synaptic proteins and synaptic protein complex densities at prefusion contact sites between membranes. We observed distinct morphologies of individual synaptic proteins and their complexes. The minimal system, consisting of neuronal SNAREs and synaptotagmin-1, produced point and long-contact prefusion states. Morphologies and populations of these states changed as the regulatory factors complexin and Munc13 were added. Complexin increased the membrane separation, along with a higher propensity of point contacts. Further inclusion of the priming factor Munc13 exclusively restricted prefusion states to point contacts, all of which efficiently fused upon Ca2+ triggering. We conclude that synaptic proteins have evolved to limit possible contact site assemblies and morphologies to those that promote fast Ca2+-triggered release.

  View details for PubMedID 28739947

• Molecular Mechanisms of Synaptic Vesicle Priming by Munc13 and Munc18 NEURON Lai, Y., Choi, U. B., Leitz, J., Rhee, H., Lee, C., Altas, B., Zhao, M., Pfuetzner, R. A., Wang, A. L., Brose, N., Rhee, J., Brunger, A. T. 2017; 95 (3): 591-+

  Abstract

  Munc13 catalyzes the transit of syntaxin from a closed complex with Munc18 into the ternary SNARE complex. Here we report a new function of Munc13, independent of Munc18: it promotes the proper syntaxin/synaptobrevin subconfiguration during assembly of the ternary SNARE complex. In cooperation with Munc18, Munc13 additionally ensures the proper syntaxin/SNAP-25 subconfiguration. In a reconstituted fusion assay with SNAREs, complexin, and synaptotagmin, inclusion of both Munc13 and Munc18 quadruples the Ca2+-triggered amplitude and achieves Ca2+ sensitivity at near-physiological concentrations. In Munc13-1/2 double-knockout neurons, expression of a constitutively open mutant of syntaxin could only minimally restore neurotransmitter release relative to Munc13-1 rescue. Together, the physiological functions of Munc13 may be related to regulation of proper SNARE complex assembly.

  View details for PubMedID 28772123

  View details for PubMedCentralID PMC5747255

• Conformational change of syntaxin linker region induced by Munc13s initiates SNARE complex formation in synaptic exocytosis EMBO JOURNAL Wang, S., Choi, U. B., Gong, J., Yang, X., Li, Y., Wang, A. L., Yang, X., Brunger, A. T., Ma, C. 2017; 36 (6): 816-829

  Abstract

  The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein syntaxin-1 adopts a closed conformation when bound to Munc18-1, preventing binding to synaptobrevin-2 and SNAP-25 to form the ternary SNARE complex. Although it is known that the MUN domain of Munc13-1 catalyzes the transition from the Munc18-1/syntaxin-1 complex to the SNARE complex, the molecular mechanism is unclear. Here, we identified two conserved residues (R151, I155) in the syntaxin-1 linker region as key sites for the MUN domain interaction. This interaction is essential for SNARE complex formation in vitro and synaptic vesicle priming in neuronal cultures. Moreover, this interaction is important for a tripartite Munc18-1/syntaxin-1/MUN complex, in which syntaxin-1 still adopts a closed conformation tightly bound to Munc18-1, whereas the syntaxin-1 linker region changes its conformation, similar to that of the LE mutant of syntaxin-1 when bound to Munc18-1. We suggest that the conformational change of the syntaxin-1 linker region induced by Munc13-1 initiates ternary SNARE complex formation in the neuronal system.

  View details for DOI 10.15252/embj.201695775

  View details for Web of Science ID 000397293500010

  View details for PubMedID 28137749

• The primed SNARE-complexin-synaptotagmin complex for neuronal exocytosis. Nature Zhou, Q. n., Zhou, P. n., Wang, A. L., Wu, D. n., Zhao, M. n., Südhof, T. C., Brunger, A. T. 2017; 548 (7668): 420–25

  Abstract

  Synaptotagmin, complexin, and neuronal SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) proteins mediate evoked synchronous neurotransmitter release, but the molecular mechanisms mediating the cooperation between these molecules remain unclear. Here we determine crystal structures of the primed pre-fusion SNARE-complexin-synaptotagmin-1 complex. These structures reveal an unexpected tripartite interface between synaptotagmin-1 and both the SNARE complex and complexin. Simultaneously, a second synaptotagmin-1 molecule interacts with the other side of the SNARE complex via the previously identified primary interface. Mutations that disrupt either interface in solution also severely impair evoked synchronous release in neurons, suggesting that both interfaces are essential for the primed pre-fusion state. Ca(2+) binding to the synaptotagmin-1 molecules unlocks the complex, allows full zippering of the SNARE complex, and triggers membrane fusion. The tripartite SNARE-complexin-synaptotagmin-1 complex at a synaptic vesicle docking site has to be unlocked for triggered fusion to start, explaining the cooperation between complexin and synaptotagmin-1 in synchronizing evoked release on the sub-millisecond timescale.

  View details for PubMedID 28813412

• 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

• C-terminal domain of mammalian complexin-1 localizes to highly curved membranes. Proceedings of the National Academy of Sciences of the United States of America Gong, J., Lai, Y., Li, X., Wang, M., Leitz, J., Hu, Y., Zhang, Y., Choi, U. B., Cipriano, D., Pfuetzner, R. A., Südhof, T. C., Yang, X., Brunger, A. T., Diao, J. 2016

  Abstract

  In presynaptic nerve terminals, complexin regulates spontaneous "mini" neurotransmitter release and activates Ca(2+)-triggered synchronized neurotransmitter release. We studied the role of the C-terminal domain of mammalian complexin in these processes using single-particle optical imaging and electrophysiology. The C-terminal domain is important for regulating spontaneous release in neuronal cultures and suppressing Ca(2+)-independent fusion in vitro, but it is not essential for evoked release in neuronal cultures and in vitro. This domain interacts with membranes in a curvature-dependent fashion similar to a previous study with worm complexin [Snead D, Wragg RT, Dittman JS, Eliezer D (2014) Membrane curvature sensing by the C-terminal domain of complexin. Nat Commun 5:4955]. The curvature-sensing value of the C-terminal domain is comparable to that of α-synuclein. Upon replacement of the C-terminal domain with membrane-localizing elements, preferential localization to the synaptic vesicle membrane, but not to the plasma membrane, results in suppression of spontaneous release in neurons. Membrane localization had no measurable effect on evoked postsynaptic currents of AMPA-type glutamate receptors, but mislocalization to the plasma membrane increases both the variability and the mean of the synchronous decay time constant of NMDA-type glutamate receptor evoked postsynaptic currents.

  View details for PubMedID 27821736

• 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

• De novo phasing with X-ray laser reveals mosquito larvicide BinAB structure. Nature Colletier, J., Sawaya, M. R., Gingery, M., Rodriguez, J. A., Cascio, D., Brewster, A. S., Michels-Clark, T., Hice, R. H., Coquelle, N., Boutet, S., Williams, G. J., Messerschmidt, M., Deponte, D. P., Sierra, R. G., Laksmono, H., Koglin, J. E., Hunter, M. S., Park, H., Uervirojnangkoorn, M., Bideshi, D. K., Brunger, A. T., Federici, B. A., Sauter, N. K., Eisenberg, D. S. 2016; 539 (7627): 43-47

  Abstract

  BinAB is a naturally occurring paracrystalline larvicide distributed worldwide to combat the devastating diseases borne by mosquitoes. These crystals are composed of homologous molecules, BinA and BinB, which play distinct roles in the multi-step intoxication process, transforming from harmless, robust crystals, to soluble protoxin heterodimers, to internalized mature toxin, and finally to toxic oligomeric pores. The small size of the crystals-50 unit cells per edge, on average-has impeded structural characterization by conventional means. Here we report the structure of Lysinibacillus sphaericus BinAB solved de novo by serial-femtosecond crystallography at an X-ray free-electron laser. The structure reveals tyrosine- and carboxylate-mediated contacts acting as pH switches to release soluble protoxin in the alkaline larval midgut. An enormous heterodimeric interface appears to be responsible for anchoring BinA to receptor-bound BinB for co-internalization. Remarkably, this interface is largely composed of propeptides, suggesting that proteolytic maturation would trigger dissociation of the heterodimer and progression to pore formation.

  View details for DOI 10.1038/nature19825

  View details for PubMedID 27680699

  View details for PubMedCentralID PMC5161637

• Expression of C1ql3 in Discrete Neuronal Populations Controls Efferent Synapse Numbers and Diverse Behaviors. Neuron Martinelli, D. C., Chew, K. S., Rohlmann, A., Lum, M. Y., Ressl, S., Hattar, S., Brunger, A. T., Missler, M., Südhof, T. C. 2016; 91 (5): 1034-1051

  Abstract

  C1ql3 is a secreted neuronal protein that binds to BAI3, an adhesion-class GPCR. C1ql3 is homologous to other gC1q-domain proteins that control synapse numbers, but a role for C1ql3 in regulating synapse density has not been demonstrated. We show in cultured neurons that C1ql3 expression is activity dependent and supports excitatory synapse density. Using newly generated conditional and constitutive C1ql3 knockout mice, we found that C1ql3-deficient mice exhibited fewer excitatory synapses and diverse behavioral abnormalities, including marked impairments in fear memories. Using circuit-tracing tools and conditional ablation of C1ql3 targeted to specific brain regions, we demonstrate that C1ql3-expressing neurons in the basolateral amygdala project to the medial prefrontal cortex, that these efferents contribute to fear memory behavior, and that C1ql3 is required for formation and/or maintenance of these synapses. Our results suggest that C1ql3 is a signaling protein essential for subsets of synaptic projections and the behaviors controlled by these projections.

  View details for DOI 10.1016/j.neuron.2016.07.002

  View details for PubMedID 27478018

• Phosphorylation of residues inside the SNARE complex suppresses secretory vesicle fusion. EMBO journal Malmersjö, S., Di Palma, S., Diao, J., Lai, Y., Pfuetzner, R. A., Wang, A. L., McMahon, M. A., Hayer, A., Porteus, M., Bodenmiller, B., Brunger, A. T., Meyer, T. 2016; 35 (16): 1810-1821

  Abstract

  Membrane fusion is essential for eukaryotic life, requiring SNARE proteins to zipper up in an α-helical bundle to pull two membranes together. Here, we show that vesicle fusion can be suppressed by phosphorylation of core conserved residues inside the SNARE domain. We took a proteomics approach using a PKCB knockout mast cell model and found that the key mast cell secretory protein VAMP8 becomes phosphorylated by PKC at multiple residues in the SNARE domain. Our data suggest that VAMP8 phosphorylation reduces vesicle fusion in vitro and suppresses secretion in living cells, allowing vesicles to dock but preventing fusion with the plasma membrane. Markedly, we show that the phosphorylation motif is absent in all eukaryotic neuronal VAMPs, but present in all other VAMPs. Thus, phosphorylation of SNARE domains is a general mechanism to restrict how much cells secrete, opening the door for new therapeutic strategies for suppression of secretion.

  View details for DOI 10.15252/embj.201694071

  View details for PubMedID 27402227

• N-terminal domain of complexin independently activates calcium-triggered fusion. Proceedings of the National Academy of Sciences of the United States of America Lai, Y., Choi, U. B., Zhang, Y., Zhao, M., Pfuetzner, R. A., Wang, A. L., Diao, J., Brunger, A. T. 2016; 113 (32): E4698-707

  Abstract

  Complexin activates Ca(2+)-triggered neurotransmitter release and regulates spontaneous release in the presynaptic terminal by cooperating with the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and the Ca(2+)-sensor synaptotagmin. The N-terminal domain of complexin is important for activation, but its molecular mechanism is still poorly understood. Here, we observed that a split pair of N-terminal and central domain fragments of complexin is sufficient to activate Ca(2+)-triggered release using a reconstituted single-vesicle fusion assay, suggesting that the N-terminal domain acts as an independent module within the synaptic fusion machinery. The N-terminal domain can also interact independently with membranes, which is enhanced by a cooperative interaction with the neuronal SNARE complex. We show by mutagenesis that membrane binding of the N-terminal domain is essential for activation of Ca(2+)-triggered fusion. Consistent with the membrane-binding property, the N-terminal domain can be substituted by the influenza virus hemagglutinin fusion peptide, and this chimera also activates Ca(2+)-triggered fusion. Membrane binding of the N-terminal domain of complexin therefore cooperates with the other fusogenic elements of the synaptic fusion machinery during Ca(2+)-triggered release.

  View details for DOI 10.1073/pnas.1604348113

  View details for PubMedID 27444020

  View details for PubMedCentralID PMC4987820

• Complexin induces a conformational change at the membrane-proximal C-terminal end of the SNARE complex ELIFE Choi, U. B., Zhao, M., Zhang, Y., Lai, Y., Brunger, A. T. 2016; 5

  Abstract

  Complexin regulates spontaneous and activates Ca(2+)-triggered neurotransmitter release, yet the molecular mechanisms are still unclear. Here we performed single molecule fluorescence resonance energy transfer experiments and uncovered two conformations of complexin-1 bound to the ternary SNARE complex. In the cis conformation, complexin-1 induces a conformational change at the membrane-proximal C-terminal end of the ternary SNARE complex that specifically depends on the N-terminal, accessory, and central domains of complexin-1. The complexin-1 induced conformation of the ternary SNARE complex may be related to a conformation that is juxtaposing the synaptic vesicle and plasma membranes. In the trans conformation, complexin-1 can simultaneously interact with a ternary SNARE complex via the central domain and a binary SNARE complex consisting of syntaxin-1A and SNAP-25A via the accessory domain. The cis conformation may be involved in activation of synchronous neurotransmitter release, whereas both conformations may be involved in regulating spontaneous release.

  View details for DOI 10.7554/eLife.16886

  View details for Web of Science ID 000379857100001

  View details for PubMedID 27253060

  View details for PubMedCentralID PMC4927292

• 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

• Recent Advances in Deciphering the Structure and Molecular Mechanism of the AAA plus ATPase N-Ethylmaleimide-Sensitive Factor (NSF) JOURNAL OF MOLECULAR BIOLOGY Zhao, M., Brunger, A. T. 2016; 428 (9): 1912-1926

  Abstract

  N-ethylmaleimide-sensitive factor (NSF), first discovered in 1988, is a key factor for eukaryotic trafficking, including protein and hormone secretion and neurotransmitter release. It is a member of the AAA+ family (ATPases associated with diverse cellular activities). NSF disassembles soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes in conjunction with soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP). Structural studies of NSF and its complex with SNAREs and SNAPs (known as 20S supercomplex) started about 20years ago. Crystal structures of individual N and D2 domains of NSF and low-resolution electron microscopy structures of full-length NSF and 20S supercomplex have been reported over the years. Nevertheless, the molecular architecture of the 20S supercomplex and the molecular mechanism of NSF-mediated SNARE complex disassembly remained unclear until recently. Here we review recent atomic-resolution or near-atomic resolution structures of NSF and of the 20S supercomplex, as well as recent insights into the molecular mechanism and energy requirements of NSF. We also compare NSF with other known AAA+ family members.

  View details for DOI 10.1016/j.jmb.2015.10.026

  View details for Web of Science ID 000376695100009

  View details for PubMedID 26546278

  View details for PubMedCentralID PMC4854814

• Simultaneous single-molecule epigenetic imaging of DNA methylation and hydroxymethylation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Song, C., Diao, J., Brunger, A. T., Quake, S. R. 2016; 113 (16): 4338-4343

  Abstract

  The modifications 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are the two major DNA epigenetic modifications in mammalian genomes and play crucial roles in development and pathogenesis. Little is known about the colocalization or potential correlation of these two modifications. Here we present an ultrasensitive single-molecule imaging technology capable of detecting and quantifying 5hmC and 5mC from trace amounts of DNA. We used this approach to perform single-molecule fluorescence resonance energy transfer (smFRET) experiments which measure the proximity between 5mC and 5hmC in the same DNA molecule. Our results reveal high levels of adjacent and opposing methylated and hydroxymethylated CpG sites (5hmC/5mCpGs) in mouse genomic DNA across multiple tissues. This identifies the previously undetectable and unappreciated 5hmC/5mCpGs as one of the major states for 5hmC in the mammalian genome and suggest that they could function in promoting gene expression.

  View details for DOI 10.1073/pnas.1600223113

  View details for Web of Science ID 000374393800042

  View details for PubMedID 27035984

  View details for PubMedCentralID PMC4843451

• Atomic resolution experimental phase information reveals extensive disorder and bound 2-methyl-2,4-pentanediol in Ca2+-calmodulin ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY Lin, J., van den Bedem, H., Brunger, A. T., Wilson, M. A. 2016; 72: 83-92

  Abstract

  Calmodulin (CaM) is the primary calcium signaling protein in eukaryotes and has been extensively studied using various biophysical techniques. Prior crystal structures have noted the presence of ambiguous electron density in both hydrophobic binding pockets of Ca(2+)-CaM, but no assignment of these features has been made. In addition, Ca(2+)-CaM samples many conformational substates in the crystal and accurately modeling the full range of this functionally important disorder is challenging. In order to characterize these features in a minimally biased manner, a 1.0 Å resolution single-wavelength anomalous diffraction data set was measured for selenomethionine-substituted Ca(2+)-CaM. Density-modified electron-density maps enabled the accurate assignment of Ca(2+)-CaM main-chain and side-chain disorder. These experimental maps also substantiate complex disorder models that were automatically built using low-contour features of model-phased electron density. Furthermore, experimental electron-density maps reveal that 2-methyl-2,4-pentanediol (MPD) is present in the C-terminal domain, mediates a lattice contact between N-terminal domains and may occupy the N-terminal binding pocket. The majority of the crystal structures of target-free Ca(2+)-CaM have been derived from crystals grown using MPD as a precipitant, and thus MPD is likely to be bound in functionally critical regions of Ca(2+)-CaM in most of these structures. The adventitious binding of MPD helps to explain differences between the Ca(2+)-CaM crystal and solution structures and is likely to favor more open conformations of the EF-hands in the crystal.

  View details for DOI 10.1107/S2059798315021609

  View details for Web of Science ID 000371707300010

  View details for PubMedID 26894537

  View details for PubMedCentralID PMC4756614

• 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

• Correction: Synaptic proteins promote calcium-triggered fast transition from point contact to full fusion. eLife Diao, J., Grob, P., Cipriano, D. J., Kyoung, M., Zhang, Y., Shah, S., Nguyen, A., Padolina, M., Srivastava, A., Vrljic, M., Shah, A., Nogales, E., Chu, S., Brunger, A. T. 2015; 4: e12289

  View details for DOI 10.7554/eLife.12289

  View details for PubMedID 26486862

• A high-transparency, micro-patternable chip for X-ray diffraction analysis of microcrystals under native growth conditions ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY Murray, T. D., Lyubimov, A. Y., Ogata, C. M., Huy Vo, H., Uervirojnangkoorn, M., Brunger, A. T., Berger, J. M. 2015; 71: 1987-1997

  Abstract

  Microcrystals present a significant impediment to the determination of macromolecular structures by X-ray diffraction methods. Although microfocus synchrotron beamlines and X-ray free-electron lasers (XFELs) can enable the collection of interpretable diffraction data from microcrystals, there is a need for efficient methods of harvesting small volumes (<2 µl) of microcrystals grown under common laboratory formats and delivering them to an X-ray beam source under native growth conditions. One approach that shows promise in overcoming the challenges intrinsic to microcrystal analysis is to pair so-called `fixed-target' sample-delivery devices with microbeam-based X-ray diffraction methods. However, to record weak diffraction patterns it is necessary to fabricate devices from X-ray-transparent materials that minimize background scattering. Presented here is the design of a new micro-diffraction device consisting of three layers fabricated from silicon nitride, photoresist and polyimide film. The chip features low X-ray scattering and X-ray absorption properties, and uses a customizable blend of hydrophobic and hydrophilic surface patterns to help localize microcrystals to defined regions. Microcrystals in their native growth conditions can be loaded into the chips with a standard pipette, allowing data collection at room temperature. Diffraction data collected from hen egg-white lysozyme microcrystals (10-15 µm) loaded into the chips yielded a complete, high-resolution (<1.6 Å) data set sufficient to determine a high-quality structure by molecular replacement. The features of the chip allow the rapid and user-friendly analysis of microcrystals grown under virtually any laboratory format at microfocus synchrotron beamlines and XFELs.

  View details for DOI 10.1107/S1399004715015011

  View details for PubMedID 26457423

  View details for PubMedCentralID PMC4601365

• 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

• ATG14 promotes membrane tethering and fusion of autophagosomes to endolysosomes. Nature Diao, J., Liu, R., Rong, Y., Zhao, M., Zhang, J., Lai, Y., Zhou, Q., Wilz, L. M., Li, J., Vivona, S., Pfuetzner, R. A., Brunger, A. T., Zhong, Q. 2015; 520 (7548): 563-566

  Abstract

  Autophagy, an important catabolic pathway implicated in a broad spectrum of human diseases, begins by forming double membrane autophagosomes that engulf cytosolic cargo and ends by fusing autophagosomes with lysosomes for degradation. Membrane fusion activity is required for early biogenesis of autophagosomes and late degradation in lysosomes. However, the key regulatory mechanisms of autophagic membrane tethering and fusion remain largely unknown. Here we report that ATG14 (also known as beclin-1-associated autophagy-related key regulator (Barkor) or ATG14L), an essential autophagy-specific regulator of the class III phosphatidylinositol 3-kinase complex, promotes membrane tethering of protein-free liposomes, and enhances hemifusion and full fusion of proteoliposomes reconstituted with the target (t)-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) syntaxin 17 (STX17) and SNAP29, and the vesicle (v)-SNARE VAMP8 (vesicle-associated membrane protein 8). ATG14 binds to the SNARE core domain of STX17 through its coiled-coil domain, and stabilizes the STX17-SNAP29 binary t-SNARE complex on autophagosomes. The STX17 binding, membrane tethering and fusion-enhancing activities of ATG14 require its homo-oligomerization by cysteine repeats. In ATG14 homo-oligomerization-defective cells, autophagosomes still efficiently form but their fusion with endolysosomes is blocked. Recombinant ATG14 homo-oligomerization mutants also completely lose their ability to promote membrane tethering and to enhance SNARE-mediated fusion in vitro. Taken together, our data suggest an autophagy-specific membrane fusion mechanism in which oligomeric ATG14 directly binds to STX17-SNAP29 binary t-SNARE complex on autophagosomes and primes it for VAMP8 interaction to promote autophagosome-endolysosome fusion.

  View details for DOI 10.1038/nature14147

  View details for PubMedID 25686604

• ATG14 promotes membrane tethering and fusion of autophagosomes to endolysosomes NATURE Diao, J., Liu, R., Rong, Y., Zhao, M., Zhang, J., Lai, Y., Zhou, Q., Wilz, L. M., Li, J., Vivona, S., Pfuetzner, R. A., Brunger, A. T., Zhong, Q. 2015; 520 (7548): 563-?

  Abstract

  Autophagy, an important catabolic pathway implicated in a broad spectrum of human diseases, begins by forming double membrane autophagosomes that engulf cytosolic cargo and ends by fusing autophagosomes with lysosomes for degradation. Membrane fusion activity is required for early biogenesis of autophagosomes and late degradation in lysosomes. However, the key regulatory mechanisms of autophagic membrane tethering and fusion remain largely unknown. Here we report that ATG14 (also known as beclin-1-associated autophagy-related key regulator (Barkor) or ATG14L), an essential autophagy-specific regulator of the class III phosphatidylinositol 3-kinase complex, promotes membrane tethering of protein-free liposomes, and enhances hemifusion and full fusion of proteoliposomes reconstituted with the target (t)-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) syntaxin 17 (STX17) and SNAP29, and the vesicle (v)-SNARE VAMP8 (vesicle-associated membrane protein 8). ATG14 binds to the SNARE core domain of STX17 through its coiled-coil domain, and stabilizes the STX17-SNAP29 binary t-SNARE complex on autophagosomes. The STX17 binding, membrane tethering and fusion-enhancing activities of ATG14 require its homo-oligomerization by cysteine repeats. In ATG14 homo-oligomerization-defective cells, autophagosomes still efficiently form but their fusion with endolysosomes is blocked. Recombinant ATG14 homo-oligomerization mutants also completely lose their ability to promote membrane tethering and to enhance SNARE-mediated fusion in vitro. Taken together, our data suggest an autophagy-specific membrane fusion mechanism in which oligomeric ATG14 directly binds to STX17-SNAP29 binary t-SNARE complex on autophagosomes and primes it for VAMP8 interaction to promote autophagosome-endolysosome fusion.

  View details for DOI 10.1038/nature14147

  View details for Web of Science ID 000353334500048

  View details for PubMedID 25686604

• 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

• Structures of C1q-like Proteins Reveal Unique Features among the C1q/TNF Superfamily STRUCTURE Ressl, S., Vu, B. K., Vivona, S., Martinelli, D. C., Suedhof, T. C., Brunger, A. T. 2015; 23 (4): 688-699

  Abstract

  C1q-like (C1QL) -1, -2, and -3 proteins are encoded by homologous genes that are highly expressed in brain. C1QLs bind to brain-specific angiogenesis inhibitor 3 (BAI3), an adhesion-type G-protein coupled receptor that may regulate dendritic morphology by organizing actin filaments. To begin to understand the function of C1QLs, we determined high-resolution crystal structures of the globular C1q-domains of C1QL1, C1QL2, and C1QL3. Each structure is a trimer, with each protomer forming a jelly-roll fold consisting of 10 β strands. Moreover, C1QL trimers may assemble into higher-order oligomers similar to adiponectin and contain four Ca(2+)-binding sites along the trimeric symmetry axis, as well as additional surface Ca(2+)-binding sites. Mutation of Ca(2+)-coordinating residues along the trimeric symmetry axis lowered the Ca(2+)-binding affinity and protein stability. Our results reveal unique structural features of C1QLs among C1q/TNF superfamily proteins that may be associated with their specific brain functions.

  View details for DOI 10.1016/j.str.2015.01.019

  View details for PubMedID 25752542

• Capture and X-ray diffraction studies of protein microcrystals in a microfluidic trap array. Acta crystallographica. Section D, Biological crystallography Lyubimov, A. Y., Murray, T. D., Koehl, A., Araci, I. E., Uervirojnangkoorn, M., Zeldin, O. B., Cohen, A. E., Soltis, S. M., Baxter, E. L., Brewster, A. S., Sauter, N. K., Brunger, A. T., Berger, J. M. 2015; 71: 928-940

  Abstract

  X-ray free-electron lasers (XFELs) promise to enable the collection of interpretable diffraction data from samples that are refractory to data collection at synchrotron sources. At present, however, more efficient sample-delivery methods that minimize the consumption of microcrystalline material are needed to allow the application of XFEL sources to a wide range of challenging structural targets of biological importance. Here, a microfluidic chip is presented in which microcrystals can be captured at fixed, addressable points in a trap array from a small volume (<10 µl) of a pre-existing slurry grown off-chip. The device can be mounted on a standard goniostat for conducting diffraction experiments at room temperature without the need for flash-cooling. Proof-of-principle tests with a model system (hen egg-white lysozyme) demonstrated the high efficiency of the microfluidic approach for crystal harvesting, permitting the collection of sufficient data from only 265 single-crystal still images to permit determination and refinement of the structure of the protein. This work shows that microfluidic capture devices can be readily used to facilitate data collection from protein microcrystals grown in traditional laboratory formats, enabling analysis when cryopreservation is problematic or when only small numbers of crystals are available. Such microfluidic capture devices may also be useful for data collection at synchrotron sources.

  View details for DOI 10.1107/S1399004715002308

  View details for PubMedID 25849403

  View details for PubMedCentralID PMC4388268

• Capture and X-ray diffraction studies of protein microcrystals in a microfluidic trap array ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY Lyubimov, A. Y., Murray, T. D., Koehl, A., Araci, I. E., Uervirojnangkoorn, M., Zeldin, O. B., Cohen, A. E., Soltis, S. M., Baxter, E. L., Brewster, A. S., Sauter, N. K., Brunger, A. T., Berger, J. M. 2015; 71: 928-940

  Abstract

  X-ray free-electron lasers (XFELs) promise to enable the collection of interpretable diffraction data from samples that are refractory to data collection at synchrotron sources. At present, however, more efficient sample-delivery methods that minimize the consumption of microcrystalline material are needed to allow the application of XFEL sources to a wide range of challenging structural targets of biological importance. Here, a microfluidic chip is presented in which microcrystals can be captured at fixed, addressable points in a trap array from a small volume (<10 µl) of a pre-existing slurry grown off-chip. The device can be mounted on a standard goniostat for conducting diffraction experiments at room temperature without the need for flash-cooling. Proof-of-principle tests with a model system (hen egg-white lysozyme) demonstrated the high efficiency of the microfluidic approach for crystal harvesting, permitting the collection of sufficient data from only 265 single-crystal still images to permit determination and refinement of the structure of the protein. This work shows that microfluidic capture devices can be readily used to facilitate data collection from protein microcrystals grown in traditional laboratory formats, enabling analysis when cryopreservation is problematic or when only small numbers of crystals are available. Such microfluidic capture devices may also be useful for data collection at synchrotron sources.

  View details for DOI 10.1107/S1399004715002308

  View details for Web of Science ID 000352507200019

  View details for PubMedID 25849403

  View details for PubMedCentralID PMC4388268

• 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

• Mechanistic insights into the recycling machine of the SNARE complex. Nature Zhao, M., Wu, S., Zhou, Q., Vivona, S., Cipriano, D. J., Cheng, Y., Brunger, A. T. 2015; 518 (7537): 61-67

  Abstract

  Evolutionarily conserved SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptors) proteins form a complex that drives membrane fusion in eukaryotes. The ATPase NSF (N-ethylmaleimide sensitive factor), together with SNAPs (soluble NSF attachment protein), disassembles the SNARE complex into its protein components, making individual SNAREs available for subsequent rounds of fusion. Here we report structures of ATP- and ADP-bound NSF, and the NSF/SNAP/SNARE (20S) supercomplex determined by single-particle electron cryomicroscopy at near-atomic to sub-nanometre resolution without imposing symmetry. Large, potentially force-generating, conformational differences exist between ATP- and ADP-bound NSF. The 20S supercomplex exhibits broken symmetry, transitioning from six-fold symmetry of the NSF ATPase domains to pseudo four-fold symmetry of the SNARE complex. SNAPs interact with the SNARE complex with an opposite structural twist, suggesting an unwinding mechanism. The interfaces between NSF, SNAPs, and SNAREs exhibit characteristic electrostatic patterns, suggesting how one NSF/SNAP species can act on many different SNARE complexes.

  View details for DOI 10.1038/nature14148

  View details for PubMedID 25581794

  View details for PubMedCentralID PMC4320033

• Mechanistic insights into the recycling machine of the SNARE complex NATURE Zhao, M., Wu, S., Zhou, Q., Vivona, S., Cipriano, D. J., Cheng, Y., Brunger, A. T. 2015; 518 (7537): 61-?

  Abstract

  Evolutionarily conserved SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptors) proteins form a complex that drives membrane fusion in eukaryotes. The ATPase NSF (N-ethylmaleimide sensitive factor), together with SNAPs (soluble NSF attachment protein), disassembles the SNARE complex into its protein components, making individual SNAREs available for subsequent rounds of fusion. Here we report structures of ATP- and ADP-bound NSF, and the NSF/SNAP/SNARE (20S) supercomplex determined by single-particle electron cryomicroscopy at near-atomic to sub-nanometre resolution without imposing symmetry. Large, potentially force-generating, conformational differences exist between ATP- and ADP-bound NSF. The 20S supercomplex exhibits broken symmetry, transitioning from six-fold symmetry of the NSF ATPase domains to pseudo four-fold symmetry of the SNARE complex. SNAPs interact with the SNARE complex with an opposite structural twist, suggesting an unwinding mechanism. The interfaces between NSF, SNAPs, and SNAREs exhibit characteristic electrostatic patterns, suggesting how one NSF/SNAP species can act on many different SNARE complexes.

  View details for DOI 10.1038/nature14148

  View details for Web of Science ID 000349098000031

  View details for PubMedID 25581794

  View details for PubMedCentralID PMC4320033

• 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 PubMedID 25664746

  View details for PubMedCentralID PMC4321488

• 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

• 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 DOI 10.7554/eLife.05421

  View details for PubMedID 25781634

• Tools to assist determination and validation of carbohydrate 3D structure data. Methods in molecular biology (Clifton, N.J.) Emsley, P., Brunger, A. T., Lutteke, T. 2015; 1273: 229-40

  Abstract

  The frequency of glycosylated protein 3D structures in the Protein Data Bank (PDB) is significantly lower than the proportion of glycoproteins in nature, and if glycan 3D structures are present, then they often exhibit a large degree of errors. There are various reasons for this, one of which is a comparably low support of carbohydrates in software tools for 3D structure determination and validation. This chapter illustrates the current features that assist crystallographers with handling glycans during 3D structure determination in Coot and CNS and with validation of the results.

  View details for DOI 10.1007/978-1-4939-2343-4_17

  View details for PubMedID 25753715

• 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

• Towards reconstitution of membrane fusion mediated by SNAREs and other synaptic proteins CRITICAL REVIEWS IN BIOCHEMISTRY AND MOLECULAR BIOLOGY Brunger, A. T., Cipriano, D. J., Diao, J. 2015; 50 (3): 231-241

  Abstract

  Proteoliposomes have been widely used for in vitro studies of membrane fusion mediated by synaptic proteins. Initially, such studies were made with large unsynchronized ensembles of vesicles. Such ensemble assays limited the insights into the SNARE-mediated fusion mechanism that could be obtained from them. Single particle microscopy experiments can alleviate many of these limitations but they pose significant technical challenges. Here we summarize various approaches that have enabled studies of fusion mediated by SNAREs and other synaptic proteins at a single-particle level. Currently available methods are described and their advantages and limitations are discussed.

  View details for DOI 10.3109/10409238.2015.1023252

  View details for Web of Science ID 000361316400005

  View details for PubMedID 25788028

  View details for PubMedCentralID PMC4673598

• 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

• Direct visualization of trans-synaptic neurexin-neuroligin interactions during synapse formation. journal of neuroscience Tsetsenis, T., Boucard, A. A., Araç, D., Brunger, A. T., Südhof, T. C. 2014; 34 (45): 15083-15096

  Abstract

  Neurexins and neuroligins are synaptic cell-adhesion molecules that are essential for normal synapse specification and function and are thought to bind to each other trans-synaptically, but such interactions have not been demonstrated directly. Here, we generated neurexin-1β and neuroligin-1 and neuroligin-2 fusion proteins containing complementary "split" GFP fragments positioned such that binding of neurexin-1β to neuroligin-1 or neuroligin-2 allowed GFP reconstitution without dramatically changing their binding affinities. GFP fluorescence was only reconstituted from split-GFP-modified neurexin-1β and neuroligin-1 if and after neurexin-1β bound to its neuroligin partner; reassociation of the split-GFP components with each other did not mediate binding. Using trans-cellular reconstitution of GFP fluorescence from split-GFP-modified neurexin-1β and neuroligins as an assay, we demonstrate that trans-synaptic neurexin/neuroligin binding indeed occurred when mouse hippocampal neurons formed synapses onto non-neuronal COS-7 cells expressing neuroligins or when mouse hippocampal neurons formed synapses with each other. This visualization of synapses by neurexin/neuroligin binding prompted us to refer to this approach as "SynView." Our data demonstrate that neurexin-1β forms a trans-synaptic complex with neuroligin-1 and neuroligin-2 and that this interaction can be used to label synapses in a specific fashion in vivo.

  View details for DOI 10.1523/JNEUROSCI.0348-14.2014

  View details for PubMedID 25378172

  View details for PubMedCentralID PMC4220035

• Deformable elastic network refinement for low-resolution macromolecular crystallography ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY Schroeder, G. F., Levitt, M., Brunger, A. T. 2014; 70: 2241-2255

  Abstract

  Crystals of membrane proteins and protein complexes often diffract to low resolution owing to their intrinsic molecular flexibility, heterogeneity or the mosaic spread of micro-domains. At low resolution, the building and refinement of atomic models is a more challenging task. The deformable elastic network (DEN) refinement method developed previously has been instrumental in the determinion of several structures at low resolution. Here, DEN refinement is reviewed, recommendations for its optimal usage are provided and its limitations are discussed. Representative examples of the application of DEN refinement to challenging cases of refinement at low resolution are presented. These cases include soluble as well as membrane proteins determined at limiting resolutions ranging from 3 to 7 Å. Potential extensions of the DEN refinement technique and future perspectives for the interpretation of low-resolution crystal structures are also discussed.

  View details for DOI 10.1107/S1399004714016496

  View details for Web of Science ID 000341819500001

  View details for PubMedCentralID PMC4157441

• Deformable elastic network refinement for low-resolution macromolecular crystallography. Acta crystallographica. Section D, Biological crystallography Schröder, G. F., Levitt, M., Brunger, A. T. 2014; 70: 2241-2255

  Abstract

  Crystals of membrane proteins and protein complexes often diffract to low resolution owing to their intrinsic molecular flexibility, heterogeneity or the mosaic spread of micro-domains. At low resolution, the building and refinement of atomic models is a more challenging task. The deformable elastic network (DEN) refinement method developed previously has been instrumental in the determinion of several structures at low resolution. Here, DEN refinement is reviewed, recommendations for its optimal usage are provided and its limitations are discussed. Representative examples of the application of DEN refinement to challenging cases of refinement at low resolution are presented. These cases include soluble as well as membrane proteins determined at limiting resolutions ranging from 3 to 7 Å. Potential extensions of the DEN refinement technique and future perspectives for the interpretation of low-resolution crystal structures are also discussed.

  View details for DOI 10.1107/S1399004714016496

  View details for PubMedID 25195739

• Structure Refinement at Low-resolution: Strategies and Validation Schroeder, G., Levitt, M., Brunger, A. INT UNION CRYSTALLOGRAPHY. 2014: C1482

  View details for DOI 10.1107/S2053273314085179

  View details for Web of Science ID 000519606301485

• Complexin inhibits spontaneous release and synchronizes Ca2+-triggered synaptic vesicle fusion by distinct mechanisms. eLife Lai, Y., Diao, J., Cipriano, D. J., Zhang, Y., Pfuetzner, R. A., Padolina, M. S., Brunger, A. T. 2014; 3

  Abstract

  Previously we showed that fast Ca(2+)-triggered vesicle fusion with reconstituted neuronal SNAREs and synaptotagmin-1 begins from an initial hemifusion-free membrane point contact, rather than a hemifusion diaphragm, using a single vesicle-vesicle lipid/content mixing assay (Diao et al., 2012). When complexin-1 was included, a more pronounced Ca(2+)-triggered fusion burst was observed, effectively synchronizing the process. Here we show that complexin-1 also reduces spontaneous fusion in the same assay. Moreover, distinct effects of several complexin-1 truncation mutants on spontaneous and Ca(2+)-triggered fusion closely mimic those observed in neuronal cultures. The very N-terminal domain is essential for synchronization of Ca(2+)-triggered fusion, but not for suppression of spontaneous fusion, whereas the opposite is true for the C-terminal domain. By systematically varying the complexin-1 concentration, we observed differences in titration behavior for spontaneous and Ca(2+)-triggered fusion. Taken together, complexin-1 utilizes distinct mechanisms for synchronization of Ca(2+)-triggered fusion and inhibition of spontaneous fusion.DOI: http://dx.doi.org/10.7554/eLife.03756.001.

  View details for DOI 10.7554/eLife.03756

  View details for PubMedID 25122624

  View details for PubMedCentralID PMC4130161

• Complexin inhibits spontaneous release and synchronizes Ca2+-triggered synaptic vesicle fusion by distinct mechanisms. eLife Lai, Y., Diao, J., Cipriano, D. J., Zhang, Y., Pfuetzner, R. A., Padolina, M. S., Brunger, A. T. 2014; 3: e03756

  Abstract

  Previously we showed that fast Ca(2+)-triggered vesicle fusion with reconstituted neuronal SNAREs and synaptotagmin-1 begins from an initial hemifusion-free membrane point contact, rather than a hemifusion diaphragm, using a single vesicle-vesicle lipid/content mixing assay (Diao et al., 2012). When complexin-1 was included, a more pronounced Ca(2+)-triggered fusion burst was observed, effectively synchronizing the process. Here we show that complexin-1 also reduces spontaneous fusion in the same assay. Moreover, distinct effects of several complexin-1 truncation mutants on spontaneous and Ca(2+)-triggered fusion closely mimic those observed in neuronal cultures. The very N-terminal domain is essential for synchronization of Ca(2+)-triggered fusion, but not for suppression of spontaneous fusion, whereas the opposite is true for the C-terminal domain. By systematically varying the complexin-1 concentration, we observed differences in titration behavior for spontaneous and Ca(2+)-triggered fusion. Taken together, complexin-1 utilizes distinct mechanisms for synchronization of Ca(2+)-triggered fusion and inhibition of spontaneous fusion.DOI: http://dx.doi.org/10.7554/eLife.03756.001.

  View details for DOI 10.7554/eLife.03756

  View details for PubMedID 25122624

  View details for PubMedCentralID PMC4130161

• Gently does it for submicron crystals. eLife Zeldin, O. B., Brunger, A. T. 2013; 2: e01662

  Abstract

  A protein structure has been refined with electron diffraction data obtained by using a very weak electron beam to collect large numbers of diffraction patterns from a few sub-micron-sized three-dimensional crystals.

  View details for DOI 10.7554/eLife.01662

  View details for PubMedID 24252881

  View details for PubMedCentralID PMC3831941

• Model morphing and sequence assignment after molecular replacement ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY Terwilliger, T. C., Read, R. J., Adams, P. D., Brunger, A. T., Afonine, P. V., Hung, L. 2013; 69: 2244-2250

  Abstract

  A procedure termed `morphing' for improving a model after it has been placed in the crystallographic cell by molecular replacement has recently been developed. Morphing consists of applying a smooth deformation to a model to make it match an electron-density map more closely. Morphing does not change the identities of the residues in the chain, only their coordinates. Consequently, if the true structure differs from the working model by containing different residues, these differences cannot be corrected by morphing. Here, a procedure that helps to address this limitation is described. The goal of the procedure is to obtain a relatively complete model that has accurate main-chain atomic positions and residues that are correctly assigned to the sequence. Residues in a morphed model that do not match the electron-density map are removed. Each segment of the resulting trimmed morphed model is then assigned to the sequence of the molecule using information about the connectivity of the chains from the working model and from connections that can be identified from the electron-density map. The procedure was tested by application to a recently determined structure at a resolution of 3.2 Å and was found to increase the number of correctly identified residues in this structure from the 88 obtained using phenix.resolve sequence assignment alone (Terwilliger, 2003) to 247 of a possible 359. Additionally, the procedure was tested by application to a series of templates with sequence identities to a target structure ranging between 7 and 36%. The mean fraction of correctly identified residues in these cases was increased from 33% using phenix.resolve sequence assignment to 47% using the current procedure. The procedure is simple to apply and is available in the Phenix software package.

  View details for DOI 10.1107/S0907444913017770

  View details for Web of Science ID 000326648900011

  View details for PubMedID 24189236

  View details for PubMedCentralID PMC3817698

• Complexin-1 Enhances the On-Rate of Vesicle Docking via Simultaneous SNARE and Membrane Interactions. Journal of the American Chemical Society Diao, J., Cipriano, D. J., Zhao, M., Zhang, Y., Shah, S., Padolina, M. S., Pfuetzner, R. A., Brunger, A. T. 2013; 135 (41): 15274-15277

  Abstract

  In synaptic terminals, complexin is thought to have inhibitory and activating roles for spontaneous "mini" release and evoked synchronized neurotransmitter release, respectively. We used single vesicle-vesicle microscopy imaging to study the effect of complexin-1 on the on-rate of docking between vesicles that mimic synaptic vesicles and the plasma membrane. We found that complexin-1 enhances the on-rate of docking of synaptic vesicle mimics containing full-length synaptobrevin-2 and full-length synaptotagmin-1 to plasma membrane-mimicking vesicles containing full-length syntaxin-1A and SNAP-25A. This effect requires the C-terminal domain of complexin-1, which binds to the membrane, the presence of PS in the membrane, and the core region of complexin-1, which binds to the SNARE complex.

  View details for DOI 10.1021/ja407392n

  View details for PubMedID 24083833

• Disassembly of all SNARE complexes by N-ethylmaleimide-sensitive factor (NSF) is initiated by a conserved 1:1 interaction between a-soluble NSF attachment protein (SNAP) and SNARE complex. journal of biological chemistry Vivona, S., Cipriano, D. J., O'Leary, S., Li, Y. H., Fenn, T. D., Brunger, A. T. 2013; 288 (34): 24984-24991

  Abstract

  Vesicle trafficking in eukaryotic cells is facilitated by SNARE-mediated membrane fusion. The ATPase NSF (N-ethylmaleimide-sensitive factor) and the adaptor protein α-SNAP (soluble NSF attachment protein) disassemble all SNARE complexes formed throughout different pathways, but the effect of SNARE sequence and domain variation on the poorly understood disassembly mechanism is unknown. By measuring SNARE-stimulated ATP hydrolysis rates, Michaelis-Menten constants for disassembly, and SNAP-SNARE binding constants for four different ternary SNARE complexes and one binary complex, we found a conserved mechanism, not influenced by N-terminal SNARE domains. α-SNAP and the ternary SNARE complex form a 1:1 complex as revealed by multiangle light scattering. We propose a model of NSF-mediated disassembly in which the reaction is initiated by a 1:1 interaction between α-SNAP and the ternary SNARE complex, followed by NSF binding. Subsequent additional α-SNAP binding events may occur as part of a processive disassembly mechanism.

  View details for DOI 10.1074/jbc.M113.489807

  View details for PubMedID 23836889

  View details for PubMedCentralID PMC3750193

• Processive ATP-driven substrate disassembly by the N-ethylmaleimide-sensitive factor (NSF) molecular machine. journal of biological chemistry Cipriano, D. J., Jung, J., Vivona, S., Fenn, T. D., Brunger, A. T., Bryant, Z. 2013; 288 (32): 23436-23445

  Abstract

  SNARE proteins promote membrane fusion by forming a 4-stranded parallel helical bundle that brings the membranes into close proximity. Post fusion, the complex is disassembled by an AAA+ ATPase called N-ethylmaleimide sensitive factor (NSF). We present evidence that NSF uses a processive unwinding mechanism to disassemble SNARE proteins. Using a real-time disassembly assay based on fluorescence dequenching, we correlate NSF-driven disassembly rates with the SNARE-activated ATPase activity of NSF. Neuronal SNAREs activate the ATPase rate of NSF by ~26-fold. One SNARE complex takes an average of ~5 seconds to disassemble in a process that consumes ~50 ATP. Investigations of substrate requirements show that NSF is capable of disassembling a truncated SNARE substrate consisting of only the core SNARE domain, but not an unrelated four-stranded coiled coil. NSF can also disassemble an engineered double-length SNARE complex, suggesting a processive unwinding mechanism. We further investigated processivity using single turnover experiments, which show that SNAREs can be unwound in a single encounter with NSF. We propose a processive helicase-like mechanism for NSF in which ~1 residue is unwound for every hydrolyzed ATP molecule.

  View details for DOI 10.1074/jbc.M113.476705

  View details for PubMedID 23775070

• Ultrahigh-resolution imaging reveals formation of neuronal SNARE/Munc18 complexes in situ. Proceedings of the National Academy of Sciences of the United States of America Pertsinidis, A., Mukherjee, K., Sharma, M., Pang, Z. P., Park, S. R., Zhang, Y., Brunger, A. T., Südhof, T. C., Chu, S. 2013; 110 (30): E2812-20

  Abstract

  Membrane fusion is mediated by complexes formed by SNAP-receptor (SNARE) and Secretory 1 (Sec1)/mammalian uncoordinated-18 (Munc18)-like (SM) proteins, but it is unclear when and how these complexes assemble. Here we describe an improved two-color fluorescence nanoscopy technique that can achieve effective resolutions of up to 7.5-nm full width at half maximum (3.2-nm localization precision), limited only by stochastic photon emission from single molecules. We use this technique to dissect the spatial relationships between the neuronal SM protein Munc18-1 and SNARE proteins syntaxin-1 and SNAP-25 (25 kDa synaptosome-associated protein). Strikingly, we observed nanoscale clusters consisting of syntaxin-1 and SNAP-25 that contained associated Munc18-1. Rescue experiments with syntaxin-1 mutants revealed that Munc18-1 recruitment to the plasma membrane depends on the Munc18-1 binding to the N-terminal peptide of syntaxin-1. Our results suggest that in a primary neuron, SNARE/SM protein complexes containing syntaxin-1, SNAP-25, and Munc18-1 are preassembled in microdomains on the presynaptic plasma membrane. Our superresolution imaging method provides a framework for investigating interactions between the synaptic vesicle fusion machinery and other subcellular systems in situ.

  View details for DOI 10.1073/pnas.1310654110

  View details for PubMedID 23821748

• Ultrahigh-resolution imaging reveals formation of neuronal SNARE/Munc18 complexes in situ PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Pertsinidis, A., Mukherjee, K., Sharma, M., Pang, Z. P., Park, S. R., Zhang, Y., Brunger, A. T., Suedhof, T. C., Chu, S. 2013; 110 (30): E2812-E2820

  Abstract

  Membrane fusion is mediated by complexes formed by SNAP-receptor (SNARE) and Secretory 1 (Sec1)/mammalian uncoordinated-18 (Munc18)-like (SM) proteins, but it is unclear when and how these complexes assemble. Here we describe an improved two-color fluorescence nanoscopy technique that can achieve effective resolutions of up to 7.5-nm full width at half maximum (3.2-nm localization precision), limited only by stochastic photon emission from single molecules. We use this technique to dissect the spatial relationships between the neuronal SM protein Munc18-1 and SNARE proteins syntaxin-1 and SNAP-25 (25 kDa synaptosome-associated protein). Strikingly, we observed nanoscale clusters consisting of syntaxin-1 and SNAP-25 that contained associated Munc18-1. Rescue experiments with syntaxin-1 mutants revealed that Munc18-1 recruitment to the plasma membrane depends on the Munc18-1 binding to the N-terminal peptide of syntaxin-1. Our results suggest that in a primary neuron, SNARE/SM protein complexes containing syntaxin-1, SNAP-25, and Munc18-1 are preassembled in microdomains on the presynaptic plasma membrane. Our superresolution imaging method provides a framework for investigating interactions between the synaptic vesicle fusion machinery and other subcellular systems in situ.

  View details for DOI 10.1073/pnas.1310654110

  View details for Web of Science ID 000322112300011

  View details for PubMedID 23821748

• Studying protein-reconstituted proteoliposome fusion with content indicators in vitro BIOESSAYS Diao, J., Zhao, M., Zhang, Y., Kyoung, M., Brunger, A. T. 2013; 35 (7): 658-665

  Abstract

  In vitro reconstitution assays are commonly used to study biological membrane fusion. However, to date, most ensemble and single-vesicle experiments involving SNARE proteins have been performed only with lipid-mixing, but not content-mixing indicators. Through simultaneous detection of lipid and small content-mixing indicators, we found that lipid mixing often occurs seconds prior to content mixing, or without any content mixing at all, during a 50-seconds observation period, for Ca(2+) -triggered fusion with SNAREs, full-length synaptotagmin-1, and complexin. Our results illustrate the caveats of commonly used bulk lipid-mixing fusion experiments. We recommend that proteoliposome fusion experiments should always employ content-mixing indicators in addition to, or in place of, lipid-mixing indicators.

  View details for DOI 10.1002/bies.201300010

  View details for Web of Science ID 000320394000012

  View details for PubMedID 23625805

  View details for PubMedCentralID PMC4453005

• Structural basis for the inhibition of botulinum neurotoxin serotype A by potent peptidomimetics Zuniga, J., Hammill, J., Drory, O., Nuss, J., Burnett, J., Gussio, R., Wipf, P., Bavari, S., Brunger, A. T. PERGAMON-ELSEVIER SCIENCE LTD. 2013: 103-104

  View details for DOI 10.1016/j.toxicon.2012.07.122

  View details for Web of Science ID 000320075500110

• Molecular mechanism of calcium-triggered vesicle fusion Kyoung, M., Srivastava, A., Zhang, Y., Diao, J., Vrljic, M., Grob, P., Nogales, E., Chu, S., Brunger, A. T. PERGAMON-ELSEVIER SCIENCE LTD. 2013: 66-67

  View details for DOI 10.1016/j.toxicon.2012.07.035

  View details for Web of Science ID 000320075500023

• Molecular dissection of botulinum neurotoxin reveals interdomain chaperone function Fischer, A., Mushrush, D. J., Sambashivan, S., Lacy, D. B., Brunger, A. T., Montal, M. PERGAMON-ELSEVIER SCIENCE LTD. 2013: 86

  View details for DOI 10.1016/j.toxicon.2012.07.080

  View details for Web of Science ID 000320075500068

• Properties of native brain a-synuclein. Nature Burré, J., Vivona, S., Diao, J., Sharma, M., Brunger, A. T., Südhof, T. C. 2013; 498 (7453): E4-6

  View details for DOI 10.1038/nature12125

  View details for PubMedID 23765500

• Properties of native brain alpha-synuclein NATURE Burre, J., Vivona, S., Diao, J., Sharma, M., Brunger, A. T., Suedhof, T. C. 2013; 498 (7453): E4-E6

  View details for DOI 10.1038/nature12125

  View details for Web of Science ID 000320283400001

• Native alpha-synuclein induces clustering of synaptic-vesicle mimics via binding to phospholipids and synaptobrevin-2/VAMP2 ELIFE Diao, J., Burre, J., Vivona, S., Cipriano, D. J., Sharma, M., Kyoung, M., Suedhof, T. C., Brunger, A. T. 2013; 2

  Abstract

  α-Synuclein is a presynaptic protein that is implicated in Parkinson's and other neurodegenerative diseases. Physiologically, native α-synuclein promotes presynaptic SNARE-complex assembly, but its molecular mechanism of action remains unknown. Here, we found that native α-synuclein promotes clustering of synaptic-vesicle mimics, using a single-vesicle optical microscopy system. This vesicle-clustering activity was observed for both recombinant and native α-synuclein purified from mouse brain. Clustering was dependent on specific interactions of native α-synuclein with both synaptobrevin-2/VAMP2 and anionic lipids. Out of the three familial Parkinson's disease-related point mutants of α-synuclein, only the lipid-binding deficient mutation A30P disrupted clustering, hinting at a possible loss of function phenotype for this mutant. α-Synuclein had little effect on Ca(2+)-triggered fusion in our reconstituted single-vesicle system, consistent with in vivo data. α-Synuclein may therefore lead to accumulation of synaptic vesicles at the active zone, providing a 'buffer' of synaptic vesicles, without affecting neurotransmitter release itself. DOI:http://dx.doi.org/10.7554/eLife.00592.001.

  View details for DOI 10.7554/eLife.00592

  View details for Web of Science ID 000328614700004

  View details for PubMedCentralID PMC3639508

• Studying calcium-triggered vesicle fusion in a single vesicle-vesicle content and lipid-mixing system. Nature protocols Kyoung, M., Zhang, Y., Diao, J., Chu, S., Brunger, A. T. 2013; 8 (1): 1-16

  Abstract

  This protocol describes a single vesicle-vesicle microscopy system to study Ca(2+)-triggered vesicle fusion. Donor vesicles contain reconstituted synaptobrevin and synaptotagmin-1. Acceptor vesicles contain reconstituted syntaxin and synaptosomal-associated protein 25 (SNAP-25), and they are tethered to a PEG-coated glass surface. Donor vesicles are mixed with the tethered acceptor vesicles and incubated for several minutes at a zero-Ca(2+) concentration, resulting in a collection of single interacting vesicle pairs. The donor vesicles also contain two spectrally distinct fluorophores that allow simultaneous monitoring of temporal changes of the content and membrane. Upon Ca(2+) injection into the sample chamber, our system therefore differentiates between hemifusion and complete fusion of interacting vesicle pairs and determines the temporal sequence of these events on a sub-100-millisecond time scale. Other factors such as complexin can be easily added. Our system is unique in that it monitors both content and lipid mixing and starts from a metastable state of interacting vesicle pairs before Ca(2+) injection.

  View details for DOI 10.1038/nprot.2012.134

  View details for PubMedID 23222454

  View details for PubMedCentralID PMC3566647

• Advances, Interactions, and Future Developments in the CNS, Phenix, and Rosetta Structural Biology Software Systems ANNUAL REVIEW OF BIOPHYSICS, VOL 42 Adams, P. D., Baker, D., Brunger, A. T., Das, R., DiMaio, F., Read, R. J., Richardson, D. C., Richardson, J. S., Terwilliger, T. C. 2013; 42: 265-287

  Abstract

  Advances in our understanding of macromolecular structure come from experimental methods, such as X-ray crystallography, and also computational analysis of the growing number of atomic models obtained from such experiments. The later analyses have made it possible to develop powerful tools for structure prediction and optimization in the absence of experimental data. In recent years, a synergy between these computational methods for crystallographic structure determination and structure prediction and optimization has begun to be exploited. We review some of the advances in the algorithms used for crystallographic structure determination in the Phenix and Crystallography & NMR System software packages and describe how methods from ab initio structure prediction and refinement in Rosetta have been applied to challenging crystallographic problems. The prospects for future improvement of these methods are discussed.

  View details for DOI 10.1146/annurev-biophys-083012-130253

  View details for Web of Science ID 000321695700013

  View details for PubMedID 23451892

• Studying calcium-triggered vesicle fusion in a single vesicle-vesicle content and lipid-mixing system NATURE PROTOCOLS Kyoung, M., Zhang, Y., Diao, J., Chu, S., Brunger, A. T. 2013; 8 (1): 1-16

  Abstract

  This protocol describes a single vesicle-vesicle microscopy system to study Ca(2+)-triggered vesicle fusion. Donor vesicles contain reconstituted synaptobrevin and synaptotagmin-1. Acceptor vesicles contain reconstituted syntaxin and synaptosomal-associated protein 25 (SNAP-25), and they are tethered to a PEG-coated glass surface. Donor vesicles are mixed with the tethered acceptor vesicles and incubated for several minutes at a zero-Ca(2+) concentration, resulting in a collection of single interacting vesicle pairs. The donor vesicles also contain two spectrally distinct fluorophores that allow simultaneous monitoring of temporal changes of the content and membrane. Upon Ca(2+) injection into the sample chamber, our system therefore differentiates between hemifusion and complete fusion of interacting vesicle pairs and determines the temporal sequence of these events on a sub-100-millisecond time scale. Other factors such as complexin can be easily added. Our system is unique in that it monitors both content and lipid mixing and starts from a metastable state of interacting vesicle pairs before Ca(2+) injection.

  View details for DOI 10.1038/nprot.2012.134

  View details for Web of Science ID 000313051300001

  View details for PubMedID 23222454

  View details for PubMedCentralID PMC3566647

• Native a-synuclein induces clustering of synaptic-vesicle mimics via binding to phospholipids and synaptobrevin-2/VAMP2. eLife Diao, J., Burré, J., Vivona, S., Cipriano, D. J., Sharma, M., Kyoung, M., Südhof, T. C., Brunger, A. T. 2013; 2

  Abstract

  α-Synuclein is a presynaptic protein that is implicated in Parkinson's and other neurodegenerative diseases. Physiologically, native α-synuclein promotes presynaptic SNARE-complex assembly, but its molecular mechanism of action remains unknown. Here, we found that native α-synuclein promotes clustering of synaptic-vesicle mimics, using a single-vesicle optical microscopy system. This vesicle-clustering activity was observed for both recombinant and native α-synuclein purified from mouse brain. Clustering was dependent on specific interactions of native α-synuclein with both synaptobrevin-2/VAMP2 and anionic lipids. Out of the three familial Parkinson's disease-related point mutants of α-synuclein, only the lipid-binding deficient mutation A30P disrupted clustering, hinting at a possible loss of function phenotype for this mutant. α-Synuclein had little effect on Ca(2+)-triggered fusion in our reconstituted single-vesicle system, consistent with in vivo data. α-Synuclein may therefore lead to accumulation of synaptic vesicles at the active zone, providing a 'buffer' of synaptic vesicles, without affecting neurotransmitter release itself. DOI:http://dx.doi.org/10.7554/eLife.00592.001.

  View details for DOI 10.7554/eLife.00592

  View details for PubMedID 23638301

  View details for PubMedCentralID PMC3639508

• Synaptic proteins promote calcium-triggered fast transition from point contact to full fusion ELIFE Diao, J., Grob, P., Cipriano, D. J., Kyoung, M., Zhang, Y., Shah, S., Amie Nguyen, A., Padolina, M., Srivastava, A., Vrljic, M., Shah, A., Nogales, E., Chu, S., Brunger, A. T. 2012; 1

  Abstract

  The molecular underpinnings of synaptic vesicle fusion for fast neurotransmitter release are still unclear. Here, we used a single vesicle-vesicle system with reconstituted SNARE and synaptotagmin-1 proteoliposomes to decipher the temporal sequence of membrane states upon Ca(2+)-injection at 250-500 μM on a 100-ms timescale. Furthermore, detailed membrane morphologies were imaged with cryo-electron microscopy before and after Ca(2+)-injection. We discovered a heterogeneous network of immediate and delayed fusion pathways. Remarkably, all instances of Ca(2+)-triggered immediate fusion started from a membrane-membrane point-contact and proceeded to complete fusion without discernible hemifusion intermediates. In contrast, pathways that involved a stable hemifusion diaphragm only resulted in fusion after many seconds, if at all. When complexin was included, the Ca(2+)-triggered fusion network shifted towards the immediate pathway, effectively synchronizing fusion, especially at lower Ca(2+)-concentration. Synaptic proteins may have evolved to select this immediate pathway out of a heterogeneous network of possible membrane fusion pathways.DOI:http://dx.doi.org/10.7554/eLife.00109.001.

  View details for DOI 10.7554/eLife.00109

  View details for Web of Science ID 000328584600005

  View details for PubMedCentralID PMC3514886

• Improved crystallographic models through iterated local density-guided model deformation and reciprocal-space refinement ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY Terwilliger, T. C., Read, R. J., Adams, P. D., Brunger, A. T., Afonine, P. V., Grosse-Kunstleve, R. W., Hung, L. 2012; 68: 861-870

  Abstract

  An approach is presented for addressing the challenge of model rebuilding after molecular replacement in cases where the placed template is very different from the structure to be determined. The approach takes advantage of the observation that a template and target structure may have local structures that can be superimposed much more closely than can their complete structures. A density-guided procedure for deformation of a properly placed template is introduced. A shift in the coordinates of each residue in the structure is calculated based on optimizing the match of model density within a 6 Å radius of the center of that residue with a prime-and-switch electron-density map. The shifts are smoothed and applied to the atoms in each residue, leading to local deformation of the template that improves the match of map and model. The model is then refined to improve the geometry and the fit of model to the structure-factor data. A new map is then calculated and the process is repeated until convergence. The procedure can extend the routine applicability of automated molecular replacement, model building and refinement to search models with over 2 Å r.m.s.d. representing 65-100% of the structure.

  View details for DOI 10.1107/S0907444912015636

  View details for Web of Science ID 000305968400015

  View details for PubMedID 22751672

  View details for PubMedCentralID PMC3388814

• Improving the accuracy of macromolecular structure refinement at 7 Å resolution. Structure Brunger, A. T., Adams, P. D., Fromme, P., Fromme, R., Levitt, M., Schröder, G. F. 2012; 20 (6): 957-966

  Abstract

  In X-ray crystallography, molecular replacement and subsequent refinement is challenging at low resolution. We compared refinement methods using synchrotron diffraction data of photosystem I at 7.4 Å resolution, starting from different initial models with increasing deviations from the known high-resolution structure. Standard refinement spoiled the initial models, moving them further away from the true structure and leading to high R(free)-values. In contrast, DEN refinement improved even the most distant starting model as judged by R(free), atomic root-mean-square differences to the true structure, significance of features not included in the initial model, and connectivity of electron density. The best protocol was DEN refinement with initial segmented rigid-body refinement. For the most distant initial model, the fraction of atoms within 2 Å of the true structure improved from 24% to 60%. We also found a significant correlation between R(free) values and the accuracy of the model, suggesting that R(free) is useful even at low resolution.

  View details for DOI 10.1016/j.str.2012.04.020

  View details for PubMedID 22681901

  View details for PubMedCentralID PMC3380535

• Improving the Accuracy of Macromolecular Structure Refinement at 7 angstrom Resolution STRUCTURE Brunger, A. T., Adams, P. D., Fromme, P., Fromme, R., Levitt, M., Schroeder, G. F. 2012; 20 (6): 957-966

  Abstract

  In X-ray crystallography, molecular replacement and subsequent refinement is challenging at low resolution. We compared refinement methods using synchrotron diffraction data of photosystem I at 7.4 Å resolution, starting from different initial models with increasing deviations from the known high-resolution structure. Standard refinement spoiled the initial models, moving them further away from the true structure and leading to high R(free)-values. In contrast, DEN refinement improved even the most distant starting model as judged by R(free), atomic root-mean-square differences to the true structure, significance of features not included in the initial model, and connectivity of electron density. The best protocol was DEN refinement with initial segmented rigid-body refinement. For the most distant initial model, the fraction of atoms within 2 Å of the true structure improved from 24% to 60%. We also found a significant correlation between R(free) values and the accuracy of the model, suggesting that R(free) is useful even at low resolution.

  View details for DOI 10.1016/j.str.2012.04.020

  View details for Web of Science ID 000305094500004

  View details for PubMedCentralID PMC3380535

• Application of DEN refinement and automated model building to a difficult case of molecular-replacement phasing: the structure of a putative succinyl-diaminopimelate desuccinylase from Corynebacterium glutamicum ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY Brunger, A. T., Das, D., Deacon, A. M., Grant, J., Terwilliger, T. C., Read, R. J., Adams, P. D., Levitt, M., Schroeder, G. F. 2012; 68: 391-403

  Abstract

  Phasing by molecular replacement remains difficult for targets that are far from the search model or in situations where the crystal diffracts only weakly or to low resolution. Here, the process of determining and refining the structure of Cgl1109, a putative succinyl-diaminopimelate desuccinylase from Corynebacterium glutamicum, at ∼3 Å resolution is described using a combination of homology modeling with MODELLER, molecular-replacement phasing with Phaser, deformable elastic network (DEN) refinement and automated model building using AutoBuild in a semi-automated fashion, followed by final refinement cycles with phenix.refine and Coot. This difficult molecular-replacement case illustrates the power of including DEN restraints derived from a starting model to guide the movements of the model during refinement. The resulting improved model phases provide better starting points for automated model building and produce more significant difference peaks in anomalous difference Fourier maps to locate anomalous scatterers than does standard refinement. This example also illustrates a current limitation of automated procedures that require manual adjustment of local sequence misalignments between the homology model and the target sequence.

  View details for DOI 10.1107/S090744491104978X

  View details for Web of Science ID 000302138400008

  View details for PubMedID 22505259

  View details for PubMedCentralID PMC3322598

• A novel evolutionarily conserved domain of cell-adhesion GPCRs mediates autoproteolysis EMBO JOURNAL Arac, D., Boucard, A. A., Bolliger, M. F., Nguyen, J., Soltis, S. M., Suedhof, T. C., Brunger, A. T. 2012; 31 (6): 1364-1378

  Abstract

  The G protein-coupled receptor (GPCR) Proteolysis Site (GPS) of cell-adhesion GPCRs and polycystic kidney disease (PKD) proteins constitutes a highly conserved autoproteolysis sequence, but its catalytic mechanism remains unknown. Here, we show that unexpectedly the ∼40-residue GPS motif represents an integral part of a much larger ∼320-residue domain that we termed GPCR-Autoproteolysis INducing (GAIN) domain. Crystal structures of GAIN domains from two distantly related cell-adhesion GPCRs revealed a conserved novel fold in which the GPS motif forms five β-strands that are tightly integrated into the overall GAIN domain. The GAIN domain is evolutionarily conserved from tetrahymena to mammals, is the only extracellular domain shared by all human cell-adhesion GPCRs and PKD proteins, and is the locus of multiple human disease mutations. Functionally, the GAIN domain is both necessary and sufficient for autoproteolysis, suggesting an autoproteolytic mechanism whereby the overall GAIN domain fine-tunes the chemical environment in the GPS to catalyse peptide bond hydrolysis. Thus, the GAIN domain embodies a unique, evolutionarily ancient and widespread autoproteolytic fold whose function is likely relevant for GPCR signalling and for multiple human diseases.

  View details for DOI 10.1038/emboj.2012.26

  View details for Web of Science ID 000302131600005

  View details for PubMedID 22333914

  View details for PubMedCentralID PMC3321182

• A grid-enabled web service for low-resolution crystal structure refinement ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY O'Donovan, D. J., Stokes-Rees, I., Nam, Y., Blacklow, S. C., Schroeder, G. F., Brunger, A. T., Sliz, P. 2012; 68: 261-267

  Abstract

  Deformable elastic network (DEN) restraints have proved to be a powerful tool for refining structures from low-resolution X-ray crystallographic data sets. Unfortunately, optimal refinement using DEN restraints requires extensive calculations and is often hindered by a lack of access to sufficient computational resources. The DEN web service presented here intends to provide structural biologists with access to resources for running computationally intensive DEN refinements in parallel on the Open Science Grid, the US cyberinfrastructure. Access to the grid is provided through a simple and intuitive web interface integrated into the SBGrid Science Portal. Using this portal, refinements combined with full parameter optimization that would take many thousands of hours on standard computational resources can now be completed in several hours. An example of the successful application of DEN restraints to the human Notch1 transcriptional complex using the grid resource, and summaries of all submitted refinements, are presented as justification.

  View details for DOI 10.1107/S0907444912001163

  View details for Web of Science ID 000300444300008

  View details for PubMedID 22349228

  View details for PubMedCentralID PMC3282622

• Beltless Translocation Domain of Botulinum Neurotoxin A Embodies a Minimum Ion-conductive Channel JOURNAL OF BIOLOGICAL CHEMISTRY Fischer, A., Sambashivan, S., Brunger, A. T., Montal, M. 2012; 287 (3): 1657-1661

  Abstract

  Botulinum neurotoxin, the causative agent of the paralytic disease botulism, is an endopeptidase composed of a catalytic domain (or light chain (LC)) and a heavy chain (HC) encompassing the translocation domain (TD) and receptor-binding domain. Upon receptor-mediated endocytosis, the LC and TD are proposed to undergo conformational changes in the acidic endocytic environment resulting in the formation of an LC protein-conducting TD channel. The mechanism of channel formation and the conformational changes in the toxin upon acidification are important but less well understood aspects of botulinum neurotoxin intoxication. Here, we have identified a minimum channel-forming truncation of the TD, the "beltless" TD, that forms transmembrane channels with ion conduction properties similar to those of the full-length TD. At variance with the holotoxin and the HC, channel formation for both the TD and the beltless TD occurs independent of a transmembrane pH gradient. Furthermore, acidification in solution induces moderate secondary structure changes. The subtle nature of the conformational changes evoked by acidification on the TD suggests that, in the context of the holotoxin, larger structural rearrangements and LC unfolding occur preceding or concurrent to channel formation. This notion is consistent with the hypothesis that although each domain of the holotoxin functions individually, each domain serves as a chaperone for the others.

  View details for DOI 10.1074/jbc.C111.319400

  View details for Web of Science ID 000299321000008

  View details for PubMedID 22158863

  View details for PubMedCentralID PMC3265847

• Synaptic proteins promote calcium-triggered fast transition from point contact to full fusion. eLife Diao, J., Grob, P., Cipriano, D. J., Kyoung, M., Zhang, Y., Shah, S., Nguyen, A., Padolina, M., Srivastava, A., Vrljic, M., Shah, A., Nogales, E., Chu, S., Brunger, A. T. 2012; 1

  Abstract

  The molecular underpinnings of synaptic vesicle fusion for fast neurotransmitter release are still unclear. Here, we used a single vesicle-vesicle system with reconstituted SNARE and synaptotagmin-1 proteoliposomes to decipher the temporal sequence of membrane states upon Ca(2+)-injection at 250-500 μM on a 100-ms timescale. Furthermore, detailed membrane morphologies were imaged with cryo-electron microscopy before and after Ca(2+)-injection. We discovered a heterogeneous network of immediate and delayed fusion pathways. Remarkably, all instances of Ca(2+)-triggered immediate fusion started from a membrane-membrane point-contact and proceeded to complete fusion without discernible hemifusion intermediates. In contrast, pathways that involved a stable hemifusion diaphragm only resulted in fusion after many seconds, if at all. When complexin was included, the Ca(2+)-triggered fusion network shifted towards the immediate pathway, effectively synchronizing fusion, especially at lower Ca(2+)-concentration. Synaptic proteins may have evolved to select this immediate pathway out of a heterogeneous network of possible membrane fusion pathways.DOI:http://dx.doi.org/10.7554/eLife.00109.001.

  View details for DOI 10.7554/eLife.00109

  View details for PubMedID 23240085

  View details for PubMedCentralID PMC3514886

• Molecular replacement and model-building using distant homology models as templates Terwilliger, T. C., DiMaio, F., Read, R. J., Baker, D., Brunger, A. T., Adams, P. D. INT UNION CRYSTALLOGRAPHY. 2012: S17-S18

  View details for DOI 10.1107/S0108767312099667

  View details for Web of Science ID 000480363300034

• Post-Translational Modifications and Lipid Binding Profile of Insect Cell-Expressed Full-Length Mammalian Synaptotagmin 1 BIOCHEMISTRY Vrljic, M., Strop, P., Hill, R. C., Hansen, K. C., Chu, S., Brunger, A. T. 2011; 50 (46): 9998-10012

  Abstract

  Synaptotagmin 1 (Syt1) is a Ca(2+) sensor for SNARE-mediated, Ca(2+)-triggered synaptic vesicle fusion in neurons. It is composed of luminal, transmembrane, linker, and two Ca(2+)-binding (C2) domains. Here we describe expression and purification of full-length mammalian Syt1 in insect cells along with an extensive biochemical characterization of the purified protein. The expressed and purified protein is properly folded and has increased α-helical content compared to the C2AB fragment alone. Post-translational modifications of Syt1 were analyzed by mass spectrometry, revealing the same modifications of Syt1 that were previously described for Syt1 purified from brain extract or mammalian cell lines, along with a novel modification of Syt1, tyrosine nitration. A lipid binding screen with both full-length Syt1 and the C2AB fragments of Syt1 and Syt3 isoforms revealed new Syt1-lipid interactions. These results suggest a conserved lipid binding mechanism in which Ca(2+)-independent interactions are mediated via a lysine rich region of the C2B domain while Ca(2+)-dependent interactions are mediated via the Ca(2+)-binding loops.

  View details for DOI 10.1021/bi200998y

  View details for Web of Science ID 000296893700007

  View details for PubMedID 21928778

  View details for PubMedCentralID PMC3217305

• A New Generation of Crystallographic Validation Tools for the Protein Data Bank STRUCTURE Read, R. J., Adams, P. D., Arendall, W. B., Brunger, A. T., Emsley, P., Joosten, R. P., Kleywegt, G. J., Krissinel, E. B., Luetteke, T., Otwinowski, Z., Perrakis, A., Richardson, J. S., Sheffler, W. H., Smith, J. L., Tickle, I. J., Vriend, G., Zwart, P. H. 2011; 19 (10): 1395-1412

  Abstract

  This report presents the conclusions of the X-ray Validation Task Force of the worldwide Protein Data Bank (PDB). The PDB has expanded massively since current criteria for validation of deposited structures were adopted, allowing a much more sophisticated understanding of all the components of macromolecular crystals. The size of the PDB creates new opportunities to validate structures by comparison with the existing database, and the now-mandatory deposition of structure factors creates new opportunities to validate the underlying diffraction data. These developments highlighted the need for a new assessment of validation criteria. The Task Force recommends that a small set of validation data be presented in an easily understood format, relative to both the full PDB and the applicable resolution class, with greater detail available to interested users. Most importantly, we recommend that referees and editors judging the quality of structural experiments have access to a concise summary of well-established quality indicators.

  View details for DOI 10.1016/j.str.2011.08.006

  View details for Web of Science ID 000296125100009

  View details for PubMedID 22000512

  View details for PubMedCentralID PMC3195755

• In vitro system capable of differentiating fast Ca2+-triggered content mixing from lipid exchange for mechanistic studies of neurotransmitter release PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kyoung, M., Srivastava, A., Zhang, Y., Diao, J., Vrljic, M., Grob, P., Nogales, E., Chu, S., Brunger, A. T. 2011; 108 (29): E304-E313

  Abstract

  Understanding the molecular principles of synaptic vesicle fusion is a long-sought goal. It requires the development of a synthetic system that allows manipulations and observations not possible in vivo. Here, we report an in vitro system with reconstituted synaptic proteins that meets the long-sought goal to produce fast content release in the millisecond time regime upon Ca(2+) triggering. Our system simultaneously monitors both content and lipid exchange, and it starts from stable interacting pairs of donor and acceptor vesicles, mimicking the readily releasable pool of synaptic vesicles prior to an action potential. It differentiates between single-vesicle interaction, hemifusion, and complete fusion, the latter mimicking quantized neurotransmitter release upon exocytosis of synaptic vesicles. Prior to Ca(2+) injection, the system is in a state in which spontaneous fusion events between donor and acceptor vesicles are rare. Upon Ca(2+) injection, a rapid burst of complete fusion events emerges, followed by a biphasic decay. The present study focuses on neuronal SNAREs, the Ca(2+) sensor synaptotagmin 1, and the modulator complexin. However, other synaptic proteins could be added and their function examined. Ca(2+) triggering is cooperative, requiring the presence of synaptotagmin, whereas SNAREs alone do not produce a fast fusion burst. Manipulations of the system mimic effects observed in vivo. These results also show that neuronal SNAREs alone do not efficiently produce complete fusion, that the combination of SNAREs with synaptotagmin lowers the activation barriers to full fusion, and that complexin enhances this kinetic control.

  View details for DOI 10.1073/pnas.1107900108

  View details for Web of Science ID 000292876900007

  View details for PubMedID 21705659

  View details for PubMedCentralID PMC3141984

• Reintroducing Electrostatics into Macromolecular Crystallographic Refinement: Application to Neutron Crystallography and DNA Hydration STRUCTURE Fenn, T. D., Schnieders, M. J., Mustyakimov, M., Wu, C., Langan, P., Pande, V. S., Brunger, A. T. 2011; 19 (4): 523-533

  Abstract

  Most current crystallographic structure refinements augment the diffraction data with a priori information consisting of bond, angle, dihedral, planarity restraints, and atomic repulsion based on the Pauli exclusion principle. Yet, electrostatics and van der Waals attraction are physical forces that provide additional a priori information. Here, we assess the inclusion of electrostatics for the force field used for all-atom (including hydrogen) joint neutron/X-ray refinement. Two DNA and a protein crystal structure were refined against joint neutron/X-ray diffraction data sets using force fields without electrostatics or with electrostatics. Hydrogen-bond orientation/geometry favors the inclusion of electrostatics. Refinement of Z-DNA with electrostatics leads to a hypothesis for the entropic stabilization of Z-DNA that may partly explain the thermodynamics of converting the B form of DNA to its Z form. Thus, inclusion of electrostatics assists joint neutron/X-ray refinements, especially for placing and orienting hydrogen atoms.

  View details for DOI 10.1016/j.str.2011.01.015

  View details for Web of Science ID 000289592600011

  View details for PubMedID 21481775

  View details for PubMedCentralID PMC3083928

• Towards Structural Biology with Single Molecules Experimental Biology Meeting 2011 Brunger, A., Strop, P., Vrljic, M., Chu, S., Weninger, K. FEDERATION AMER SOC EXP BIOL. 2011

  View details for Web of Science ID 000310708402359

• Three-dimensional molecular modeling with single molecule FRET JOURNAL OF STRUCTURAL BIOLOGY Brunger, A. T., Strop, P., Vrljic, M., Chu, S., Weninger, K. R. 2011; 173 (3): 497-505

  Abstract

  Single molecule fluorescence energy transfer experiments enable investigations of macromolecular conformation and folding by the introduction of fluorescent dyes at specific sites in the macromolecule. Multiple such experiments can be performed with different labeling site combinations in order to map complex conformational changes or interactions between multiple molecules. Distances that are derived from such experiments can be used for determination of the fluorophore positions by triangulation. When combined with a known structure of the macromolecule(s) to which the fluorophores are attached, a three-dimensional model of the system can be determined. However, care has to be taken to properly derive distance from fluorescence energy transfer efficiency and to recognize the systematic or random errors for this relationship. Here we review the experimental and computational methods used for three-dimensional modeling based on single molecule fluorescence resonance transfer, and describe recent progress in pushing the limits of this approach to macromolecular complexes.

  View details for DOI 10.1016/j.jsb.2010.09.004

  View details for Web of Science ID 000287681200010

  View details for PubMedID 20837146

  View details for PubMedCentralID PMC3051805

• Challenges for refinement at low resolution Brunger, A. T., Schroder, G. F., Levitt, M. INT UNION CRYSTALLOGRAPHY. 2011: C187

  View details for DOI 10.1107/S010876731109533X

  View details for Web of Science ID 000484219400467

• Refinement of X-ray Crystal Structures COMPREHENSIVE BIOPHYSICS, VOL 1: BIOPHYSICAL TECHNIQUES FOR STRUCTURAL CHARACTERIZATION OF MACROMOLECULES Brunger, A. T., Adams, P. D., Egelman, E. H. 2011: 105-115

  View details for DOI 10.1016/B978-0-12-374920-8.00108-9

  View details for Web of Science ID 000333766600007

• A smooth and differentiable bulk-solvent model for macromolecular diffraction ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY Fenn, T. D., Schnieders, M. J., Brunger, A. T. 2010; 66: 1024-1031

  Abstract

  Inclusion of low-resolution data in macromolecular crystallography requires a model for the bulk solvent. Previous methods have used a binary mask to accomplish this, which has proven to be very effective, but the mask is discontinuous at the solute-solvent boundary (i.e. the mask value jumps from zero to one) and is not differentiable with respect to atomic parameters. Here, two algorithms are introduced for computing bulk-solvent models using either a polynomial switch or a smoothly thresholded product of Gaussians, and both models are shown to be efficient and differentiable with respect to atomic coordinates. These alternative bulk-solvent models offer algorithmic improvements, while showing similar agreement of the model with the observed amplitudes relative to the binary model as monitored using R, R(free) and differences between experimental and model phases. As with the standard solvent models, the alternative models improve the agreement primarily with lower resolution (>6 A) data versus no bulk solvent. The models are easily implemented into crystallographic software packages and can be used as a general method for bulk-solvent correction in macromolecular crystallography.

  View details for DOI 10.1107/S0907444910031045

  View details for Web of Science ID 000281635500008

  View details for PubMedID 20823553

  View details for PubMedCentralID PMC2935282

• Iterative Structure-Based Peptide-Like Inhibitor Design against the Botulinum Neurotoxin Serotype A PLOS ONE Zuniga, J. E., Hammill, J. T., Drory, O., Nuss, J. E., Burnett, J. C., Gussio, R., Wipf, P., Bavari, S., Brunger, A. T. 2010; 5 (6)

  Abstract

  The botulinum neurotoxin serotype A light chain (BoNT/A LC) protease is the catalytic component responsible for the neuroparalysis that is characteristic of the disease state botulism. Three related peptide-like molecules (PLMs) were designed using previous information from co-crystal structures, synthesized, and assayed for in vitro inhibition against BoNT/A LC. Our results indicate these PLMS are competitive inhibitors of the BoNT/A LC protease and their K(i) values are in the nM-range. A co-crystal structure for one of these inhibitors was determined and reveals that the PLM, in accord with the goals of our design strategy, simultaneously involves both ionic interactions via its P1 residue and hydrophobic contacts by means of an aromatic group in the P2' position. The PLM adopts a helical conformation similar to previously determined co-crystal structures of PLMs, although there are also major differences to these other structures such as contacts with specific BoNT/A LC residues. Our structure further demonstrates the remarkable plasticity of the substrate binding cleft of the BoNT/A LC protease and provides a paradigm for iterative structure-based design and development of BoNT/A LC inhibitors.

  View details for DOI 10.1371/journal.pone.0011378

  View details for Web of Science ID 000279370000009

  View details for PubMedID 20614028

  View details for PubMedCentralID PMC2894858

• Polarizable Atomic Multipole X-Ray Refinement: Hydration Geometry and Application to Macromolecules BIOPHYSICAL JOURNAL Fenn, T. D., Schnieders, M. J., Brunger, A. T., Pande, V. S. 2010; 98 (12): 2984-2992

  Abstract

  We recently developed a polarizable atomic multipole refinement method assisted by the AMOEBA force field for macromolecular crystallography. Compared to standard refinement procedures, the method uses a more rigorous treatment of x-ray scattering and electrostatics that can significantly improve the resultant information contained in an atomic model. We applied this method to high-resolution lysozyme and trypsin data sets, and validated its utility for precisely describing biomolecular electron density, as indicated by a 0.4-0.6% decrease in the R- and R(free)-values, and a corresponding decrease in the relative energy of 0.4-0.8 Kcal/mol/residue. The re-refinements illustrate the ability of force-field electrostatics to orient water networks and catalytically relevant hydrogens, which can be used to make predictions regarding active site function, activity, and protein-ligand interaction energies. Re-refinement of a DNA crystal structure generates the zigzag spine pattern of hydrogen bonding in the minor groove without manual intervention. The polarizable atomic multipole electrostatics model implemented in the AMOEBA force field is applicable and informative for crystal structures solved at any resolution.

  View details for DOI 10.1016/j.bpj.2010.02.057

  View details for Web of Science ID 000278913500027

  View details for PubMedID 20550911

  View details for PubMedCentralID PMC2884231

• The Longin SNARE VAMP7/TI-VAMP Adopts a Closed Conformation JOURNAL OF BIOLOGICAL CHEMISTRY Vivona, S., Liu, C. W., Strop, P., Rossi, V., Filippini, F., Brunger, A. T. 2010; 285 (23): 17965-17973

  Abstract

  SNARE protein complexes are key mediators of exocytosis by juxtaposing opposing membranes, leading to membrane fusion. SNAREs generally consist of one or two core domains that can form a four-helix bundle with other SNARE core domains. Some SNAREs, such as syntaxin target-SNAREs and longin vesicular-SNAREs, have independent, folded N-terminal domains that can interact with their respective SNARE core domains and thereby affect the kinetics of SNARE complex formation. This autoinhibition mechanism is believed to regulate the role of the longin VAMP7/TI-VAMP in neuronal morphogenesis. Here we use nuclear magnetic resonance spectroscopy to study the longin-SNARE core domain interaction for VAMP7. Using complete backbone resonance assignments, chemical shift perturbations analysis, and hydrogen/deuterium exchange experiments, we conclusively show that VAMP7 adopts a preferentially closed conformation in solution. Taken together, the closed conformation of longins is conserved, in contrast to the syntaxin family of SNAREs for which mixtures of open and closed states have been observed. This may indicate different regulatory mechanisms for SNARE complexes containing syntaxins and longins, respectively.

  View details for DOI 10.1074/jbc.M110.120972

  View details for Web of Science ID 000278133400072

  View details for PubMedID 20378544

  View details for PubMedCentralID PMC2878558

• Neurexins Physically and Functionally Interact with GABA(A) Receptors NEURON Zhang, C., Atasoy, D., Arac, D., Yang, X., Fucillo, M. V., Robison, A. J., Ko, J., Brunger, A. T., Sudhof, T. C. 2010; 66 (3): 403-416

  Abstract

  Neurexins are presynaptic cell-adhesion molecules that form trans-synaptic complexes with postsynaptic neuroligins. When overexpressed in nonneuronal cells, neurexins induce formation of postsynaptic specializations in cocultured neurons, suggesting that neurexins are synaptogenic. However, we find that when overexpressed in neurons, neurexins do not increase synapse density, but instead selectively suppressed GABAergic synaptic transmission without decreasing GABAergic synapse numbers. This suppression was mediated by all subtypes of neurexins tested, in a cell-autonomous and neuroligin-independent manner. Strikingly, addition of recombinant neurexin to cultured neurons at submicromolar concentrations induced the same suppression of GABAergic synaptic transmission as neurexin overexpression. Moreover, experiments with native brain proteins and purified recombinant proteins revealed that neurexins directly and stoichiometrically bind to GABA(A) receptors, suggesting that they decrease GABAergic synaptic responses by interacting with GABA(A) receptors. Our findings suggest that besides their other well-documented interactions, presynaptic neurexins directly act on postsynaptic GABA(A) receptors, which may contribute to regulate the excitatory/inhibitory balance in brain.

  View details for DOI 10.1016/j.neuron.2010.04.008

  View details for PubMedID 20471353

• Super-resolution biomolecular crystallography with low-resolution data NATURE Schroeder, G. F., Levitt, M., Brunger, A. T. 2010; 464 (7292): 1218-U146

  Abstract

  X-ray diffraction plays a pivotal role in the understanding of biological systems by revealing atomic structures of proteins, nucleic acids and their complexes, with much recent interest in very large assemblies like the ribosome. As crystals of such large assemblies often diffract weakly (resolution worse than 4 A), we need methods that work at such low resolution. In macromolecular assemblies, some of the components may be known at high resolution, whereas others are unknown: current refinement methods fail as they require a high-resolution starting structure for the entire complex. Determining the structure of such complexes, which are often of key biological importance, should be possible in principle as the number of independent diffraction intensities at a resolution better than 5 A generally exceeds the number of degrees of freedom. Here we introduce a method that adds specific information from known homologous structures but allows global and local deformations of these homology models. Our approach uses the observation that local protein structure tends to be conserved as sequence and function evolve. Cross-validation with R(free) (the free R-factor) determines the optimum deformation and influence of the homology model. For test cases at 3.5-5 A resolution with known structures at high resolution, our method gives significant improvements over conventional refinement in the model as monitored by coordinate accuracy, the definition of secondary structure and the quality of electron density maps. For re-refinements of a representative set of 19 low-resolution crystal structures from the Protein Data Bank, we find similar improvements. Thus, a structure derived from low-resolution diffraction data can have quality similar to a high-resolution structure. Our method is applicable to the study of weakly diffracting crystals using X-ray micro-diffraction as well as data from new X-ray light sources. Use of homology information is not restricted to X-ray crystallography and cryo-electron microscopy: as optical imaging advances to subnanometre resolution, it can use similar tools.

  View details for DOI 10.1038/nature08892

  View details for Web of Science ID 000276891100043

  View details for PubMedID 20376006

  View details for PubMedCentralID PMC2859093

• Single-molecule FRET-derived model of the synaptotagmin 1-SNARE fusion complex NATURE STRUCTURAL & MOLECULAR BIOLOGY Choi, U. B., Strop, P., Vrljic, M., Chu, S., Brunger, A. T., Weninger, K. R. 2010; 17 (3): 318-U84

  Abstract

  Synchronous neurotransmission is triggered when Ca(2+) binds to synaptotagmin 1 (Syt1), a synaptic-vesicle protein that interacts with SNAREs and membranes. We used single-molecule fluorescence resonance energy transfer (FRET) between synaptotagmin's two C2 domains to determine that their conformation consists of multiple states with occasional transitions, consistent with domains in random relative motion. SNARE binding results in narrower intrasynaptotagmin FRET distributions and less frequent transitions between states. We obtained an experimentally determined model of the elusive Syt1-SNARE complex using a multibody docking approach with 34 FRET-derived distances as restraints. The Ca(2+)-binding loops point away from the SNARE complex, so they may interact with the same membrane. The loop arrangement is similar to that of the crystal structure of SNARE-induced Ca(2+)-bound Syt3, suggesting a common mechanism by which the interaction between synaptotagmins and SNAREs aids in Ca(2+)-triggered fusion.

  View details for DOI 10.1038/nsmb.1763

  View details for Web of Science ID 000275182700012

  View details for PubMedID 20173763

  View details for PubMedCentralID PMC2922927

• Molecular mechanism of the synaptotagmin-SNARE interaction in Ca2+-triggered vesicle fusion NATURE STRUCTURAL & MOLECULAR BIOLOGY Vrljic, M., Strop, P., Ernst, J. A., Sutton, R. B., Chu, S., Brunger, A. T. 2010; 17 (3): 325-U92

  Abstract

  In neurons, SNAREs, synaptotagmin and other factors catalyze Ca(2+)-triggered fusion of vesicles with the plasma membrane. The molecular mechanism of this process, especially the interaction between synaptotagmin and SNAREs, remains an enigma. Here we characterized this interaction by single-molecule fluorescence microscopy and crystallography. The two rigid Ca(2+)-binding domains of synaptotagmin 3 (Syt3) undergo large relative motions in solution. Interaction with SNARE complex amplifies a particular state of the two domains that is further enhanced by Ca(2+). This state is represented by the first SNARE-induced Ca(2+)-bound crystal structure of a synaptotagmin fragment containing both domains. The arrangement of the Ca(2+)-binding loops of this structure of Syt3 matches that of SNARE-bound Syt1, suggesting a conserved feature of synaptotagmins. The loops resemble the membrane-interacting loops of certain viral fusion proteins in the postfusion state, suggesting unexpected similarities between both fusion systems.

  View details for DOI 10.1038/nsmb.1764

  View details for Web of Science ID 000275182700013

  View details for PubMedID 20173762

  View details for PubMedCentralID PMC2928146

• Warren L. DeLano 21 June 1972-3 November 2009 OBITUARY NATURE STRUCTURAL & MOLECULAR BIOLOGY Brunger, A. T., Wells, J. A. 2009; 16 (12): 1202-1203

  View details for Web of Science ID 000272609200002

  View details for PubMedID 19956203

• Neuroligin-1 performs neurexin-dependent and neurexin-independent functions in synapse validation EMBO JOURNAL Ko, J., Zhang, C., Arac, D., Boucard, A. A., Brunger, A. T., Suedhof, T. C. 2009; 28 (20): 3244-3255

  Abstract

  Postsynaptic neuroligins are thought to perform essential functions in synapse validation and synaptic transmission by binding to, and dimerizing, presynaptic alpha- and beta-neurexins. To test this hypothesis, we examined the functional effects of neuroligin-1 mutations that impair only alpha-neurexin binding, block both alpha- and beta-neurexin binding, or abolish neuroligin-1 dimerization. Abolishing alpha-neurexin binding abrogated neuroligin-induced generation of neuronal synapses onto transfected non-neuronal cells in the so-called artificial synapse-formation assay, even though beta-neurexin binding was retained. Thus, in this assay, neuroligin-1 induces apparent synapse formation by binding to presynaptic alpha-neurexins. In transfected neurons, however, neither alpha- nor beta-neurexin binding was essential for the ability of postsynaptic neuroligin-1 to dramatically increase synapse density, suggesting a neurexin-independent mechanism of synapse formation. Moreover, neuroligin-1 dimerization was not required for either the non-neuronal or the neuronal synapse-formation assay. Nevertheless, both alpha-neurexin binding and neuroligin-1 dimerization were essential for the increase in apparent synapse size that is induced by neuroligin-1 in transfected neurons. Thus, neuroligin-1 performs diverse synaptic functions by mechanisms that include as essential components of alpha-neurexin binding and neuroligin dimerization, but extend beyond these activities.

  View details for DOI 10.1038/emboj.2009.249

  View details for PubMedID 19730411

• Receptor and substrate interactions of clostridial neurotoxins 6th International Conference on Basic and Therapeutic Aspects of Botulinum and Tetanus Toxins Brunger, A. T., Rummel, A. PERGAMON-ELSEVIER SCIENCE LTD. 2009: 550–60

  Abstract

  The high potency of clostridial neurotoxins relies predominantly on their neurospecific binding and specific hydrolysis of SNARE proteins. Their multi-step mode of mechanism can be ascribed to their multi-domain three-dimensional structure. The C-terminal H(CC)-domain interacts subsequently with complex polysialo-gangliosides such as GT1b and a synaptic vesicle protein receptor via two neighbouring binding sites, resulting in highly specific uptake of the neurotoxins at synapses of cholinergic motoneurons. After its translocation the enzymatically active light chain specifically hydrolyses specific SNARE proteins, preventing SNARE complex assembly and thereby blocking exocytosis of neurotransmitter.

  View details for DOI 10.1016/j.toxicon.2008.12.027

  View details for Web of Science ID 000269965400002

  View details for PubMedID 19268493

  View details for PubMedCentralID PMC2756235

• Polarizable atomic multipole X-ray refinement: application to peptide crystals ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY Schnieders, M. J., Fenn, T. D., Pande, V. S., Brunger, A. T. 2009; 65: 952-965

  Abstract

  Recent advances in computational chemistry have produced force fields based on a polarizable atomic multipole description of biomolecular electrostatics. In this work, the Atomic Multipole Optimized Energetics for Biomolecular Applications (AMOEBA) force field is applied to restrained refinement of molecular models against X-ray diffraction data from peptide crystals. A new formalism is also developed to compute anisotropic and aspherical structure factors using fast Fourier transformation (FFT) of Cartesian Gaussian multipoles. Relative to direct summation, the FFT approach can give a speedup of more than an order of magnitude for aspherical refinement of ultrahigh-resolution data sets. Use of a sublattice formalism makes the method highly parallelizable. Application of the Cartesian Gaussian multipole scattering model to a series of four peptide crystals using multipole coefficients from the AMOEBA force field demonstrates that AMOEBA systematically underestimates electron density at bond centers. For the trigonal and tetrahedral bonding geometries common in organic chemistry, an atomic multipole expansion through hexadecapole order is required to explain bond electron density. Alternatively, the addition of interatomic scattering (IAS) sites to the AMOEBA-based density captured bonding effects with fewer parameters. For a series of four peptide crystals, the AMOEBA-IAS model lowered R(free) by 20-40% relative to the original spherically symmetric scattering model.

  View details for DOI 10.1107/S0907444909022707

  View details for Web of Science ID 000269350000009

  View details for PubMedID 19690373

  View details for PubMedCentralID PMC2733883

• Mechanistic insights into active site-associated polyubiquitination by the ubiquitin-conjugating enzyme Ube2g2 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Li, W., Tu, D., Li, L., Wollert, T., Ghirlando, R., Brunger, A. T., Ye, Y. 2009; 106 (10): 3722-3727

  Abstract

  Lys-48-linked polyubiquitination regulates a variety of cellular processes by targeting ubiquitinated proteins to the proteasome for degradation. Although polyubiquitination had been presumed to occur by transferring ubiquitin molecules, one at a time, from an E2 active site to a substrate, we recently showed that the endoplasmic reticulum-associated RING finger ubiquitin ligase gp78 can mediate the preassembly of Lys-48-linked polyubiquitin chains on the catalytic cysteine of its cognate E2 Ube2g2 and subsequent transfer to a substrate. Active site-linked polyubiquitin chains are detected in cells on Ube2g2 and its yeast homolog Ubc7p, but how these chains are assembled is unclear. Here, we show that gp78 forms an oligomer via 2 oligomerization sites, one of which is a hydrophobic segment located in the gp78 cytosolic domain. We further demonstrate that a gp78 oligomer can simultaneously associate with multiple Ube2g2 molecules. This interaction is mediated by a novel Ube2g2 surface distinct from the predicted RING binding site. Our data suggest that a large gp78-Ube2g2 heterooligomer brings multiple Ube2g2 molecules into close proximity, allowing ubiquitin moieties to be transferred between neighboring Ube2g2s to form active site-linked polyubiquitin chains.

  View details for DOI 10.1073/pnas.0808564106

  View details for Web of Science ID 000264036900017

  View details for PubMedID 19223579

  View details for PubMedCentralID PMC2644258

• SINGLE MOLECULE STUDIES OF THE SYNAPTIC VESICLE FUSION MACHINERY 40th Annual Meeting of the American-Society-for-Neurochemistry Brunger, A. T., Weninger, K., Vrljic, M., Choi, U. B., Bowen, M. A., Chu, S. WILEY-BLACKWELL. 2009: 55–55

  View details for Web of Science ID 000263336800116

• X-ray structure determination at low resolution ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY Brunger, A. T., DeLaBarre, B., Davies, J. M., Weis, W. I. 2009; 65: 128-133

  Abstract

  As an example of structure determination in the 3.5-4.5 A resolution range, crystal structures of the ATPase p97/VCP, consisting of an N-terminal domain followed by a tandem pair of ATPase domains (D1 and D2), are discussed. The structures were originally solved by molecular replacement with the high-resolution structure of the N-D1 fragment of p97/VCP, whereas the D2 domain was manually built using its homology to the D1 domain as a guide. The structure of the D2 domain alone was subsequently solved at 3 A resolution. The refined model of D2 and the high-resolution structure of the N-D1 fragment were then used as starting models for re-refinement against the low-resolution diffraction data for full-length p97. The re-refined full-length models showed significant improvement in both secondary structure and R values. The free R values dropped by as much as 5% compared with the original structure refinements, indicating that refinement is meaningful at low resolution and that there is information in the diffraction data even at approximately 4 A resolution that objectively assesses the quality of the model. It is concluded that de novo model building is problematic at low resolution and refinement should start from high-resolution crystal structures whenever possible.

  View details for DOI 10.1107/S0907444908043795

  View details for Web of Science ID 000263557900004

  View details for PubMedID 19171967

  View details for PubMedCentralID PMC2631637

• Single-Molecule Studies of the Neuronal SNARE Fusion Machinery ANNUAL REVIEW OF BIOCHEMISTRY Brunger, A. T., Weninger, K., Bowen, M., Chu, S. 2009; 78: 903-928

  Abstract

  SNAREs are essential components of the machinery for Ca(2+)-triggered fusion of synaptic vesicles with the plasma membrane, resulting in neurotransmitter release into the synaptic cleft. Although much is known about their biophysical and structural properties and their interactions with accessory proteins such as the Ca(2+) sensor synaptotagmin, their precise role in membrane fusion remains an enigma. Ensemble studies of liposomes with reconstituted SNAREs have demonstrated that SNAREs and accessory proteins can trigger lipid mixing/fusion, but the inability to study individual fusion events has precluded molecular insights into the fusion process. Thus, this field is ripe for studies with single-molecule methodology. In this review, we discuss applications of single-molecule approaches to observe reconstituted SNAREs, their complexes, associated proteins, and their effect on biological membranes. Some of the findings are provocative, such as the possibility of parallel and antiparallel SNARE complexes or of vesicle docking with only syntaxin and synaptobrevin, but have been confirmed by other experiments.

  View details for DOI 10.1146/annurev.biochem.77.070306.103621

  View details for Web of Science ID 000268069200032

  View details for PubMedID 19489736

  View details for PubMedCentralID PMC2854664

• A Potent Peptidomimetic Inhibitor of Botulinum Neurotoxin Serotype A Has a Very Different Conformation than SNAP-25 Substrate STRUCTURE Zuniga, J. E., Schmidt, J. J., Fenn, T., Burnett, J. C., Arac, D., Gussio, R., Stafford, R. G., Badie, S. S., Bavari, S., Brunger, A. T. 2008; 16 (10): 1588-1597

  Abstract

  Botulinum neurotoxin serotype A is the most lethal of all known toxins. Here, we report the crystal structure, along with SAR data, of the zinc metalloprotease domain of BoNT/A bound to a potent peptidomimetic inhibitor (K(i)=41 nM) that resembles the local sequence of the SNAP-25 substrate. Surprisingly, the inhibitor adopts a helical conformation around the cleavage site, in contrast to the extended conformation of the native substrate. The backbone of the inhibitor's P1 residue displaces the putative catalytic water molecule and concomitantly interacts with the "proton shuttle" E224. This mechanism of inhibition is aided by residue contacts in the conserved S1' pocket of the substrate binding cleft and by the induction of new hydrophobic pockets, which are not present in the apo form, especially for the P2' residue of the inhibitor. Our inhibitor is specific for BoNT/A as it does not inhibit other BoNT serotypes or thermolysin.

  View details for DOI 10.1016/j.str.2008.07.011

  View details for Web of Science ID 000259930800017

  View details for PubMedID 18940613

  View details for PubMedCentralID PMC2716802

• Highly specific interactions between botulinum neurotoxins and synaptic vesicle proteins CELLULAR AND MOLECULAR LIFE SCIENCES Brunger, A. T., Jin, R., Breidenbach, M. A. 2008; 65 (15): 2296-2306

  Abstract

  Despite its extreme toxicity, botulinum neurotoxin is widely utilized in low doses as a treatment for several neurological disorders; higher doses cause the neuroparalytic syndrome botulism. The toxin blocks neurotransmitter release by preferentially attaching to pre-synaptic membrane receptors at neuromuscular junctions and subsequently delivering a Zn2+-dependent protease component to presynaptic neuronal cytosol. These highly specialized enzymes exclusively hydrolyze peptide bonds within SNARE (soluble N-ethylmaleiamide sensitive factor attachment protein receptor) proteins. In this review we discuss the structural basis for botulinum toxin's exquisite specificity for its neuronal cell-surface receptors and intracellular SNARE targets.

  View details for DOI 10.1007/s00018-008-8088-0

  View details for Web of Science ID 000258275900005

  View details for PubMedID 18425411

• Botulinum neurotoxin interactions with substrate 6th International Conference on Basic and Therapeutic Aspects of Botulinum and Tetanus Toxins Brunger, A. PERGAMON-ELSEVIER SCIENCE LTD. 2008: 2–2

  View details for Web of Science ID 000257192200004

• 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

• Accessory proteins stabilize the acceptor complex for synaptobrevin, the 1 : 1 syntaxin/SNAP-25 complex STRUCTURE Weninger, K., Bowen, M. E., Choi, U. B., Chu, S., Brunger, A. T. 2008; 16 (2): 308-320

  Abstract

  Syntaxin/SNAP-25 interactions precede assembly of the ternary SNARE complex that is essential for neurotransmitter release. This binary complex has been difficult to characterize by bulk methods because of the prevalence of a 2:1 dead-end species. Here, using single-molecule fluorescence, we find the structure of the 1:1 syntaxin/SNAP-25 binary complex is variable, with states changing on the second timescale. One state corresponds to a parallel three-helix bundle, whereas other states show one of the SNAP-25 SNARE domains dissociated. Adding synaptobrevin suppresses the dissociated helix states. Remarkably, upon addition of complexin, Munc13, Munc18, or synaptotagmin, a similar effect is observed. Thus, the 1:1 binary complex is a dynamic acceptor for synaptobrevin binding, and accessory proteins stabilize this acceptor. In the cellular environment the binary complex is actively maintained in a configuration where it can rapidly interact with synaptobrevin, so formation is not likely a limiting step for neurotransmitter release.

  View details for DOI 10.1016/j.str.2007.12.010

  View details for Web of Science ID 000253219400015

  View details for PubMedID 18275821

  View details for PubMedCentralID PMC2856644

• Rab and arl GTPase family members cooperate in the localization of the golgin GCC185 CELL Burguete, A. S., Fenn, T. D., Brunger, A. T., Pfeffer, S. R. 2008; 132 (2): 286-298

  Abstract

  GCC185 is a large coiled-coil protein at the trans Golgi network that is required for receipt of transport vesicles inbound from late endosomes and for anchoring noncentrosomal microtubules that emanate from the Golgi. Here, we demonstrate that recruitment of GCC185 to the Golgi is mediated by two Golgi-localized small GTPases of the Rab and Arl families. GCC185 binds Rab6, and mutation of residues needed for Rab binding abolishes Golgi localization. The crystal structure of Rab6 bound to the GCC185 Rab-binding domain reveals that Rab6 recognizes a two-fold symmetric surface on a coiled coil immediately adjacent to a C-terminal GRIP domain. Unexpectedly, Rab6 binding promotes association of Arl1 with the GRIP domain. We present a structure-derived model for dual GTPase membrane attachment that highlights the potential ability of Rab GTPases to reach binding partners at a significant distance from the membrane via their unstructured and membrane-anchored, hypervariable domains.

  View details for DOI 10.1016/j.cell.2007.11.048

  View details for Web of Science ID 000253427700014

  View details for PubMedID 18243103

  View details for PubMedCentralID PMC2344137

• The structure of the yeast plasma membrane SNARE complex reveals destabilizing water-filled cavities JOURNAL OF BIOLOGICAL CHEMISTRY Strop, P., Kaiser, S. E., Vrljic, M., Brunger, A. T. 2008; 283 (2): 1113-1119

  Abstract

  SNARE proteins form a complex that leads to membrane fusion between vesicles, organelles, and plasma membrane in all eukaryotic cells. We report the 1.7A resolution structure of the SNARE complex that mediates exocytosis at the plasma membrane in the yeast Saccharomyces cerevisiae. Similar to its neuronal and endosomal homologues, the S. cerevisiae SNARE complex forms a parallel four-helix bundle in the center of which is an ionic layer. The S. cerevisiae SNARE complex exhibits increased helix bending near the ionic layer, contains water-filled cavities in the complex core, and exhibits reduced thermal stability relative to mammalian SNARE complexes. Mutagenesis experiments suggest that the water-filled cavities contribute to the lower stability of the S. cerevisiae complex.

  View details for DOI 10.1074/jbc.M707912200

  View details for Web of Science ID 000252128100051

  View details for PubMedID 17956869

• Structures of neuroligin-1 and the Neuroligin-l/Neurexin-1 beta complex reveal specificprotein-protein and protein-Ca2+ interactions NEURON Arac, D., Boucard, A. A., Ozkan, E., Strop, P., Newell, E., Sudhof, T. C., Brunger, A. T. 2007; 56 (6): 992-1003

  Abstract

  Neurexins and neuroligins provide trans-synaptic connectivity by the Ca2+-dependent interaction of their alternatively spliced extracellular domains. Neuroligins specify synapses in an activity-dependent manner, presumably by binding to neurexins. Here, we present the crystal structures of neuroligin-1 in isolation and in complex with neurexin-1 beta. Neuroligin-1 forms a constitutive dimer, and two neurexin-1 beta monomers bind to two identical surfaces on the opposite faces of the neuroligin-1 dimer to form a heterotetramer. The neuroligin-1/neurexin-1 beta complex exhibits a nanomolar affinity and includes a large binding interface that contains bound Ca2+. Alternatively spliced sites in neurexin-1 beta and in neuroligin-1 are positioned nearby the binding interface, explaining how they regulate the interaction. Structure-based mutations of neuroligin-1 at the interface disrupt binding to neurexin-1 beta, but not the folding of neuroligin-1 and confirm the validity of the binding interface of the neuroligin-1/neurexin-1 beta complex. Our results provide molecular insights for understanding the role of cell-adhesion proteins in synapse function.

  View details for DOI 10.1016/j.neuron.2007.12.002

  View details for PubMedID 18093522

• Transglutaminase 2 undergoes a large conformational change upon activation PLOS BIOLOGY Pinkas, D. M., Strop, P., Brunger, A. T., Khosla, C. 2007; 5 (12): 2788-2796

  Abstract

  Human transglutaminase 2 (TG2), a member of a large family of enzymes that catalyze protein crosslinking, plays an important role in the extracellular matrix biology of many tissues and is implicated in the gluten-induced pathogenesis of celiac sprue. Although vertebrate transglutaminases have been studied extensively, thus far all structurally characterized members of this family have been crystallized in conformations with inaccessible active sites. We have trapped human TG2 in complex with an inhibitor that mimics inflammatory gluten peptide substrates and have solved, at 2-A resolution, its x-ray crystal structure. The inhibitor stabilizes TG2 in an extended conformation that is dramatically different from earlier transglutaminase structures. The active site is exposed, revealing that catalysis takes place in a tunnel, bridged by two tryptophan residues that separate acyl-donor from acyl-acceptor and stabilize the tetrahedral reaction intermediates. Site-directed mutagenesis was used to investigate the acyl-acceptor side of the tunnel, yielding mutants with a marked increase in preference for hydrolysis over transamidation. By providing the ability to visualize this activated conformer, our results create a foundation for understanding the catalytic as well as the non-catalytic roles of TG2 in biology, and for dissecting the process by which the autoantibody response to TG2 is induced in celiac sprue patients.

  View details for DOI 10.1371/journal.pbio.0050327

  View details for Web of Science ID 000251874900010

  View details for PubMedID 18092889

  View details for PubMedCentralID PMC2140088

• Combining efficient conformational sampling with a deformable elastic network model facilitates structure refinement at low resolution STRUCTURE Schroeder, G. F., Brunger, A. T., Levitt, M. 2007; 15 (12): 1630-1641

  Abstract

  Structural studies of large proteins and protein assemblies are a difficult and pressing challenge in molecular biology. Experiments often yield only low-resolution or sparse data that are not sufficient to fully determine atomistic structures. We have developed a general geometry-based algorithm that efficiently samples conformational space under constraints imposed by low-resolution density maps obtained from electron microscopy or X-ray crystallography experiments. A deformable elastic network (DEN) is used to restrain the sampling to prior knowledge of an approximate structure. The DEN restraints dramatically reduce over-fitting, especially at low resolution. Cross-validation is used to optimally weight the structural information and experimental data. Our algorithm is robust even for noise-added density maps and has a large radius of convergence for our test case. The DEN restraints can also be used to enhance reciprocal space simulated annealing refinement.

  View details for DOI 10.1016/j.str.2007.09.021

  View details for Web of Science ID 000251655400015

  View details for PubMedID 18073112

  View details for PubMedCentralID PMC2213367

• Structure and function of the yeast U-box-containing ubiquitin ligase Ufd2p PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Tu, D., Li, W., Ye, Y., Brunger, A. T. 2007; 104 (40): 15599-15606

  Abstract

  Proteins conjugated by Lys-48-linked polyubiquitin chains are preferred substrates of the eukaryotic proteasome. Polyubiquitination requires an activating enzyme (E1), a conjugating enzyme (E2), and a ligase (E3). Occasionally, these enzymes only assemble short ubiquitin oligomers, and their extension to full length involves a ubiquitin elongating factor termed E4. Ufd2p, as the first E4 identified to date, is involved in the degradation of misfolded proteins of the endoplasmic reticulum and of a ubiquitin-beta-GAL fusion substrate in Saccharomyces cerevisiae. The mechanism of action of Ufd2p is unknown. Here we describe the crystal structure of the full-length yeast Ufd2p protein. Ufd2p has an elongated shape consisting of several irregular Armadillo-like repeats with two helical hairpins protruding from it and a U-box domain flexibly attached to its C terminus. The U-box of Ufd2p has a fold similar to that of the RING (Really Interesting New Gene) domain that is present in certain ubiquitin ligases. Accordingly, Ufd2p has all of the hallmarks of a RING finger-containing ubiquitin ligase: it associates with its cognate E2 Ubc4p via its U-box domain and catalyzes the transfer of ubiquitin from the E2 active site to Ufd2p itself or to an acceptor ubiquitin molecule to form unanchored diubiquitin oligomers. Thus, Ufd2p can function as a bona fide E3 ubiquitin ligase to promote ubiquitin chain elongation on a substrate.

  View details for DOI 10.1073/pnas.0701369104

  View details for Web of Science ID 000249942700004

  View details for PubMedID 17890322

  View details for PubMedCentralID PMC2000413

• Structural and biochemical studies of botulinum neurotoxin serotype C1 light chain protease: Implications for dual substrate specificity BIOCHEMISTRY Jin, R., Sikorra, S., Stegmann, C. M., Pich, A., Binz, T., Brunger, A. T. 2007; 46 (37): 10685-10693

  Abstract

  Clostridial neurotoxins are the causative agents of the neuroparalytic disease botulism and tetanus. They block neurotransmitter release through specific proteolysis of one of the three soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs) SNAP-25, syntaxin, and synaptobrevin, which constitute part of the synaptic vesicle fusion machinery. The catalytic component of the clostridial neurotoxins is their light chain (LC), a Zn2+ endopeptidase. There are seven structurally and functionally related botulinum neurotoxins (BoNTs), termed serotype A to G, and tetanus neurotoxin (TeNT). Each of them exhibits unique specificity for their target SNAREs and peptide bond(s) they cleave. The mechanisms of action for substrate recognition and target cleavage are largely unknown. Here, we report structural and biochemical studies of BoNT/C1-LC, which is unique among BoNTs in that it exhibits dual specificity toward both syntaxin and SNAP-25. A distinct pocket (S1') near the active site likely achieves the correct register for the cleavage site by only allowing Ala as the P1' residue for both SNAP-25 and syntaxin. Mutations of this SNAP-25 residue dramatically reduce enzymatic activity. The remote alpha-exosite that was previously identified in the complex of BoNT/A-LC and SNAP-25 is structurally conserved in BoNT/C1. However, mutagenesis experiments show that the alpha-exosite of BoNT/C1 plays a less stringent role in substrate discrimination in comparison to that of BoNT/A, which could account for its dual substrate specificity.

  View details for DOI 10.1021/bi701162d

  View details for Web of Science ID 000249433100028

  View details for PubMedID 17718519

• Botulinum neurotoxin heavy chain belt as an intramolecular chaperone for the light chain PLOS PATHOGENS Brunger, A. T., Breidenbach, M. A., Jin, R., Fischer, A., Santos, J. S., Montal, M. 2007; 3 (9): 1191-1194

  View details for DOI 10.1371/journal.ppat.0020113.g001

  View details for Web of Science ID 000249768300002

  View details for PubMedID 17907800

  View details for PubMedCentralID PMC1994969

• Crystallography - Crystallographic evidence for deviating C3b structure? NATURE Janssen, B. C., Read, R. J., Bruenger, A. T., Gros, P. 2007; 448 (7154): E1-E2

  Abstract

  Activation of the protein C3 into C3b in the complement pathway is a crucial step in the complement immune response against pathogenic, immunogenic and apoptotic particles. Ajees et al. describe a crystal structure for C3b that deviates from the one reported by Janssen et al. and by Wiesmann et al.. We have reanalysed the data deposited by Ajees et al. and have discovered features that are inconsistent with the known physical properties of macromolecular structures and their diffraction data. Our findings therefore call into question the crystal structure for C3b reported by Ajees et al..

  View details for DOI 10.1038/nature06102

  View details for Web of Science ID 000248598000035

  View details for PubMedID 17687277

  View details for PubMedCentralID PMC2504710

• Crystal structure of a hyperactive Escherichia coli glycerol kinase mutant Gly230 -> Asp obtained using microfluidic crystallization devices BIOCHEMISTRY Anderson, M. J., DeLaBarre, B., Raghunathan, A., Palsson, B. O., Brunger, A. T., Quake, S. R. 2007; 46 (19): 5722-5731

  Abstract

  The crystal structure of an Escherichia coli glycerol kinase mutant Gly230 --> Asp (GKG230D) was determined to 2.0 A resolution using a microfluidics based crystallization platform. The crystallization strategy involved a suite of microfluidic devices that characterized the solubility trends of GKG230D, performed nanoliter volume free interface diffusion crystallization experiments, and produced diffraction-quality crystals for in situ data collection. GKG230D displays increased enzymatic activity and decreased allosteric regulation by the glycolytic pathway intermediate fructose 1,6-bisphosphate (FBP) compared to wild-type GK (GKWT). Structural analysis revealed that the decreased allosteric regulation is a result of the altered FBP binding loop conformations in GKG230D that interfere with the wild-type FBP binding site. The altered FBP binding loop conformations in GKG230D are supported through a series of intramolecular loop interactions. The appearance of Asp230 in the FBP binding loops also repositions the wild-type FBP binding residues away from the FBP binding site. Light scattering analysis confirmed GKG230D is a dimer and is resistant to tetramer formation in the presence of FBP, whereas GKWT dimers are converted into putatively inactive tetramers in the presence of FBP. GKG230D also provides the first structural evidence for multiple GK monomer conformations in the presence of glycerol and in the absence of a nucleotide substrate and verifies that glycerol binding is not responsible for locking GK into the closed conformation necessary for GK activity.

  View details for DOI 10.1021/bi700096p

  View details for Web of Science ID 000246283600010

  View details for PubMedID 17441732

• A ubiquitin ligase transfers preformed polyubiquitin chains from a conjugating enzyme to a substrate NATURE Li, W., Tu, D., Brunger, A. T., Ye, Y. 2007; 446 (7133): 333-337

  Abstract

  In eukaryotic cells, many short-lived proteins are conjugated with Lys 48-linked ubiquitin chains and degraded by the proteasome. Ubiquitination requires an activating enzyme (E1), a conjugating enzyme (E2) and a ligase (E3). Most ubiquitin ligases use either a HECT (homologous to E6-associated protein C terminus) or a RING (really interesting new gene) domain to catalyse polyubiquitination, but the mechanism of E3 catalysis is poorly defined. Here we dissect this process using mouse Ube2g2 (E2; identical at the amino acid level to human Ube2g2) and human gp78 (E3), an endoplasmic reticulum (ER)-associated conjugating system essential for the degradation of misfolded ER proteins. We demonstrate by expressing recombinant proteins in Escherichia coli that Ube2g2/gp78-mediated polyubiquitination involves preassembly of Lys 48-linked ubiquitin chains at the catalytic cysteine of Ube2g2. The growth of Ube2g2-anchored ubiquitin chains seems to be mediated by an aminolysis-based transfer reaction between two Ube2g2 molecules that each carries a ubiquitin moiety in its active site. Intriguingly, polyubiquitination of a substrate can be achieved by transferring preassembled ubiquitin chains from Ube2g2 to a lysine residue in a substrate.

  View details for DOI 10.1038/nature05542

  View details for Web of Science ID 000244892900049

  View details for PubMedID 17310145

• Inhibition of metalloprotease botulinum serotype A from a pseudo-peptide binding mode to a small molecule that is active in primary neurons JOURNAL OF BIOLOGICAL CHEMISTRY Burnett, J. C., Ruthel, G., Stegmann, C. M., Panchal, R. G., Nguyen, T. L., Hermone, A. R., Stafford, R. G., Lane, D. J., Kenny, T. A., McGrath, C. F., Wipf, P., Stahl, A. M., Schmidt, J. J., Gussio, R., Brunger, A. T., Bavari, S. 2007; 282 (7): 5004-5014

  Abstract

  An efficient research strategy integrating empirically guided, structure-based modeling and chemoinformatics was used to discover potent small molecule inhibitors of the botulinum neurotoxin serotype A light chain. First, a modeled binding mode for inhibitor 2-mercapto-3-phenylpropionyl-RATKML (K(i) = 330 nM) was generated, and required the use of a molecular dynamic conformer of the enzyme displaying the reorientation of surface loops bordering the substrate binding cleft. These flexible loops are conformationally variable in x-ray crystal structures, and the model predicted that they were pivotal for providing complementary binding surfaces and solvent shielding for the pseudo-peptide. The docked conformation of 2-mercapto-3-phenylpropionyl-RATKML was then used to refine our pharmacophore for botulinum serotype A light chain inhibition. Data base search queries derived from the pharmacophore were employed to mine small molecule (non-peptidic) inhibitors from the National Cancer Institute's Open Repository. Four of the inhibitors possess K(i) values ranging from 3.0 to 10.0 microM. Of these, NSC 240898 is a promising lead for therapeutic development, as it readily enters neurons, exhibits no neuronal toxicity, and elicits dose-dependent protection of synaptosomal-associated protein (of 25 kDa) in a primary culture of embryonic chicken neurons. Isothermal titration calorimetry showed that the interaction between NSC 240898 and the botulinum A light chain is largely entropy-driven, and occurs with a 1:1 stoichiometry and a dissociation constant of 4.6 microM.

  View details for DOI 10.1074/jbc.M608166200

  View details for Web of Science ID 000244482000083

  View details for PubMedID 17092934

• Ab initio molecular-replacement phasing for symmetric helical membrane proteins ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY Strop, P., Brzustowicz, M. R., Brunger, A. T. 2007; 63: 188-196

  Abstract

  Obtaining phases for X-ray diffraction data can be a rate-limiting step in structure determination. Taking advantage of constraints specific to membrane proteins, an ab initio molecular-replacement method has been developed for phasing X-ray diffraction data for symmetric helical membrane proteins without prior knowledge of their structure or heavy-atom derivatives. The described method is based on generating all possible orientations of idealized transmembrane helices and using each model in a molecular-replacement search. The number of models is significantly reduced by taking advantage of geometrical and structural restraints specific to membrane proteins. The top molecular-replacement results are evaluated based on noncrystallographic symmetry (NCS) map correlation, OMIT map correlation and R(free) value after refinement of a polyalanine model. The feasibility of this approach is illustrated by phasing the mechanosensitive channel of large conductance (MscL) with only 4 A diffraction data. No prior structural knowledge was used other than the number of transmembrane helices. The search produced the correct spatial organization and the position in the asymmetric unit of all transmembrane helices of MscL. The resulting electron-density maps were of sufficient quality to automatically build all helical segments of MscL including the cytoplasmic domain. The method does not require high-resolution diffraction data and can be used to obtain phases for symmetrical helical membrane proteins with one or two helices per monomer.

  View details for DOI 10.1107/S0907444906045793

  View details for Web of Science ID 000243495700008

  View details for PubMedID 17242512

  View details for PubMedCentralID PMC2483470

• Single molecule studies of SNARE-dependent fusion 51st Annual Meeting of the Biophysical-Society Brunger, A., Chu, S., Bowen, M., Weninger, K., Vrljic, M. CELL PRESS. 2007: 375A–375A

  View details for Web of Science ID 000243972402305

• Version 1.2 of the Crystallography and NMR system NATURE PROTOCOLS Brunger, A. T. 2007; 2 (11): 2728-2733

  Abstract

  Version 1.2 of the software system, termed Crystallography and NMR system (CNS), for crystallographic and NMR structure determination has been released. Since its first release, the goals of CNS have been (i) to create a flexible computational framework for exploration of new approaches to structure determination, (ii) to provide tools for structure solution of difficult or large structures, (iii) to develop models for analyzing structural and dynamical properties of macromolecules and (iv) to integrate all sources of information into all stages of the structure determination process. Version 1.2 includes an improved model for the treatment of disordered solvent for crystallographic refinement that employs a combined grid search and least-squares optimization of the bulk solvent model parameters. The method is more robust than previous implementations, especially at lower resolution, generally resulting in lower R values. Other advances include the ability to apply thermal factor sharpening to electron density maps. Consistent with the modular design of CNS, these additions and changes were implemented in the high-level computing language of CNS.

  View details for DOI 10.1038/nprot.2007.406

  View details for Web of Science ID 000253140000009

  View details for PubMedID 18007608

• Botulinum neurotoxin B recognizes its protein receptor with high affinity and specificity NATURE Jin, R., Rummel, A., Binz, T., Brunger, A. T. 2006; 444 (7122): 1092-1095

  Abstract

  Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum and cause the neuroparalytic syndrome of botulism. With a lethal dose of 1 ng kg(-1), they pose a biological hazard to humans and a serious potential bioweapon threat. BoNTs bind with high specificity at neuromuscular junctions and they impair exocytosis of synaptic vesicles containing acetylcholine through specific proteolysis of SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors), which constitute part of the synaptic vesicle fusion machinery. The molecular details of the toxin-cell recognition have been elusive. Here we report the structure of a BoNT in complex with its protein receptor: the receptor-binding domain of botulinum neurotoxin serotype B (BoNT/B) bound to the luminal domain of synaptotagmin II, determined at 2.15 A resolution. On binding, a helix is induced in the luminal domain which binds to a saddle-shaped crevice on a distal tip of BoNT/B. This crevice is adjacent to the non-overlapping ganglioside-binding site of BoNT/B. Synaptotagmin II interacts with BoNT/B with nanomolar affinity, at both neutral and acidic endosomal pH. Biochemical and neuronal ex vivo studies of structure-based mutations indicate high specificity and affinity of the interaction, and high selectivity of BoNT/B among synaptotagmin I and II isoforms. Synergistic binding of both synaptotagmin and ganglioside imposes geometric restrictions on the initiation of BoNT/B translocation after endocytosis. Our results provide the basis for the rational development of preventive vaccines or inhibitors against these neurotoxins.

  View details for DOI 10.1038/nature05387

  View details for Web of Science ID 000242971100061

  View details for PubMedID 17167421

• Structural and functional comparisons of nucleotide pyrophosphatase/phosphodiesterase and alkaline phosphatase: Implications for mechanism and evolution BIOCHEMISTRY Zalatan, J. G., Fenn, T. D., Brunger, A. T., Herschlag, D. 2006; 45 (32): 9788-9803

  Abstract

  The rapid expansion of the amount of genomic and structural data has provided many examples of enzymes with evolutionarily related active sites that catalyze different reactions. Functional comparisons of these active sites can provide insight into the origins of the enormous catalytic proficiency of enzymes and the evolutionary changes that can lead to different enzyme activities. The alkaline phosphatase (AP) superfamily is an ideal system to use in making such comparisons given the extensive data available on both nonenzymatic and enzymatic phosphoryl transfer reactions. Some superfamily members, such as AP itself, preferentially hydrolyze phosphate monoesters, whereas others, such as nucleotide pyrophosphatase/phosphodiesterase (NPP), preferentially hydrolyze phosphate diesters. We have measured rate constants for NPP-catalyzed hydrolysis of phosphate diesters and monoesters. NPP preferentially catalyzes diester hydrolysis by factors of 10(2)-10(6), depending on the identity of the diester substrate. To identify features of the NPP active site that could lead to preferential phosphate diester hydrolysis, we have determined the structure of NPP in the absence of ligands and in complexes with vanadate and AMP. Comparisons to existing structures of AP reveal bimetallo cores that are structurally indistinguishable, but there are several distinct structural features outside of the conserved bimetallo site. The structural and functional data together suggest that some of these distinct functional groups provide specific substrate binding interactions, whereas others tune the properties of the bimetallo active site itself to discriminate between phosphate diester and monoester substrates.

  View details for DOI 10.1021/bi060847t

  View details for Web of Science ID 000239596600016

  View details for PubMedID 16893180

• Ensemble molecular dynamics yields submillisecond kinetics and intermediates of membrane fusion PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kasson, P. M., Kelley, N. W., Singhal, N., Vrljic, M., Brunger, A. T., Pande, V. S. 2006; 103 (32): 11916-11921

  Abstract

  Lipid membrane fusion is critical to cellular transport and signaling processes such as constitutive secretion, neurotransmitter release, and infection by enveloped viruses. Here, we introduce a powerful computational methodology for simulating membrane fusion from a starting configuration designed to approximate activated prefusion assemblies from neuronal and viral fusion, producing results on a time scale and degree of mechanistic detail not previously possible to our knowledge. We use an approach to the long time scale simulation of fusion by constructing a Markovian state model with large-scale distributed computing, yielding an understanding of fusion mechanisms on time scales previously impossible to simulate to our knowledge. Our simulation data suggest a branched pathway for fusion, in which a common stalk-like intermediate can either rapidly form a fusion pore or remain in a metastable hemifused state that slowly forms fully fused vesicles. This branched reaction pathway provides a mechanistic explanation both for the biphasic fusion kinetics and the stable hemifused intermediates previously observed experimentally. Our distributed computing and Markovian state model approaches provide sufficient sampling to detect rare transitions, a systematic process for analyzing reaction pathways, and the ability to develop quantitative approximations of reaction kinetics for fusion.

  View details for DOI 10.1073/pnas.0601597103

  View details for Web of Science ID 000239701900019

  View details for PubMedID 16880392

  View details for PubMedCentralID PMC1567673

• Considerations for the refinement of low-resolution crystal structures ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY DeLaBarre, B., Brunger, A. T. 2006; 62: 923-932

  Abstract

  It is often assumed that crystal structures have to be obtained at sufficiently high resolution in order to perform macromolecular refinement. In several recent structures, the threshold of what is considered ;acceptable' has been pushed to lower diffraction resolutions. Here, considerations and modifications to standard refinement protocols are described that were used to solve and refine a particular set of low-resolution structures for the ATPase p97/VCP. It was found that reasonable R(free) values and good geometry can be achieved upon refinement that includes experimental phase information along with judicious use of restraints at diffraction limits as low as 4.7 A. At this resolution, the topology and the backbone-chain trace are mostly defined, some side-chain positions can be unambiguously assigned and ligands within known binding sites can be identified. Furthermore, large conformational changes can be discerned when structures in different states are available, information that is not easily obtainable by other means.

  View details for DOI 10.1107/S0907444906012650

  View details for Web of Science ID 000239119800012

  View details for PubMedID 16855310

• Conformation of the synaptobrevin transmembrane domain PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bowen, M., Brunger, A. T. 2006; 103 (22): 8378-8383

  Abstract

  The synaptic vesicle protein synaptobrevin (also called VAMP, vesicle-associated membrane protein) forms part of the SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) complex, which is essential for vesicle fusion. Additionally, the synaptobrevin transmembrane domain can promote lipid mixing independently of complex formation. Here, the conformation of the transmembrane domain was studied by using circular dichroism and attenuated total reflection Fourier-transform infrared spectroscopy. The synaptobrevin transmembrane domain has an alpha-helical structure that breaks in the juxtamembrane region, leaving the cytoplasmic domain unstructured. In phospholipid bilayers, infrared dichroism data indicate that the transmembrane domain adopts a 36 degrees angle with respect to the membrane normal, similar to that reported for viral fusion peptides. A conserved aromatic/basic motif in the juxtamembrane region may be causing this relatively high insertion angle.

  View details for DOI 10.1073/pnas.0602644103

  View details for Web of Science ID 000238206800016

  View details for PubMedID 16709671

  View details for PubMedCentralID PMC1463247

• Central pore residues mediate the p97/VCP activity required for ERAD MOLECULAR CELL DeLaBarre, B., Christianson, J. C., Kopito, R. R., Brunger, A. T. 2006; 22 (4): 451-462

  Abstract

  The AAA-ATPase p97/VCP facilitates protein dislocation during endoplasmic reticulum-associated degradation (ERAD). To understand how p97/VCP accomplishes dislocation, a series of point mutants was made to disrupt distinguishing structural features of its central pore. Mutants were evaluated in vitro for ATPase activity in the presence and absence of synaptotagmin I (SytI) and in vivo for ability to process the ERAD substrate TCRalpha. Synaptotagmin induces a 4-fold increase in the ATPase activity of wild-type p97/VCP (p97/VCP(wt)), but not in mutants that showed an ERAD impairment. Mass spectrometry of crosslinked synaptotagmin . p97/VCP revealed interactions near Trp551 and Phe552. Additionally, His317, Arg586, and Arg599 were found to be essential for substrate interaction and ERAD. Except His317, which serves as an interaction nexus, these residues all lie on prominent loops within the D2 pore. These data support a model of substrate dislocation facilitated by interactions with p97/VCP's D2 pore.

  View details for DOI 10.1016/j.molcel.2006.03.036

  View details for Web of Science ID 000237813300007

  View details for PubMedID 16713576

• Towards an understanding of the molecular mechanism of neurotransmitter release Experimental Biology 2006 Annual Meeting Brunger, A. T. FEDERATION AMER SOC EXP BIOL. 2006: A1309–A1309

  View details for Web of Science ID 000236326204248

• The structure of nucleotide pyrophosphatase/phosphodiesterase and implications for enzyme evolution in the alkaline phosphatase superfamily Experimental Biology 2006 Annual Meeting Zalatan, J., Fenn, T., Brunger, A., Herschlag, D. FEDERATION AMER SOC EXP BIOL. 2006: A477–A477

  View details for Web of Science ID 000236206504055

• Neuronal SNAREs do not trigger fusion between synthetic membranes but do promote PEG-mediated membrane fusion BIOPHYSICAL JOURNAL Dennison, S. M., Bowen, M. E., Brunger, A. T., Lentz, B. R. 2006; 90 (5): 1661-1675

  Abstract

  At low surface concentrations that permit formation of impermeable membranes, neuronal soluble N-ethyl maleimide sensitive factor attachment protein receptor (SNARE) proteins form a stable, parallel, trans complex when vesicles are brought into contact by a low concentration of poly(ethylene glycol) (PEG). Surprisingly, formation of a stable SNARE complex does not trigger fusion under these conditions. However, neuronal SNAREs do promote fusion at low protein/lipid ratios when triggered by higher concentrations of PEG. Promotion of PEG-triggered fusion required phosphatidylserine and depended only on the surface concentration of SNAREs and not on the formation of a trans SNARE complex. These results were obtained at protein surface concentrations reported for synaptobrevin in synaptic vesicles and with an optimally fusogenic lipid composition. At a much higher protein/lipid ratio, vesicles joined by SNARE complex slowly mixed lipids at 37 degrees C in the absence of PEG, in agreement with earlier reports. However, vesicles containing syntaxin at a high protein/lipid ratio (>or=1:250) lost membrane integrity. We conclude that the neuronal SNARE complex promotes fusion by joining membranes and that the individual proteins syntaxin and synaptobrevin disrupt membranes so as to favor formation of a stalk complex and to promote conversion of the stalk to a fusion pore. These effects are similar to the effects of viral fusion peptides and transmembrane domains, but they are not sufficient by themselves to produce fusion in our in vitro system at surface concentrations documented to occur in synaptic vesicles. Thus, it is likely that proteins or factors other than the SNARE complex must trigger fusion in vivo.

  View details for DOI 10.1529/biophysj.105.069617

  View details for Web of Science ID 000235235600020

  View details for PubMedID 16339880

  View details for PubMedCentralID PMC1367317

• Automated crystallographic ligand building using the medial axis transform of an electron-density isosurface ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY Aishima, J., Russel, D. S., Guibas, L. J., Adams, P. D., Brunger, A. T. 2005; 61: 1354-1363

  Abstract

  Automatic fitting methods that build molecules into electron-density maps usually fail below 3.5 A resolution. As a first step towards addressing this problem, an algorithm has been developed using an approximation of the medial axis to simplify an electron-density isosurface. This approximation captures the central axis of the isosurface with a graph which is then matched against a graph of the molecular model. One of the first applications of the medial axis to X-ray crystallography is presented here. When applied to ligand fitting, the method performs at least as well as methods based on selecting peaks in electron-density maps. Generalization of the method to recognition of common features across multiple contour levels could lead to powerful automatic fitting methods that perform well even at low resolution.

  View details for DOI 10.1107/S0907444905023152

  View details for Web of Science ID 000232353500006

  View details for PubMedID 16204887

• A hybrid machine-learning approach for segmentation of protein localization data BIOINFORMATICS Kasson, P. M., Huppa, J. B., DAVIS, M. M., Brunger, A. T. 2005; 21 (19): 3778-3786

  Abstract

  Subcellular protein localization data are critical to the quantitative understanding of cellular function and regulation. Such data are acquired via observation and quantitative analysis of fluorescently labeled proteins in living cells. Differentiation of labeled protein from cellular artifacts remains an obstacle to accurate quantification. We have developed a novel hybrid machine-learning-based method to differentiate signal from artifact in membrane protein localization data by deriving positional information via surface fitting and combining this with fluorescence-intensity-based data to generate input for a support vector machine.We have employed this classifier to analyze signaling protein localization in T-cell activation. Our classifier displayed increased performance over previously available techniques, exhibiting both flexibility and adaptability: training on heterogeneous data yielded a general classifier with good overall performance; training on more specific data yielded an extremely high-performance specific classifier. We also demonstrate accurate automated learning utilizing additional experimental data.

  View details for DOI 10.1093/bioinformatics/bti615

  View details for Web of Science ID 000232596100012

  View details for PubMedID 16091410

• SNARE complex between reconstituted vesicles does not trigger but does promote PEG-triggered fusion 230th National Meeting of the American-Chemical-Society Lentz, B., Dennison, M., Bowen, M. E., Brunger, A. T. AMER CHEMICAL SOC. 2005: U1075–U1075

  View details for Web of Science ID 000236797302158

• Single-molecule studies of synaptotagmin and complexin binding to the SNARE complex BIOPHYSICAL JOURNAL Bowen, M. E., Weninger, K., Ernst, J., Chu, S., Brunger, A. T. 2005; 89 (1): 690-702

  Abstract

  The assembly of multiprotein complexes at the membrane interface governs many signaling processes in cells. However, very few methods exist for obtaining biophysical information about protein complex formation at the membrane. We used single molecule fluorescence resonance energy transfer to study complexin and synaptotagmin interactions with the SNARE complex in deposited lipid bilayers. Using total internal reflectance microscopy, individual binding events at the membrane could be resolved despite an excess of unbound protein in solution. Fluorescence resonance energy transfer (FRET)-efficiency derived distances for the complexin-SNARE interaction were consistent with the crystal structure of the complexin-SNARE complex. The unstructured N-terminal region of complexin showed broad distributions of FRET efficiencies to the SNARE complex, suggesting that information on conformational variability can be obtained from FRET efficiency distributions. The low-affinity interaction of synaptotagmin with the SNARE complex changed dramatically upon addition of Ca2+ with high FRET efficiency interactions appearing between the C2B domain and linker domains of synaptotagmin and the membrane proximal portion of the SNARE complex. These results demonstrate that single molecule FRET can be used as a "spectroscopic ruler" to simultaneously gain structural and kinetic information about transient multiprotein complexes at the membrane interface.

  View details for DOI 10.1529/biophysj.104.054064

  View details for Web of Science ID 000230114500067

  View details for PubMedID 15821166

  View details for PubMedCentralID PMC1366567

• X-ray scattering from unilamellar lipid vesicles JOURNAL OF APPLIED CRYSTALLOGRAPHY Brzustowicz, M. R., Brunger, A. T. 2005; 38: 126-131

  View details for DOI 10.1107/S0021889804029206

  View details for Web of Science ID 000226414500014

• Structure and function of SNARE and SNARE-interacting proteins QUARTERLY REVIEWS OF BIOPHYSICS Brunger, A. T. 2005; 38 (1): 1-47

  Abstract

  This review focuses on the so-called SNARE (soluble N-ethyl maleimide sensitive factor attachment protein receptor) proteins that are involved in exocytosis at the pre-synpatic plasma membrane. SNAREs play a role in docking and fusion of synaptic vesicles to the active zone, as well as in the Ca2+-triggering step itself, most likely in combination with the Ca2+ sensor synaptotagmin. Different SNARE domains are involved in different processes, such as regulation, docking, and fusion. SNAREs exhibit multiple configurational, conformational, and oliogomeric states. These different states allow SNAREs to interact with their matching SNARE partners, auxiliary proteins, or with other SNARE domains, often in a mutually exclusive fashion. SNARE core domains undergo progressive disorder to order transitions upon interactions with other proteins, culminating with the fully folded post-fusion (cis) SNARE complex. Physiological concentrations of neuronal SNAREs can juxtapose membranes, and promote fusion in vitro under certain conditions. However, significantly more work will be required to reconstitute an in vitro system that faithfully mimics the Ca2+-triggered fusion of a synaptic vesicle at the active zone.

  View details for DOI 10.1017/S0033583505004051

  View details for Web of Science ID 000236307800001

  View details for PubMedID 16336742

• Structure of a human A-type potassium channel accelerating factor DPPX, a member of the dipeptidyl aminopeptidase family 49th Annual Meeting of the Biophysical-Society Strop, P., Bankovich, A. J., Hansen, K. C., Garcia, K. C., Brunger, A. T. CELL PRESS. 2005: 456A–456A

  View details for Web of Science ID 000226378502221

• X-ray scattering from unilamellar lipid vesicles 49th Annual Meeting of the Biophysical-Society Brzustowicz, M. R., Brunger, A. T. CELL PRESS. 2005: 17A–17A

  View details for Web of Science ID 000226378500074

• Deformable modeling for improved calculation of molecular velocities from single-particle tracking IEEE Computational Systems Bioinformatics Conference Kasson, P., Davis, M. M., Brunger, A. T. IEEE COMPUTER SOC. 2005: 208–211

  Abstract

  Single-particle tracking provides a powerful technique for measuring dynamic cellular processes on the level of individual molecules. Much recent work has been devoted to using single particle tracking to measure long-range movement of particles on the cell surface, including methods for automated localization and tracking of particles [1-3]. However, most particle tracking studies to date ignore cell surface curvature and dynamic cellular deformation, factors frequently present in physiologically relevant situations. In this report, we perform quantitative evaluation of single-particle tracking on curved and deforming cell surfaces. We also introduce a new hybrid method that uses non-rigid cellular modeling for improved computation of single-particle tracking trajectories on the surfaces of cells undergoing deformation. This method combines single-molecule and bulk fluorescence measurements in an automated manner to enable more accurate and robust characterization of dynamic cell physiology and regulation.

  View details for Web of Science ID 000231800100025

  View details for PubMedID 16447978

• Observation of single liposome-bilayer fusion induced by SNARE proteins Meeting of the Division of Chemical Toxicology of the American-Chemical-Society held at the 228th National Meeting of the American-Chemical-Society Weninger, K. R., Bowen, M. E., Brunger, A. T., Chu, S. AMER CHEMICAL SOC. 2004: U207–U207

  View details for Web of Science ID 000223713801003

• Effect of SNARE complex on PEG-mediated membrane fusion 48th Annual Meeting of the Biophysical-Society Dennison, S. M., Bowen, M., Brunger, A., Lentz, B. R. CELL PRESS. 2004: 518A–518A

  View details for Web of Science ID 000187971202673

• Quantitative analysis of membrane protein localization and signalling IEEE Computational Systems Bioinformatics Conference (CSB 2004) Kasson, P. M., Huppa, J. B., DAVIS, M. M., Brunger, A. T. IEEE COMPUTER SOC. 2004: 540–541

  View details for Web of Science ID 000224127800077

• Structure of N-ethyl maleimide sensitive factor by electron cryo-microscopy at 11 angstrom resolution 48th Annual Meeting of the Biophysical-Society Furst, J., Sutton, R. B., Chen, J., Brunger, A. T., Grigorieff, N. CELL PRESS. 2004: 78A–78A

  View details for Web of Science ID 000187971200405

• Quantitative analysis of lymphocyte membrane protein redistribution from fluorescence microscopy International Conference on Image Processing (ICIP 2004) Kasson, P. M., Huppa, J. B., DAVIS, M. M., Brunger, A. T. IEEE. 2004: 2933–2936

  View details for Web of Science ID 000228043504023

• Single molecule studies of SNARE effector binding 48th Annual Meeting of the Biophysical-Society Bowen, M. E., Chu, S., Weninger, K., Brunger, A. T. CELL PRESS. 2004: 237A–238A

  View details for Web of Science ID 000187971201222

• 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

• SNARE proteins alter poly(ethylene glycol) (PEG)-mediated fusion of model membranes 47th Annual Meeting of the Biophysical-Society Dennison, S. M., Bowen, M., Brunger, A., Lentz, B. CELL PRESS. 2003: 195A–195A

  View details for Web of Science ID 000183123800956

• Conformational changes of the multifunction VCP/p97 AAA ATPase during the ATPase cycle 42nd Annual Meeting of the American-Society-for-Cell-Biology Rouiller, I., DeLaBarre, B., May, A. P., Weiss, W. I., Brunger, A. T., Milligan, R. A., Wilson-Kubalek, E. M. AMER SOC CELL BIOLOGY. 2002: 263A–263A

  View details for Web of Science ID 000179569101479

• Macromolecular assemblages machines and networks - Editorial overview CURRENT OPINION IN STRUCTURAL BIOLOGY Saibil, H. R., Brunger, A. T. 2002; 12 (2): 215-216

  View details for Web of Science ID 000175043000011

• Mutational analysis of synaptobrevin transmembrane domain oligomerization Bowen, M. E., Engelman, D. M., Brunger, A. T. AMER SOC CELL BIOLOGY. 2001: 473A–473A

  View details for Web of Science ID 000172372502599

• Biochemical studies of calcium dependent exocytosis: interactions of synaptotagmin with the SNARE complex Ernst, J. A., Brunger, A. T. AMER SOC CELL BIOLOGY. 2001: 473A

  View details for Web of Science ID 000172372502601

• The 1.0 angstrom crystal structure of Ca+2-bound calmodulin: An analysis of disorder and implications for functionally relevant plasticity Wilson, M. A., Brunger, A. T. CELL PRESS. 2001: 398A–398A

  View details for Web of Science ID 000166692201813

• Biophysical methods - New approaches to study macromolecular structure and function - Editorial overview CURRENT OPINION IN STRUCTURAL BIOLOGY Brunger, A. T., Laue, E. D. 2000; 10 (5): 557-557

  View details for Web of Science ID 000089983100008

• Algorithmic challenges in computational molecular biophysics JOURNAL OF COMPUTATIONAL PHYSICS Schlick, T., Skeel, R. D., Brunger, A. T., Kale, L. V., Board, J. A., Hermans, J., Schulten, K. 1999; 151 (1): 9-48

  View details for Web of Science ID 000080181500002

• Phase improvement by multi-start simulated annealing refinement and structure-factor averaging JOURNAL OF APPLIED CRYSTALLOGRAPHY Rice, L. M., Shamoo, Y., Brunger, A. T. 1998; 31: 798-805

  View details for Web of Science ID 000077183100019

• New applications of simulated annealing in crystallographic refinement NATO Advanced Study Institute on Direct Methods for Solving Macromolecular Structures Brunger, A. T., Adams, P. D., Rice, L. M. SPRINGER. 1998: 143–157

  View details for Web of Science ID 000074740800014

• Site-directed dichroism as a method for obtaining rotational and orientational constraints for oriented polymers JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Arkin, I. T., MacKenzie, K. R., Brunger, A. T. 1997; 119 (38): 8973-8980

  View details for Web of Science ID A1997XX82000025

• Crystallographic refinement by simulated annealing: Methods and applications MACROMOLECULAR CRYSTALLOGRAPHY, PT B Brunger, A. T., Rice, L. M. 1997; 277: 243-269

  View details for Web of Science ID A1997BJ57S00013

  View details for PubMedID 18488313

• Free R value: Cross-validation in crystallography MACROMOLECULAR CRYSTALLOGRAPHY, PT B Brunger, A. T., Carter, C. W., Sweet, R. M. 1997; 277: 366-396

  View details for DOI 10.1016/S0076-6879(97)77021-6

  View details for Web of Science ID A1997BJ57S00019

  View details for PubMedID 18488318

• REFLNEMEENTS OF HEAVY-ATOM AND PROTEIN PARAMETERS AGAINST A HIGHLY ACCURATE MAD DATA SET. Brunger, A. T., Burling, F., Flaherty, K. M., Weis, W. I. INT UNION CRYSTALLOGRAPHY. 1996: C55

  View details for DOI 10.1107/S0108767396096833

  View details for Web of Science ID 000410481900216

• ACCURATE HIGH-RESOLUTION PHASES FROM MAD ANALYSIS: EXPERIMENT AND COMPARISON OF PHASING METHODS. Weis, W. I., Flaherty, K. M., Burling, F., Brunger, A. T. INT UNION CRYSTALLOGRAPHY. 1996: C17

  View details for DOI 10.1107/S0108767396098339

  View details for Web of Science ID 000410481900067

• STRUCTURAL MODEL OF THE PHOSPHOLAMBAN ION-CHANNEL IN MEMBRANES 1995 Miami Bio/Technology Winter Symposium - Advances in Gene Technology: Protein Engineering and Structural Biology Arkin, I. T., Adams, P. D., LUDLAM, C. F., Aimoto, S., Rothschild, K. J., Brunger, A. T., Engelman, D. M., Smith, S. O. OXFORD UNIV PRESS. 1995: 44–44

  View details for Web of Science ID A1995RL74200068

• DIMERIZATION OF GLYCOPHORIN-A TRANSMEMBRANE HELICES - MUTAGENESIS AND MODELING ENGELMAN, D. M., ADAIR, B. D., BRUNGER, A., FLANAGAN, J. M., HUNT, J. F., LEMMON, M. A., TREUTLEIN, H., ZHANG, J., Reuss, L., Russell, J. M., Jennings, M. L. ROCKEFELLER UNIV PRESS. 1993: 11-21

  View details for Web of Science ID A1993BY17M00002

  View details for PubMedID 8503039

• FREE R-VALUE - A NOVEL STATISTICAL QUANTITY FOR ASSESSING THE ACCURACY OF CRYSTAL-STRUCTURES NATURE BRUNGER, A. T. 1992; 355 (6359): 472-475

  Abstract

  The determination of macromolecular structure by crystallography involves fitting atomic models to the observed diffraction data. The traditional measure of the quality of this fit, and presumably the accuracy of the model, is the R value. Despite stereochemical restraints, it is possible to overfit or 'misfit' the diffraction data: an incorrect model can be refined to fairly good R values as several recent examples have shown. Here I propose a reliable and unbiased indicator of the accuracy of such models. By analogy with the cross-validation method of testing statistical models I define a statistical quantity (R(free) (T) that measures the agreement between observed and computed structure factor amplitudes for a 'test' set of reflections that is omitted in the modelling and refinement process. As examples show, there is a high correlation between R(free) (T) and the accuracy of the atomic model phases. This is useful because experimental phase information is usually inaccurate, incomplete or unavailable. I expect that R(free) (T) will provide a measure of the information content of recently proposed models of thermal motion and disorder, time-averaging and bulk solvent.

  View details for DOI 10.1038/355472a0

  View details for Web of Science ID A1992HB53000078

  View details for PubMedID 18481394

• MOLECULAR SWITCH FOR SIGNAL TRANSDUCTION - STRUCTURAL DIFFERENCES BETWEEN ACTIVE AND INACTIVE FORMS OF PROTOONCOGENIC RAS PROTEINS SCIENCE MILBURN, M. V., TONG, L., DEVOS, A. M., BRUNGER, A., YAMAIZUMI, Z., NISHIMURA, S., KIM, S. H. 1990; 247 (4945): 939-945

  Abstract

  Ras proteins participate as a molecular switch in the early steps of the signal transduction pathway that is associated with cell growth and differentiation. When the protein is in its GTP complexed form it is active in signal transduction, whereas it is inactive in its GDP complexed form. A comparison of eight three-dimensional structures of ras proteins in four different crystal lattices, five with a nonhydrolyzable GTP analog and three with GDP, reveals that the "on" and "off" states of the switch are distinguished by conformational differences that span a length of more than 40 A, and are induced by the gamma-phosphate. The most significant differences are localized in two regions: residues 30 to 38 (the switch I region) in the second loop and residues 60 to 76 (the switch II region) consisting of the fourth loop and the short alpha-helix that follows the loop. Both regions are highly exposed and form a continuous strip on the molecular surface most likely to be the recognition sites for the effector and receptor molecule(or molecules). The conformational differences also provide a structural basis for understanding the biological and biochemical changes of the proteins due to oncogenic mutations, autophosphorylation, and GTP hydrolysis, and for understanding the interactions with other proteins.

  View details for DOI 10.1126/science.2406906

  View details for Web of Science ID A1990CP90600033

  View details for PubMedID 2406906

• DETERMINATION OF THE BACKBONE CONFORMATION OF SECRETIN BY RESTRAINED MOLECULAR-DYNAMICS ON THE BASIS OF INTERPROTON DISTANCE DATA EUROPEAN JOURNAL OF BIOCHEMISTRY CLORE, G. M., NILGES, M., BRUNGER, A., GRONENBORN, A. M. 1988; 171 (3): 479-484

  Abstract

  The backbone conformation of the 27-residue polypeptide hormone secretin has been investigated using nuclear magnetic resonance spectroscopy and restrained molecular dynamics calculations under conditions where it adopts a fully ordered structure (40% v/v trifluoroethanol). The basis for the restrained molecular dynamics calculations consists of 52 nuclear-Overhauser-enhancement-derived interproton distance restraints involving the NH, C alpha H and C beta H protons. It is shown that convergence to similar extended structures is achieved starting from four different initial structures, namely an alpha helix, a mixed alpha/beta structure, a beta strand and a polyproline helix. The converged structures are made up of short N- and C-terminal strand-like regions and a central region comprising two irregular helices connected by a 'half-turn'.

  View details for DOI 10.1111/j.1432-1033.1988.tb13814.x

  View details for Web of Science ID A1988M118800008

  View details for PubMedID 2831051

• SOLUTION OF A PROTEIN CRYSTAL-STRUCTURE WITH A MODEL OBTAINED FROM NMR INTERPROTON DISTANCE RESTRAINTS SCIENCE BRUNGER, A. T., CAMPBELL, R. L., CLORE, G. M., GRONENBORN, A. M., KARPLUS, M., PETSKO, G. A., TEETER, M. M. 1987; 235 (4792): 1049-1053

  Abstract

  Model calculations were performed to test the possibility of solving crystal structures of proteins by Patterson search techniques with three-dimensional structures obtained from nuclear magnetic resonance (NMR) interproton distance restraints. Structures for crambin obtained from simulated NMR data were used as the test system; the root-mean-square deviations of the NMR structures from the x-ray structure were 1.5 to 2.2 A for backbone atoms and 2.0 to 2.8 A for side-chain atoms. Patterson searches were made to determine the orientation and position of the NMR structures in the unit cell. The correct solution was obtained by comparing the rotation function results of several of the NMR structures and the average structure derived from them. Conventional refinement techniques reduced the R factor from 0.43 at 4 A resolution to 0.27 at 2 A resolution without inclusion of water molecules. The partially refined structure has root-mean-square backbone and side-chain atom deviations from the x-ray structure of 0.5 and 1.3 A, respectively.

  View details for DOI 10.1126/science.235.4792.1049

  View details for Web of Science ID A1987G168200039

  View details for PubMedID 17782253

• CRYSTALLOGRAPHIC R-FACTOR REFINEMENT BY MOLECULAR-DYNAMICS SCIENCE BRUNGER, A. T., KURIYAN, J., KARPLUS, M. 1987; 235 (4787): 458-460

  Abstract

  Molecular dynamics was used to refine macromolecular structures by incorporating the difference between the observed crystallographic structure factor amplitude and that calculated from an assumed atomic model into the total energy of the system. The method has a radius of convergence that is larger than that of conventional restrained least-squares refinement. Test cases showed that the need for manual corrections during refinement of macromolecular crystal structures is reduced. In crambin, the dynamics calculation moved residues that were misplaced by more than 3 angstroms into the correct positions without human intervention.

  View details for DOI 10.1126/science.235.4787.458

  View details for Web of Science ID A1987F689100024

  View details for PubMedID 17810339

• THE 3-DIMENSIONAL STRUCTURE OF ALPHA-1-PUROTHIONIN IN SOLUTION - COMBINED USE OF NUCLEAR-MAGNETIC-RESONANCE, DISTANCE GEOMETRY AND RESTRAINED MOLECULAR-DYNAMICS EMBO JOURNAL CLORE, G. M., NILGES, M., SUKUMARAN, D. K., BRUNGER, A. T., KARPLUS, M., GRONENBORN, A. M. 1986; 5 (10): 2729-2735

  Abstract

  The determination of the three-dimensional solution structure of alpha1-purothionin using a combination of metric matrix distance geometry and restrained molecular dynamics calculations based on n.m.r. data is presented. The experimental data comprise complete sequence-specific proton resonance assignments, a set of 310 approximate interproton distance restraints derived from nuclear Overhauser effects, 27 Ø backbone torsion angle restraints derived from vicinal coupling constants, 4 distance restraints from hydrogen bonds and 12 distance restraints from disulphide bridges. The average atomic rms difference between the final nine converged structures and the mean structure obtained by averaging their coordinates is 1.5 +/- 0.1 å for the backbone atoms and 2.0 +/- 0.1 å for all atoms. The overall shape of alpha1-purothionin is that of the capital letter L, similar to that of crambin, with the longer arm comprising two approximately parallel alpha-helices and the shorter arm a strand and a mini anti-parallel beta sheet.

  View details for DOI 10.1002/j.1460-2075.1986.tb04557.x

  View details for Web of Science ID A1986E419000042

  View details for PubMedID 16453716

  View details for PubMedCentralID PMC1167175

• ACTIVE-SITE DYNAMICS IN PROTEIN MOLECULES - A STOCHASTIC BOUNDARY MOLECULAR-DYNAMICS APPROACH BIOPOLYMERS BROOKS, C. L., BRUNGER, A., KARPLUS, M. 1985; 24 (5): 843-865

  View details for DOI 10.1002/bip.360240509

  View details for Web of Science ID A1985AGF7200008

  View details for PubMedID 2410050

• CONTINUOUS FLUORESCENCE MICROPHOTOLYSIS - A SENSITIVE METHOD FOR STUDY OF DIFFUSION-PROCESSES IN SINGLE CELLS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES PETERS, R., BRUNGER, A., SCHULTEN, K. 1981; 78 (2): 962-966

  Abstract

  Continuous fluorescence microphotolysis is a sensitive method for the study of translational diffusion in the plasma membrane of single living cells and related systems. In this communication the conceptual basis of the method and its theoretical framework and experimental realization, as well as applications, are reported. In continuous fluorescence microphotolysis a microscopic membrane area of a single fluorescently labeled cell is irradiated by a laser beam while the fluorescence emitted from the area is monitored. The decay of the measuring signal reflects the competition of two processes: (i) the elimination of fluorophores by irreversible photolysis, and (ii) the entrance of new fluorophores into the area by diffusion. Rate constants for the two processes can be derived from the measuring data by mathematical analysis. As compared to our initial approach, fluorescence microphotolysis [Peters, R., Peters, J., Tews, K. H. & Bähr, W. (1974) Biochim. Biophys. Acta 367, 282-294], the main advantage of the method described here is an improvement of data quality and detection limit by orders of magnitude. From the practical point of view the main advantage is a simplification of the experimental setup. Results obtained by this method are encouraging and support the contention that continuous fluorescence microphotolysis may disclose new aspects of diffusion processes in biological systems.

  View details for DOI 10.1073/pnas.78.2.962

  View details for Web of Science ID A1981LF67000055

  View details for PubMedID 16592981

  View details for PubMedCentralID PMC319925