David B. McKay

All Publications

• Structure and Function of Steroid Receptor RNA Activator Protein, the Proposed Partner of SRA Noncoding RNA JOURNAL OF MOLECULAR BIOLOGY McKay, D. B., Xi, L., Barthel, K. K., Cech, T. R. 2014; 426 (8): 1766-1785

  Abstract

  In a widely accepted model, the steroid receptor RNA activator protein (SRA protein; SRAP) modulates the transcriptional regulatory activity of SRA RNA by binding a specific stem-loop of SRA. We first confirmed that SRAP is present in the nucleus as well as the cytoplasm of MCF-7 breast cancer cells, where it is expressed at the level of about 10(5) molecules per cell. However, our SRAP-RNA binding experiments, both in vitro with recombinant protein and in cultured cells with plasmid-expressed protein and RNA, did not reveal a specific interaction between SRAP and SRA. We determined the crystal structure of the carboxy-terminal domain of human SRAP and found that it does not have the postulated RRM (RNA recognition motif). The structure is a five-helix bundle that is distinct from known RNA-binding motifs and instead is similar to the carboxy-terminal domain of the yeast spliceosome protein PRP18, which stabilizes specific protein-protein interactions within a multisubunit mRNA splicing complex. SRA binding experiments with this domain gave negative results. Transcriptional regulation by SRA/SRAP was examined with siRNA knockdown. Effects on both specific estrogen-responsive genes and genes identified by RNA-seq as candidates for regulation were examined in MCF-7 cells. Only a small effect (~20% change) on one gene resulting from depletion of SRA/SRAP could be confirmed. We conclude that the current model for SRAP function must be reevaluated; we suggest that SRAP may function in a different context to stabilize specific intermolecular interactions in the nucleus.

  View details for DOI 10.1016/j.jmb.2014.01.006

  View details for Web of Science ID 000334131700011

  View details for PubMedID 24486609

• Structure of the RNA Binding Domain of a DEAD-Box Helicase Bound to Its Ribosomal RNA Target Reveals a Novel Mode of Recognition by an RNA Recognition Motif JOURNAL OF MOLECULAR BIOLOGY Hardin, J. W., Hu, Y. X., McKay, D. B. 2010; 402 (2): 412-427

  Abstract

  DEAD-box RNA helicases of the bacterial DbpA subfamily are localized to their biological substrate when a carboxy-terminal RNA recognition motif domain binds tightly and specifically to a segment of 23S ribosomal RNA (rRNA) that includes hairpin 92 of the peptidyl transferase center. A complex between a fragment of 23S rRNA and the RNA binding domain (RBD) of the Bacillus subtilis DbpA protein YxiN was crystallized and its structure was determined to 2.9 A resolution, revealing an RNA recognition mode that differs from those observed with other RNA recognition motifs. The RBD is bound between two RNA strands at a three-way junction. Multiple phosphates of the RNA backbone interact with an electropositive band generated by lysines of the RBD. Nucleotides of the single-stranded loop of hairpin 92 interact with the RBD, including the guanosine base of G2553, which forms three hydrogen bonds with the peptide backbone. A G2553U mutation reduces the RNA binding affinity by 2 orders of magnitude, confirming that G2553 is a sequence specificity determinant in RNA binding. Binding of the RBD to 23S rRNA in the late stages of ribosome subunit maturation would position the ATP-binding duplex destabilization fragment of the protein for interaction with rRNA in the peptidyl transferase cleft of the subunit, allowing it to "melt out" unstable secondary structures and allow proper folding.

  View details for DOI 10.1016/j.jmb.2010.07.040

  View details for Web of Science ID 000283208700010

  View details for PubMedID 20673833

  View details for PubMedCentralID PMC2942969

• The Bacillus subtilis RNA helicase YxiN is distended in solution. Biophysical journal wang, s., Overgaard, M. T., Hu, Y., McKay, D. B. 2008; 94 (1): L01-3

  Abstract

  The Bacillus subtilis YxiN protein is a modular three-domain RNA helicase of the DEx(D/H)-box protein family. The first two domains form the highly conserved helicase core, and the third domain confers RNA target binding specificity. Small angle x-ray scattering on YxiN and two-domain fragments thereof shows that the protein has a distended structure in solution, in contrast to helicases involved in replication processes. These data are consistent with a chaperone activity in which the carboxy-terminal domain of YxiN tethers the protein to the vicinity of its targets and the helicase core is free to transiently interact with RNA duplexes, possibly to melt out misfolded elements of secondary structure.

  View details for PubMedID 17951299

• The periplasmic bacterial molecular chaperone SurA adapts its structure to bind peptides in different conformations to assert a sequence preference for aromatic residues JOURNAL OF MOLECULAR BIOLOGY Xu, X., wang, s., Hu, Y., McKay, D. B. 2007; 373 (2): 367-381

  Abstract

  The periplasmic molecular chaperone protein SurA facilitates correct folding and maturation of outer membrane proteins in Gram-negative bacteria. It preferentially binds peptides that have a high fraction of aromatic amino acids. Phage display selections, isothermal titration calorimetry and crystallographic structure determination have been used to elucidate the basis of the binding specificity. The peptide recognition is imparted by the first peptidyl-prolyl isomerase (PPIase) domain of SurA. Crystal structures of complexes between peptides of sequence WEYIPNV and NFTLKFWDIFRK with the first PPIase domain of the Escherichia coli SurA protein at 1.3 A resolution, and of a complex between the dodecapeptide and a SurA fragment lacking the second PPIase domain at 3.4 A resolution, have been solved. SurA binds as a monomer to the heptapeptide in an extended conformation. It binds as a dimer to the dodecapeptide in an alpha-helical conformation, predicated on a substantial structural rearrangement of the SurA protein. In both cases, side-chains of aromatic residues of the peptides contribute a large fraction of the binding interactions. SurA therefore asserts a recognition preference for aromatic amino acids in a variety of sequence configurations by adopting alternative tertiary and quaternary structures to bind peptides in different conformations.

  View details for DOI 10.1016/j.jmb.2007.07.069

  View details for Web of Science ID 000250302900012

  View details for PubMedID 17825319

  View details for PubMedCentralID PMC2040117

• Structure of the second domain of the Bacillus subtilis DEAD-box RNA helicase YxiN ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS Caruthers, J. M., Hu, Y., McKay, D. B. 2006; 62: 1191-1195

  Abstract

  The Bacillus subtilis RNA helicase YxiN is a modular three-domain protein. The first two domains form a conserved helicase core that couples an ATPase activity to an RNA duplex-destabilization activity, while the third domain recognizes a stem-loop of 23S ribosomal RNA with high affinity and specificity. The structure of the second domain, amino-acid residues 207-368, has been solved to 1.95 A resolution, revealing a parallel alphabeta-fold. The crystallographic asymmetric unit contains two protomers; superposition shows that they differ substantially in two segments of peptide that overlap the conserved helicase sequence motifs V and VI, while the remainder of the domain is isostructural. The conformational variability of these segments suggests that induced fit is intrinsic to the recognition of ligands (ATP and RNA) and the coupling of the ATPase activity to conformational changes.

  View details for DOI 10.1107/S1744309106044642

  View details for Web of Science ID 000242429700005

  View details for PubMedID 17142894

  View details for PubMedCentralID PMC2225381

• Retention of core catalytic functions by a conserved minimal ribonuclease E peptide that lacks the domain required for tetramer formation JOURNAL OF BIOLOGICAL CHEMISTRY Caruthers, J. M., Feng, Y., McKay, D. B., Cohen, S. N. 2006; 281 (37): 27046-27051

  Abstract

  Ribonuclease E (RNase E) is a multifunctional endoribonuclease that has been evolutionarily conserved in both Gram-positive and Gram-negative bacteria. X-ray crystallography and biochemical studies have concluded that the Escherichia coli RNase E protein functions as a homotetramer formed by Zn linkage of dimers within a region extending from amino acid residues 416 through 529 of the 116-kDa protein. Using fragments of RNase E proteins from E. coli and Haemophilus influenzae, we show here that RNase E derivatives that are as short as 395 amino acid residues and that lack the Zn-link region shown previously to be essential for tetramer formation (i.e. amino acid residues 400-415) are catalytically active enzymes that retain the 5' to 3' scanning ability and cleavage site specificity characteristic of full-length RNase E and that also confer colony forming ability on rne null mutant bacteria. Further truncation leads to loss of these properties. Our results, which identify a minimal catalytically active RNase E sequence, indicate that contrary to current models, a tetrameric quaternary structure is not required for RNase E to carry out its core enzymatic functions.

  View details for DOI 10.1074/jbc.M602467200

  View details for Web of Science ID 000240397700031

  View details for PubMedID 16854990

• The domain of the Bacillus subtilis DEAD-box helicase YxiN that is responsible for specific binding of 23S rRNA has an RNA recognition motif fold RNA-A PUBLICATION OF THE RNA SOCIETY wang, s., Hu, Y., Overgaard, M. T., Karginov, F. V., Uhlenbeck, O. C., McKay, D. B. 2006; 12 (6): 959-967

  Abstract

  The YxiN protein of Bacillus subtilis is a member of the DbpA subfamily of prokaryotic DEAD-box RNA helicases. Like DbpA, it binds with high affinity and specificity to segments of 23S ribosomal RNA as short as 32 nucleotides (nt) that include hairpin 92. Several experiments have shown that the 76-residue carboxy-terminal domain of YxiN is responsible for the high-affinity RNA binding. The domain has been crystallized and its structure has been solved to 1.7 Angstroms resolution. The structure reveals an RNA recognition motif (RRM) fold that is found in many eukaryotic RNA binding proteins; the RRM fold was not apparent from the amino acid sequence. The domain has two solvent exposed aromatic residues at sites that correspond to the aromatic residues of the ribonucleoprotein (RNP) motifs RNP1 and RNP2 that are essential for RNA binding in many RRMs. However, mutagenesis of these residues (Tyr404 and Tyr447) to alanine has little effect on RNA affinity, suggesting that the YxiN domain binds target RNAs in a manner that differs from the binding mode commonly found in many eukaryotic RRMs.

  View details for DOI 10.1261/rna.5906

  View details for Web of Science ID 000237740600004

  View details for PubMedID 16611943

  View details for PubMedCentralID PMC1464845

• Structure of the Escherichia coli FlhDC complex, a prokaryotic heteromeric regulator of transcription JOURNAL OF MOLECULAR BIOLOGY Wang, S. Y., Fleming, R. T., Westbrook, E. M., Matsumura, P., McKay, D. B. 2006; 355 (4): 798-808

  Abstract

  The hetero-oligomeric complex of the FlhD and FlhC proteins (FlhDC) regulates transcription from several flagellar and non-flagellar operons in bacteria. The crystallographic structure of the Escherichia coli FlhDC complex has been solved to 3.0 A resolution, revealing a hexameric FlhD4FlhC2 assembly. In the complex, each FlhC protomer binds an FlhD2 dimer; the conformation of the dimer in the complex differs significantly from its conformation in the absence of FlhC. FlhC has a novel tertiary fold that includes a heretofore unrecognized zinc-binding site in which the ion is ligated by four cysteine residues. Gel shift experiments show that binding of the FlhDC complex to a cognate promoter bends the DNA by approximately 111 degrees . The structure of the FlhDC complex is compatible with models in which a fragment of operator DNA, at least 48 base-pairs in length, wraps around the complex and bends significantly when binding.

  View details for DOI 10.1016/j.jmb.2005.11.020

  View details for Web of Science ID 000234693300018

  View details for PubMedID 16337229

• YxiN is a modular protein combining a DEx(D)/(H) core and a specific RNA-binding domain JOURNAL OF BIOLOGICAL CHEMISTRY Karginov, F. V., Caruthers, J. M., Hu, Y. X., McKay, D. B., Uhlenbeck, O. C. 2005; 280 (42): 35499-35505

  Abstract

  DEx(D/H) proteins, typically described as RNA helicases, participate in rearrangement of RNA-RNA and possibly RNA-protein complexes in the cell. Aside from the conserved DEx(D/H) core, members of this protein family often contain N- and C-terminal extensions that are responsible for additional functions. The Bacillus subtilis DEx(D/H)-box protein YxiN and its Escherichia coli ortholog DbpA contain an approximately 80 amino acid C-terminal extension that has been proposed to specifically interact with a region of 23 S ribosomal RNA including hairpin 92. In this study, the DEx(D/H)-box core and the C-terminal domain of YxiN were expressed and characterized as separate proteins. The isolated DEx(D/H)-box core, YxCat, had weak, nonspecific RNA binding activity and showed RNA-stimulated ATPase activity with a Km(ATP) that resembled several non-specific DEx(D/H) proteins. The isolated C-terminal domain, YxRBD, bound RNA with the high affinity and specificity seen with full-length YxiN. Thus, YxiN is a modular protein combining the activities of the YxCat and YxRBD domains. Footprinting of YxiN and YxRBD on a 172-nucleotide fragment of 23 S rRNA was used to identify the sites of interaction of the C-terminal and helicase domains with the RNA.

  View details for DOI 10.1074/jbc.M506815200

  View details for Web of Science ID 000232561200050

  View details for PubMedID 16118224

• Binding of phage-display-selected peptides to the periplasmic chaperone protein SurA mimics binding of unfolded outer membrane proteins FEBS LETTERS Bitto, E., McKay, D. B. 2004; 568 (1-3): 94-98

  Abstract

  SurA is a periplasmic chaperone protein that facilitates maturation of integral outer membrane proteins (OMPs). Short peptides that bind SurA have previously been characterized. In this work, an enzyme-linked immunoabsorbent assay-based competition assay is utilized to demonstrate that binding of such peptides, presented by peptide-tagged phage, mimics binding of biological substrates. Two representative unfolded OMPs, OmpF and OmpG, bind SurA and a core structural fragment thereof in competition with peptide-tagged phage, and with the same order-of-magnitude affinity as the peptides. Additionally, unfolded OmpF and OmpG bind SurA more tightly than an unfolded water-soluble protein, while folded proteins have no measurable affinity, demonstrating a specificity of SurA for OMP polypeptides.

  View details for DOI 10.1016/j.febslet.2004.05.014

  View details for Web of Science ID 000222205200019

  View details for PubMedID 15196927

• Kinetics of protein substrate degradation by HslUV 5th International Conference on AAA(plus) Proteins Kwon, A. R., Trame, C. B., McKay, D. B. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2004: 141–47

  Abstract

  The HslUV protease-chaperone complex degrades specific protein substrates in an ATP-dependent reaction. Current models propose that the HslU chaperone, a AAA protein of the Clp/Hsp100 family, binds and unfolds substrates and translocates the polypeptide into the catalytic cavity of the HslV protease. These processes are being characterized using substrates that are targeted to HslUV with a carboxy-terminal fusion of the natural substrate SulA or the carboxy-terminal 11 amino acid residues thereof. In a tandem fusion of green fluorescent protein with SulA, HslUV degrades the SulA moiety but not green fluorescent protein. Wild type and mutant Arc repressor variants are degraded; over a range of substrate stabilities, the specific rate of degradation and its dependence on substrate stability is similar to that of ClpXP. For a hyperstable Arc variant having an intermolecular disulfide bond, the rate of degradation by HslUV is an order of magnitude slower than by ClpXP. Similarity in degradation rates for a subset of substrates by HslUV and ClpXP suggests a similarity in mechanism of the apparent rate-limiting steps of unfolding and translocation by the chaperone components HslU and ClpX. The fall-off in degradation by HslUV for the more stable substrates that are degraded by ClpXP is consistent with the two systems acting on different spectra of biological substrates.

  View details for DOI 10.1016/j.jsb.2003.11.003

  View details for Web of Science ID 000220548600015

  View details for PubMedID 15037245

• The periplasmic molecular chaperone protein SurA binds a peptide motif that is characteristic of integral outer membrane proteins JOURNAL OF BIOLOGICAL CHEMISTRY Bitto, E., McKay, D. B. 2003; 278 (49): 49316-49322

  Abstract

  The Escherichia coli SurA protein is a periplasmic molecular chaperone that facilitates correct folding of outer membrane porins. The peptide binding specificity of SurA has been characterized using phage display of heptameric peptides of random sequence. The consensus binding pattern of aromatic-polar-aromatic-nonpolar-proline amino acids emerges for both SurA and a SurA "core domain," which remains after deletion of a peripheral peptidyl-proline isomerase domain. Isothermal titration calorimetry with a high affinity heptameric peptide of sequence WEYIPNV yields peptide affinities in the range of 1-14 microm for both SurA and its core domain. Although the peptide consensus aromatic-polar-aromatic-nonpolar-proline occurs infrequently in E. coli proteins, the less restrictive tripeptide motif aromatic-random-aromatic appears with greater-than-random frequency in outer membrane proteins and is prevalent in the "aromatic bands" of the porin beta barrel structures. Thus, SurA recognizes a peptide motif that is characteristic of integral outer membrane proteins.

  View details for DOI 10.1074/jbc.M308853200

  View details for Web of Science ID 000186829000099

  View details for PubMedID 14506253

• Crystal structure of the leadzyme at 1.8 angstrom resolution: Metal ion binding and the implications for catalytic mechanism and allo site ion regulation BIOCHEMISTRY Wedekind, J. E., McKay, D. B. 2003; 42 (32): 9554-9563

  Abstract

  The leadzyme is a small ribozyme, derived from in vitro selection, which catalyzes site specific, Pb(2+)-dependent RNA cleavage. Pb(2+) is required for activity; Mg(2+) inhibits activity, while many divalent and trivalent ions enhance it. The leadzyme structure consists of an RNA duplex interrupted by a trinucleotide bulge. Here, crystal structures determined to 1.8 A resolution, both with Mg(2+) as the sole divalent counterion and with Mg(2+) and Sr(2+) (which mimics Pb(2+) with respect to binding but not catalysis), reveal the metal ion interactions with both the ground state and precatalytic conformations of the leadzyme. Mg(H(2)O)(6)(2+) ions bridge complementary strands of the duplex at multiple locations by binding tandem purines of one RNA strand in the major groove. At one site, Mg(H(2)O)(6)(2+) ligates the phosphodiester backbone of the trinucleotide bulge in the ground state conformation, but not in the precatalytic conformation, suggesting (a) Mg(2+) may inhibit leadzyme activity by stabilizing the ground state and (b) metal ions which displace Mg(2+) from this site may activate the leadzyme. Binding of Sr(2+) to the presumed catalytic Pb(2+) site in the precatalytic leadzyme induces local structural changes in a manner that would facilitate alignment of the catalytic ribose 2'-hydroxyl with the scissile bond for cleavage. These data support a model wherein binding of a catalytic ion to a precatalytic conformation of the leadzyme, in conjunction with the flexibility of the trinucleotide bulge, may facilitate structural rearrangements around the scissle phosphodiester bond favoring configurations that allow bond cleavage.

  View details for DOI 10.1021/bi0300783

  View details for Web of Science ID 000184763000005

  View details for PubMedID 12911297

• Structure and reactivity of an asymmetric complex between HslV and I-domain deleted HslU, a prokaryotic homolog of the eukaryotic proteasome JOURNAL OF MOLECULAR BIOLOGY Kwon, A. R., Kessler, B. M., Overkleeft, H. S., McKay, D. B. 2003; 330 (2): 185-195

  Abstract

  In the prokaryotic homolog of the eukaryotic proteasome, HslUV, the "double donut" HslV protease is allosterically activated by HslU, an AAA protein of the Clp/Hsp100 family consisting of three (amino-terminal, carboxy-terminal, and intermediate) domains. The intermediate domains of HslU, which extend like tentacles from the hexameric ring formed by the amino-terminal and carboxy-terminal domains, have been deleted; an asymmetric HslU(DeltaI)(6)HslV(12) complex has been crystallized; and the structure has been solved to 2.5A resolution, revealing an assembly in which a HslU(DeltaI) hexamer binds one end of the HslV dodecamer. The conformation of the protomers of the HslU(DeltaI)-complexed HslV hexamer is similar to that in the symmetric wild-type HslUV complex, while the protomer conformation of the uncomplexed HslV hexamer is similar to that of HslV alone. Reaction in the crystals with a vinyl sulfone inhibitor reveals that the HslU(DeltaI)-complexed HslV hexamer is active, while the uncomplexed HslV hexamer is inactive. These results confirm that HslV can be activated by binding of a hexameric HslU(DeltaI)(6) ring lacking the I domains, that activation is effected through a conformational change in HslV rather than through alteration of the size of the entry channel into the protease catalytic cavity, and that the two HslV(6) rings in the protease dodecamer are activated independently rather than cooperatively.

  View details for DOI 10.1016/S0022-2836(03)00580-1

  View details for Web of Science ID 000183824900002

  View details for PubMedID 12823960

• Structure of the Yersinia enterocolitica molecular-chaperone protein SycE ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY Trame, C. B., McKay, D. B. 2003; 59: 389-392

  Abstract

  The crystal structure of the Yersinia enterocolitica molecular-chaperone protein SycE, which specifically binds the YopE protein, has been solved to 2.0 A resolution by molecular replacement. The crystal contains two SycE dimers per asymmetric unit; a novel feature of this crystal, when compared with closely related SycE structures, is a well ordered carboxy-terminal peptide in one protomer of each dimer. The peptide binds a hydrophobic patch of a neighboring molecule in a manner similar to that seen in a SycE-YopE chaperone-target complex, suggestive of low-affinity 'self-binding' through which the carboxy-terminal peptide might suppress counterproductive interactions with non-target proteins in vivo.

  View details for DOI 10.1107/S0907444902020826

  View details for Web of Science ID 000180641900032

  View details for PubMedID 12554962

• Crystallographic structure of SurA, a molecular chaperone that facilitates folding of outer membrane porins STRUCTURE Bitto, E., McKay, D. B. 2002; 10 (11): 1489-1498

  Abstract

  The SurA protein facilitates correct folding of outer membrane proteins in gram-negative bacteria. The sequence of Escherichia coli SurA presents four segments, two of which are peptidyl-prolyl isomerases (PPIases); the crystal structure reveals an asymmetric dumbbell, in which the amino-terminal, carboxy-terminal, and first PPIase segments of the sequence form a core structural module, and the second PPIase segment is a satellite domain tethered approximately 30 A from this module. The core module, which is implicated in membrane protein folding, has a novel fold that includes an extended crevice. Crystal contacts show that peptides bind within the crevice, suggesting a model for chaperone activity whereby segments of polypeptide may be repetitively sequestered and released during the membrane protein-folding process.

  View details for Web of Science ID 000179175300007

  View details for PubMedID 12429090

• Crystal structure of HsIUV complexed with a vinyl sulfone inhibitor: Corroboration of a proposed mechanism of allosteric activation of HsIV by HsIU JOURNAL OF MOLECULAR BIOLOGY Sousa, M. C., Kessler, B. M., Overkleeft, H. S., McKay, D. B. 2002; 318 (3): 779-785

  Abstract

  On the basis of the structure of a HslUV complex, a mechanism of allosteric activation of the HslV protease, wherein binding of the HslU chaperone propagates a conformational change to the active site cleft of the protease, has been proposed. Here, the 3.1 A X-ray crystallographic structure of Haemophilus influenzae HslUV complexed with a vinyl sulfone inhibitor is described. The inhibitor, which reacts to form a covalent linkage to Thr1 of HslV, binds in an "antiparallel beta" manner, with hydrogen-bond interactions between the peptide backbone of the protease and that of the inhibitor, and with two leucinyl side chains of the inhibitor binding in the S1 and S3 specificity pockets of the protease. Comparison of the structure of the HslUV-inhibitor complex with that of HslV without inhibitor and in the absence of HslU reveals that backbone interactions would correctly position a substrate for cleavage in the HslUV complex, but not in the HslV protease alone, corroborating the proposed mechanism of allosteric activation. This activation mechanism differs from that of the eukaryotic proteasome, for which binding of activators opens a gated channel that controls access of substrates to the protease, but does not perturb the active site environment.

  View details for DOI 10.1016/S0022-2836(02)00145-6

  View details for Web of Science ID 000175767700014

  View details for PubMedID 12054822

• Helicase structure and mechanism CURRENT OPINION IN STRUCTURAL BIOLOGY Caruthers, J. M., McKay, D. B. 2002; 12 (1): 123-133

  Abstract

  Structural information on helicase proteins has expanded recently beyond the DNA helicases Rep and PcrA, and the hepatitis C virus RNA helicase to include UvrB, members of the DEA(D/H)-box RNA helicase family, examples of DnaB-related helicases and RuvB. The expanding database of structures has clarified the structural 'theme and variations' that relate the different helicase families. Furthermore, information is emerging on the functions of the conserved helicase motifs and their participation in the mechanisms by which these proteins catalyze the remodeling of DNA and RNA in ATP-dependent activities.

  View details for Web of Science ID 000173884800017

  View details for PubMedID 11839499

• Refined crystallographic structure of Pseudomonas aeruginosa exotoxin A and its implications for the molecular mechanism of toxicity JOURNAL OF MOLECULAR BIOLOGY Wedekind, J. E., Trame, C. B., Dorywalska, M., Koehl, P., Raschke, T. M., McKee, M., Fitzgerald, D., Collier, R. J., McKay, D. B. 2001; 314 (4): 823-837

  Abstract

  Exotoxin A of Pseudomonas aeruginosa asserts its cellular toxicity through ADP-ribosylation of translation elongation factor 2, predicated on binding to specific cell surface receptors and intracellular trafficking via a complex pathway that ultimately results in translocation of an enzymatic activity into the cytoplasm. In early work, the crystallographic structure of exotoxin A was determined to 3.0 A resolution, revealing a tertiary fold having three distinct structural domains; subsequent work has shown that the domains are individually responsible for the receptor binding (domain I), transmembrane targeting (domain II), and ADP-ribosyl transferase (domain III) activities, respectively. Here, we report the structures of wild-type and W281A mutant toxin proteins at pH 8.0, refined with data to 1.62 A and 1.45 A resolution, respectively. The refined models clarify several ionic interactions within structural domains I and II that may modulate an obligatory conformational change that is induced by low pH. Proteolytic cleavage by furin is also obligatory for toxicity; the W281A mutant protein is substantially more susceptible to cleavage than the wild-type toxin. The tertiary structures of the furin cleavage sites of the wild-type and W281 mutant toxins are similar; however, the mutant toxin has significantly higher B-factors around the cleavage site, suggesting that the greater susceptibility to furin cleavage is due to increased local disorder/flexibility at the site, rather than to differences in static tertiary structure. Comparison of the refined structures of full-length toxin, which lacks ADP-ribosyl transferase activity, to that of the enzymatic domain alone reveals a salt bridge between Arg467 of the catalytic domain and Glu348 of domain II that restrains the substrate binding cleft in a conformation that precludes NAD+ binding. The refined structures of exotoxin A provide precise models for the design and interpretation of further studies of the mechanism of intoxication.

  View details for DOI 10.1006/jmbi.2001.5195

  View details for Web of Science ID 000173469400016

  View details for PubMedID 11734000

• Structure of the universal stress protein of Haemophilus influenzae STRUCTURE Sousa, M. C., McKay, D. B. 2001; 9 (12): 1135-1141

  Abstract

  The universal stress protein UspA is a small cytoplasmic bacterial protein whose expression is enhanced several-fold when cellular viability is challenged with heat shock, nutrient starvation, stress agents which arrest cell growth, or DNA-damaging agents. UspA enhances the rate of cell survival during prolonged exposure to such conditions, suggesting that it asserts a general "stress endurance" activity. However, neither the structure of UspA nor the biochemical mechanism by which it protects cells from the broad spectrum of stress agents is known.The crystal structure of Haemophilus influenzae UspA reveals an asymmetric dimer with a tertiary alpha/beta fold similar to that of the Methanococcus jannaschi MJ0577 protein, a protein whose crystal structure revealed a novel ATP binding motif. UspA differs significantly from the MJ0577 structure in several details, including the triphosphate binding loop of the ATP binding motif; UspA shows no ATP binding activity.Within the universal stress protein family that is delineated by sequence similarity, UspA is the only member which has been correlated with a cellular activity, and MJ0577 is the only member which has been assigned a biochemical activity, i.e., ATP binding. UspA has a similar fold to the MJ0577 protein but does not bind ATP. This suggests that members of this protein family will segregate into two groups, based on whether or not they bind ATP. By implication, one subset of the universal stress proteins presumably has an ATP-dependent function, while another subset functions in ATP-independent activities.

  View details for Web of Science ID 000172658700002

  View details for PubMedID 11738040

• Structure of Haemophilus influenzae HslV protein at 1.9 angstrom resolution, revealing a cation-binding site near the catalytic site ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY Sousa, M. C., McKay, D. B. 2001; 57: 1950-1954

  Abstract

  The structure of the Haemophilus influenzae HslV protease of the HslUV 'prokaryotic proteasome' has been solved by molecular replacement and refined with data to 1.9 A resolution. The protease is a 'double donut' of hexameric rings; two alternative sets of intermolecular interactions between protomers in the rings result in 'quasi-equivalent' packing within the assembly. Anomalous scattering data from crystals with potassium present in the mother liquor reveal a K(+) ion bound with octahedral coordination near the active-site Thr1 residue. The site also binds Na(+) ions and is likely to bind Mg(2+), suggesting that monovalent and divalent metal ions may influence the catalytic activity of the protease.

  View details for Web of Science ID 000172322000040

  View details for PubMedID 11717526

• Structure of Haemophilus influenzae HslU protein in crystals with one-dimensional disorder twinning ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY Trame, C. B., McKay, D. B. 2001; 57: 1079-1090

  Abstract

  The structure of the Haemophilus influenzae HslU protein, a molecular chaperone of the Clp/Hsp100 family, has been solved to 2.3 A by molecular replacement using a model of the homologous Escherichia coli protein. The crystals in which the structure was solved have an unusual twinning, or one-dimensional disorder, in which each successive crystal-packing layer is displaced laterally relative to the one below it. A model for the twinning and an algorithm for detwinning the data are described. It is known from other work that when the HslU hexamer binds its cognate protease HslV, the carboxy-terminal helices of HslU protomers distend and bind between HslV subunits. Comparison of HslU alone with its structure in the HslUV complex reveals several conserved amino-acid residues whose side-chain interactions differ between the two structures, suggesting that they may be part of a conformational switch that facilitates the release of the HslU carboxy-terminal helices when HslV binds.

  View details for Web of Science ID 000170041200004

  View details for PubMedID 11468391

• Crystal structure of yeast initiation factor 4A, a DEAD-box RNA helicase PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Caruthers, J. M., Johnson, E. R., McKay, D. B. 2000; 97 (24): 13080-13085

  Abstract

  The eukaryotic translation initiation factor 4A (eIF4A) is a member of the DEA(D/H)-box RNA helicase family, a diverse group of proteins that couples an ATPase activity to RNA binding and unwinding. Previous work has provided the structure of the amino-terminal, ATP-binding domain of eIF4A. Extending those results, we have solved the structure of the carboxyl-terminal domain of eIF4A with data to 1.75 A resolution; it has a parallel alpha-beta topology that superimposes, with minor variations, on the structures and conserved motifs of the equivalent domain in other, distantly related helicases. Using data to 2.8 A resolution and molecular replacement with the refined model of the carboxyl-terminal domain, we have completed the structure of full-length eIF4A; it is a "dumbbell" structure consisting of two compact domains connected by an extended linker. By using the structures of other helicases as a template, compact structures can be modeled for eIF4A that suggest (i) helicase motif IV binds RNA; (ii) Arg-298, which is conserved in the DEA(D/H)-box RNA helicase family but is absent from many other helicases, also binds RNA; and (iii) motifs V and VI "link" the carboxyl-terminal domain to the amino-terminal domain through interactions with ATP and the DEA(D/H) motif, providing a mechanism for coupling ATP binding and hydrolysis with conformational changes that modulate RNA binding.

  View details for Web of Science ID 000165476300035

  View details for PubMedID 11087862

  View details for PubMedCentralID PMC27181

• Crystal and solution structures of an HsIUV protease-chaperone complex CELL Sousa, M. C., Trame, C. B., Tsuruta, H., Wilbanks, S. M., Reddy, V. S., McKay, D. B. 2000; 103 (4): 633-643

  Abstract

  HslUV is a "prokaryotic proteasome" composed of the HslV protease and the HslU ATPase, a chaperone of the Clp/Hsp100 family. The 3.4 A crystal structure of an HslUV complex is presented here. Two hexameric ATP binding rings of HslU bind intimately to opposite sides of the HslV protease; the HslU "intermediate domains" extend outward from the complex. The solution structure of HslUV, derived from small angle X-ray scattering data under conditions where the complex is assembled and active, agrees with this crystallographic structure. When the complex forms, the carboxy-terminal helices of HslU distend and bind between subunits of HslV, and the apical helices of HslV shift substantially, transmitting a conformational change to the active site region of the protease.

  View details for Web of Science ID 000165348000008

  View details for PubMedID 11106733

• Purification, crystallization, and X-ray diffraction analysis of small ribozymes RNA-LIGAND INTERACTIONS PT A Wedekind, J. E., McKay, D. B. 2000; 317: 149-168

  View details for Web of Science ID 000087898000011

  View details for PubMedID 10829279

• Crystallographic structure of the amino terminal domain of yeast initiation factor 4A, a representative DEAD-box RNA helicase RNA-A PUBLICATION OF THE RNA SOCIETY Johnson, E. R., McKay, D. B. 1999; 5 (12): 1526-1534

  Abstract

  The eukaryotic translation initiation factor 4A (elF4A) is a representative of the DEAD-box RNA helicase protein family. We have solved the crystallographic structure of the amino-terminal domain (residues 1-223) of yeast elF4A. The domain is built around a core scaffold, a parallel alpha-beta motif with five beta strands, that is found in other RNA and DNA helicases, as well as in the RecA protein. The amino acid sequence motifs that are conserved within the helicase family are localized to the beta strand-->alpha helix junctions within the core. The core of the amino terminal domain of elF4A is amplified with additional structural elements that differ from those of other helicases. The phosphate binding loop (the Walker A motif) is in an unusual closed conformation. The crystallographic structure reveals specific interactions between amino acid residues of the phosphate binding loop, the DEAD motif, and the SAT motif, whose alteration is known to impair coupling between the ATPase cycle and the RNA unwinding activity of elF4A.

  View details for Web of Science ID 000084122000002

  View details for PubMedID 10606264

  View details for PubMedCentralID PMC1369875

• Mapping the role of active site residues for transducing an ATP-induced conformational change in the bovine 70-kDa heat shock cognate protein BIOCHEMISTRY Johnson, E. R., McKay, D. B. 1999; 38 (33): 10823-10830

  Abstract

  ATP binding induces a conformational change in 70-kDa heat shock proteins (Hsp70s) that facilitates release of bound polypeptides. Using the bovine heat shock cognate protein (Hsc70) as a representative of the Hsp70 family, we have characterized the effect of mutations on the coupling between ATP binding and the nucleotide-induced conformational change. Steady-state solution small-angle X-ray scattering and kinetic fluorescence measurements on a 60-kDa fragment of Hsc70 show that point mutations K71M, E175S, D199S, and D206S in the nucleotide binding cleft impair the ability of ATP to induce a conformational change. A secondary mutation in the peptide binding domain, E543K, "rescues" the ATP-induced transition for three of these mutations (E175S/E543K, D199S/E543K, and D206S/E543K) but not for K71M/E543K. Analysis of kinetics of the ATPase cycle confirm that these effects do not result from unexpectedly rapid ATP hydrolysis or slow ATP binding. Crystallographic structures of E175S, D199S, and D206S mutant ATPase fragment proteins show that the mutations do not perturb the tertiary structure of the protein but do significantly alter the protein-ligand interactions, due in part to an apparent charge compensation effect whereby mutating a (probably) negatively charged carboxyl group to a neutral serine displaces a K+ ion from the nucleotide binding cleft in two out of three cases (E175S and D199S but not D206S).

  View details for Web of Science ID 000082334700024

  View details for PubMedID 10451379

• Crystal structure of a lead-dependent ribozyme revealing metal binding sites revelant to catalysis NATURE STRUCTURAL BIOLOGY Wedekind, J. E., McKay, D. B. 1999; 6 (3): 261-268

  Abstract

  The leadzyme is a small RNA motif that catalyzes a site-specific, Pb2+-dependent cleavage reaction. As such, it is an example of a metal-dependent RNA enzyme. Here we describe the X-ray crystallographic structure of the leadzyme, which reveals two independent molecules per asymmetric unit. Both molecules feature an internal loop in which a bulged purine base stack twists away from the helical stem. This kinks the backbone, rendering the phosphodiester bond susceptible to cleavage. The independent molecules have different conformations: one leadzyme copy coordinates Mg2+, whereas the other binds only Ba2+ or Pb2+. In the active site of the latter molecule, a single Ba2+ ion coordinates the 2'-OH nucleophile, and appears to mimic the binding of catalytic lead. These observations allow a bond cleavage reaction to be modeled, which reveals the minimal structural features necessary for catalysis by this small ribozyme.

  View details for Web of Science ID 000078970100014

  View details for PubMedID 10074945

• The hydroxyl of threonine 13 of the bovine 70-kDa heat shock cognate protein is essential for transducing the ATP-induced conformational change BIOCHEMISTRY Sousa, M. C., McKay, D. B. 1998; 37 (44): 15392-15399

  Abstract

  The mechanism by which ATP binding transduces a conformational change in 70-kDa heat shock proteins that results in release of bound peptides remains obscure. Wei and Hendershot demonstrated that mutating Thr37 of hamster BiP to glycine impeded the ATP-induced conformational change, as monitored by proteolysis [(1995) J. Biol. Chem. 270, 26670-26676]. We have mutated the equivalent resitude of the bovine heat shock cognate protein (Hsc70), Thr13, to serine, valine, and glycine. Solution small-angle X-ray scattering experiments on a 60-kDa fragment of Hsc70 show that ATP binding induces a conformational change in the T13S mutant but not the T13V or T13G mutants. The kinetics of ATP-induced tryptophan fluorescence intensity changes in the 60-kDa proteins is biphasic for the T13S mutant but monophasic for T13V or T13G, consistent with a conformational change following initial ATP binding in the T13S mutant but not the other two. Crystallographic structures of the ATPase fragments of the T13S and T13G mutants at 1.7 A resolution show that the mutations do not disrupt the ATP binding site and that the serine hydroxyl mimics the threonine hydroxyl in the wild-type structure. We conclude that the hydroxyl of Thr13 is essential for coupling ATP binding to a conformational change in Hsc70. Molecular modeling suggests this may result from the threonine hydroxyl hydrogen-bonding to a gamma-phosphate oxygen of ATP, thereby inducing a structural shift within the ATPase domain that couples to its interactions with the peptide binding domain.

  View details for Web of Science ID 000076887400017

  View details for PubMedID 9799500

• Structural replacement of active site monovalent cations by the epsilon-amino group of lysine in the ATPase fragment of bovine Hsc70 BIOCHEMISTRY Wilbanks, S. M., McKay, D. B. 1998; 37 (20): 7456-7462

  Abstract

  We have assessed the ability of the epsilon-amino group of a non-native lysine chain to substitute for a monovalent cation in an enzyme active site. In the bovine Hsc70 ATPase fragment, mutation of cysteine 17 or aspartic acid 206 to lysine potentially allows the replacement of an active site potassium ion with the epsilon-amino nitrogen. We examined the ATP hydrolysis kinetics and crystal structures of isolated mutant ATPase domains. The introduced epsilon-amino nitrogen in the C17K mutant occupies a significantly different position than the potassium ion. The introduced epsilon-amino nitrogen in the D206K mutant occupies a position indistinguishable from that of the potassium in the wild-type structure. Each mutant retains <5% ATPase activity when compared to the wild type under physiological conditions (potassium buffer) although substrate binding is tighter, probably as a consequence of slower release. It is possible to construct a very good structural mimic of bound cation which suffices for substrate binding but not for catalytic activity.

  View details for Web of Science ID 000073768300046

  View details for PubMedID 9585559

• Crystallographic structures of the hammerhead ribozyme: Relationship to ribozyme folding and catalysis ANNUAL REVIEW OF BIOPHYSICS AND BIOMOLECULAR STRUCTURE Wedekind, J. E., McKay, D. B. 1998; 27: 475-502

  Abstract

  The hammerhead ribozyme is a small catalytic RNA that cleaves a target phosphodiester bond in a reaction dependent on divalent metal ions. Crystal structures of the hammerhead reveal the tertiary fold of an enzymatic "ground state" of the molecule; however, they do not clarify the catalytic mechanism of the ribozyme, presumably because a significant conformational rearrangement is required to reach an enzymatic transition state. The structural domains seen in the hammerhead can be related to sequence or structural motifs in transfer and ribosomal RNAs, suggesting that they represent tertiary building blocks that will be found in large, complex RNAs.

  View details for Web of Science ID 000074324000017

  View details for PubMedID 9646875

• Destabilization of peptide binding and interdomain communication by an E543K mutation in the bovine 70-kDa heat shock cognate protein, a molecular chaperone JOURNAL OF BIOLOGICAL CHEMISTRY Ha, J. H., Hellman, U., Johnson, E. R., Li, L. S., McKay, D. B., Sousa, M. C., Takeda, S., Wernstedt, C., Wilbanks, S. M. 1997; 272 (44): 27796-27803

  Abstract

  We have compared 70-kDa heat shock cognate protein (Hsc70) isolated from bovine brain with recombinant wild type protein and mutant E543K protein (previously studied as wild type in our laboratory). Wild type bovine and recombinant protein differ by posttranslational modification of lysine 561 but interact similarly with a short peptide (fluorescein-labeled FYQLALT) and with denatured staphylococcal nuclease-(Delta135-149). Mutation E543K results in 4. 5-fold faster release of peptide and lower stability of complexes with staphylococcal nuclease-(Delta135-149). ATP hydrolysis rates of the wild type proteins are enhanced 6-10-fold by the addition of peptide. The E543K mutant has a peptide-stimulated hydrolytic rate similar to that of wild type protein but a higher unstimulated rate, yielding a mere 2-fold enhancement. All three versions of Hsc70 possess similar ATP-dependent conformational shifts, and all show potassium ion dependence. These data support the following model: (i) in the presence of K+, Mg2+, and ATP, the peptide binding domain inhibits the ATPase; (ii) binding of peptide relieves this inhibition; and (iii) the E543K mutation significantly attenuates the inhibition by the peptide binding domain and destabilizes Hsc70-peptide complexes.

  View details for Web of Science ID A1997YD47300050

  View details for PubMedID 9346924

• Lysine 71 of the chaperone protein Hsc70 is essential for ATP hydrolysis JOURNAL OF BIOLOGICAL CHEMISTRY OBRIEN, M. C., Flaherty, K. M., McKay, D. B. 1996; 271 (27): 15874-15878

  Abstract

  It has been proposed that lysine 71 of the bovine 70-kDa heat shock cognate protein might participate in catalysis of ATP hydrolysis by stabilizing an H2O molecule or an OH- ion for nucleophilic attack on the gamma-phosphate of the nucleotide (Flaherty, K. M., Wilbanks, S. M., DeLuca-Flaherty, C., and McKay, D. B. (1994) J. Biol. Chem. 12899-12907; Wilbanks, S. M., DeLuca-Flaherty, C., and McKay, D. B. (1994) J. Biol. Chem. 269, 12893-12898). To test this hypothesis, lysine 71 of the ATPase fragment 70-kDa heat shock cognate protein has been mutated to glutamic acid, methionine, and alanine; and the kinetic and structural properties of the mutant proteins have been determined. All three mutant proteins are devoid of measurable ATP hydrolysis activity. Crystal structures of the mutant proteins have been determined to a resolution of 1.7 A; all three have ATP in the nucleotide binding site. These data identify lysine 71 as a residue that is essential for chemical hydrolysis of ATP.

  View details for Web of Science ID A1996UW35200008

  View details for PubMedID 8663302

• Structure and function of the hammerhead ribozyme: An unfinished story RNA-A PUBLICATION OF THE RNA SOCIETY McKay, D. B. 1996; 2 (5): 395-403

  View details for Web of Science ID A1996UQ58600001

  View details for PubMedID 8665407

  View details for PubMedCentralID PMC1369381

• Kinetics of peptide binding to the bovine 70 kDa heat shock cognate protein, a molecular chaperone BIOCHEMISTRY Takeda, S., McKay, D. B. 1996; 35 (14): 4636-4644

  Abstract

  We have measured the kinetics of binding and release of a fluorescently labeled seven-residue peptide (fluorescein-FYQLALT) to recombinant bovine heat shock cognate protein (Hsc70); additionally, we have determined the effect of peptide binding on the kinetic rate constants of individual steps of the Hsc70 ATPase cycle. In the presence of MgADP, peptide binding is a two-step process; the first step results in a low-affinity peptide-Hsc70 complex (Kd calcd approximately 14 microM), while the second step locks the peptide into a higher-affinity complex (Kd = 4.3 microM). In the presence of MgATP, peptide binding is a one-step process which yields a peptide-Hsc70 complex with an affinity of approximately 40-50 microM. The bimolecular rates of initial peptide-Hsc70 association differ less than 2-fold in the presence of MgADP and MgATP. Peptide binding increases the rates of ATP hydrolysis and product release in the Hsc70 ATPase cycle. Taken together with earlier results, these data suggest a model for the interaction of Hsc70 with peptides in which (i) with MgATP there is significant interaction between the carboxy terminal peptide binding domain and the amino terminal ATPase domain of Hsc70 such that the effect of peptide binding is transmitted to the ATPase domain (resulting in increased rates of ATP hydrolysis and product release) and, reciprocally, the ATPase domain constrains the peptide binding domain to a low-peptide affinity conformation; and (ii) with MgADP, the peptide binding domain is less constrained by the ATPase domain, allowing capture of peptides in complexes with significantly slower "off " rates than in the presence of MgATP.

  View details for Web of Science ID A1996UE61200043

  View details for PubMedID 8605215

• The metzincins: A superfamily of structurally related metalloproteinases Meeting of the Study Group Physiology of the German-Zoological-Society Bode, W., Grams, F., Reinemer, P., GOMISRUTH, F. X., Baumann, U., McKay, D. B., STOCKER, W. ELSEVIER GMBH, URBAN & FISCHER VERLAG. 1996: 237–46

  View details for Web of Science ID A1996WV70300003

• The metzincin-superfamily of zinc-peptidases 10th International Conference on Intracellular Protein Catabolism Bode, W., Grams, F., Reinemer, P., GOMISRUTH, F. X., Baumann, U., McKay, D. B., STOCKER, W. PLENUM PRESS DIV PLENUM PUBLISHING CORP. 1996: 1–11

  View details for Web of Science ID A1996BF18P00001

  View details for PubMedID 8860988

• SOLUTION SMALL-ANGLE X-RAY-SCATTERING STUDY OF THE MOLECULAR CHAPERONE HSC70 AND ITS SUBFRAGMENTS BIOCHEMISTRY Wilbanks, S. M., Chen, L. L., Tsuruta, H., Hodgson, K. O., McKay, D. B. 1995; 34 (38): 12095-12106

  Abstract

  Solution X-ray scattering experiments have been carried out on recombinant bovine Hsc70 (with 650 amino acid residues), a 60 kDa subfragment (residues 1-554) which has ATPase- and peptide-binding activities, a 44kDa subfragment (residues 1-386) which has only ATPase activity, and a peptide-binding fragment (residues 388-554). Modeling based on steady-state values of radii of gyration (Rg's) and P(r) functions shows that the 44 kDa and peptide-binding domains are oblate fragments while Hsc70 and the 60 kDa fragment are prolate and relatively elongated. Rg values decrease significantly in the presence of MgATP relative to their values in the presence of MgADP (delta Rg approximately 4-5 A) for Hsc70 and the 60 kDa fragment; in contrast, they are essentially equal in the presence of either nucleotide for the 44 kDa ATPase fragment. The kinetics of the change of Rg for Hsc70 and the 60 kDa fragment under single-ATPase cycle conditions show that the transition to the ATP-induced Rg occurs significantly more rapidly than ATP hydrolysis while the reverse transition to the larger Rg value does not occur before product release. Altogether, the solution scattering data support a model in which a conformational change in Hsc70 (presumably to the low-peptide-affinity state) is predicated on ATP binding while the reverse transition is predicated on product release.

  View details for Web of Science ID A1995RX23500002

  View details for PubMedID 7547949

• KINETICS OF NUCLEOTIDE-INDUCED CHANGES IN THE TRYPTOPHAN FLUORESCENCE OF THE MOLECULAR CHAPERONE HSC70 AND ITS SUBFRAGMENTS SUGGEST THE ATP-INDUCED CONFORMATIONAL CHANGE FOLLOWS INITIAL ATP BINDING BIOCHEMISTRY Ha, J. H., McKay, D. B. 1995; 34 (36): 11635-11644

  Abstract

  The kinetics of nucleotide-induced changes of tryptophan fluorescence have been measured for recombinant bovine 70 kDa heat shock cognate protein (Hsc70), a 60 kDa subfragment (amino acid residues 1-554) which has ATPase and peptide binding activities, and a 44 kDa subfragment (residues 1-386) which has only ATPase activity. The fluorescence changes resulting from ATP binding to Hsc70 and the 60 kDa fragment are biphasic, and can be interpreted as arising from a two-step process in which ATP initially binds in a bimolecular reaction, followed by a conformational change of the protein-MgATP complex. Fluorescence changes resulting from ADP binding indicate a single-step, bimolecular process. Under single-cycle conditions of the ATPase reaction, a fluorescence change is observed whose rate constant correlates with product release in Hsc70, and with product release/ATP hydrolysis (which are kinetically indistinguishable under single-cycle conditions) in the 60 kDa fragment. These data support a scheme for Hsc70 in which a conformational transition is induced after initial ATP binding but prior to hydrolysis, and the reverse transition is induced by product release. The 60 kDa fragment shows behavior that is quantitatively similar to that of Hsc70. The 44 kDa ATPase fragment does not show biphasic kinetics for ATP binding, and does not show fluorescence changes that suggest conformational changes of the type seen in Hsc70 and the 60 kDa fragment.

  View details for Web of Science ID A1995RV81000040

  View details for PubMedID 7547895

• THE METZINCINS - TOPOLOGICAL AND SEQUENTIAL RELATIONS BETWEEN THE ASTACINS, ADAMALYSINS, SERRALYSINS, AND MATRIXINS (COLLAGENASES) DEFINE A SUPERFAMILY OF ZINC-PEPTIDASES PROTEIN SCIENCE STOCKER, W., Grams, F., Baumann, U., Reinemer, P., GOMISRUTH, F. X., McKay, D. B., Bode, W. 1995; 4 (5): 823-840

  Abstract

  The three-dimensional structures of the zinc endopeptidases human neutrophil collagenase, adamalysin II from rattle snake venom, alkaline proteinase from Pseudomonas aeruginosa, and astacin from crayfish are topologically similar, with respect to a five-stranded beta-sheet and three alpha-helices arranged in typical sequential order. The four proteins exhibit the characteristic consensus motif HEXXHXXGXXH, whose three histidine residues are involved in binding of the catalytically essential zinc ion. Moreover, they all share a conserved methionine residue beneath the active site metal as part of a superimposable "Met-turn." This structural relationship is supported by a sequence alignment performed on the basis of topological equivalence showing faint but distinct sequential similarity. The alkaline proteinase is about equally distant (26% sequence identity) to both human neutrophil collagenase and astacin and a little further away from adamalysin II (17% identity). The pairs astacin/adamalysin II, astacin/human neutrophil collagenase, and adamalysin II/human neutrophil collagenase exhibit sequence identities of 16%, 14%, and 13%, respectively. Therefore, the corresponding four distinct families of zinc peptidases, the astacins, the matrix metalloproteinases (matrixins, collagenases), the adamalysins/reprolysins (snake venom proteinases/reproductive tract proteins), and the serralysins (large bacterial proteases from Serratia, Erwinia, and Pseudomonas) appear to have originated by divergent evolution from a common ancestor and form a superfamily of proteolytic enzymes for which the designation "metzincins" has been proposed. There is also a faint but significant structural relationship of the metzincins to the thermolysin-like enzymes, which share the truncated zinc-binding motif HEXXH and, moreover, similar topologies in their N-terminal domains.

  View details for Web of Science ID A1995QW98100002

  View details for PubMedID 7663339

• HOW POTASSIUM AFFECTS THE ACTIVITY OF THE MOLECULAR CHAPERONE HSC70 .2. POTASSIUM BINDS SPECIFICALLY IN THE ATPASE ACTIVE-SITE JOURNAL OF BIOLOGICAL CHEMISTRY Wilbanks, S. M., McKay, D. B. 1995; 270 (5): 2251-2257

  Abstract

  Crystallographic anomalous scattering from potassium at 1.7 A resolution reveals two monovalent ions that interact with MgADP and P(i) in the nucleotide binding cleft of wild-type recombinant bovine Hsc70 ATPase fragment. K+ at site 1 interacts with oxygens of the beta-phosphate of ADP, whereas K+ at site 2 interacts with an oxygen of P(i). Both K+ ions also interact with specific H2O molecules in the first hydration shell of the octahedrally coordinated Mg2+ ion and with specific protein ligands. In crystals that have Na+ present, K+ is replaced by a Na+ ion at site 1 and by a Na(+)-H2O pair at site 2. The K+ ions are positioned where they could stabilize binding of a beta,gamma-bidentate MgATP complex with Hsc70, as well as a transition state during ATP hydrolysis, suggesting that monovalent ions act as specific metal cofactors in the ATPase reaction of Hsc70.

  View details for Web of Science ID A1995QE49300043

  View details for PubMedID 7836458

• HOW POTASSIUM AFFECTS THE ACTIVITY OF THE MOLECULAR CHAPERONE HSC70 .1. POTASSIUM IS REQUIRED FOR OPTIMAL ATPASE ACTIVITY JOURNAL OF BIOLOGICAL CHEMISTRY OBRIEN, M. C., McKay, D. B. 1995; 270 (5): 2247-2250

  Abstract

  Several functions of the 70-kilodalton heat shock cognate protein (Hsc70), such as peptide binding/release and clathrin uncoating, have been shown to require potassium ions. We have examined the effect of monovalent ions on the ATPase activity of Hsc70. The steady-state ATPase activities of Hsc70 and its amino-terminal 44-kDa ATPase fragment are minimal in the absence of K+ and reach a maximum at approximately 0.1 M [K+]. Activation of the ATPase turnover correlates with the ionic radii of monovalent ions; those that are at least 0.3 A smaller (Na+ and Li+) or larger (Cs+) than K+ show negligible activation, whereas ions with radii differing only approximately 0.1 A from that of K+ (NH4+ and Rb+) activate to approximately half the turnover rate observed with K+. Single turnover experiments with Hsc70 demonstrate that ATP hydrolysis is 5-fold slower with Na+ than with K+. The equilibrium binding of ADP or ATP to Hsc70 is unperturbed when K+ is replaced with Na+. These results are consistent with a role for monovalent ions as specific cofactors in the enzymatic hydrolysis of ATP.

  View details for Web of Science ID A1995QE49300042

  View details for PubMedID 7836457

• ATPASE KINETICS OF RECOMBINANT BOVINE 70 KDA HEAT-SHOCK COGNATE PROTEIN AND ITS AMINO-TERMINAL ATPASE DOMAIN BIOCHEMISTRY Ha, J. H., McKay, D. B. 1994; 33 (48): 14625-14635

  Abstract

  Steady-state kinetic, pre-steady-state kinetic, and equilibrium binding measurements have been applied to determine the rate constants of individual steps of the ATPase cycle for the recombinant bovine 70 kDa heat shock cognate protein and its amino-terminal 44 kDa ATPase fragment. At 25 degrees C, pH 7.0, in the presence of 75 mM KCl and 4.5 mM Mg2+, the measured association rate constants for MgATP approximately hsc70 and MgADP approximately hsc70 are (2.7 +/- 0.5) x 10(5) and (4.1 +/- 0.5) x 10(5) M-1 s-1, respectively, while the dissociation rate constants are 0.0114 (+/- 0.0002) and 0.0288 (+/- 0.0018) s-1, respectively. MgATP (Kd = 0.042 microM) therefore binds to hsc70 more tightly than MgADP (Kd = 0.11 microM). ADP release is inhibited by inorganic phosphate (Pi), suggesting that product dissociation is ordered with Pi released first and ADP second. The rate of chemical hydrolysis of ATP is 0.0030 (+/- 0.0003) s-1 for hsc70 and 0.0135 (+/- 0.0033) s-1 for the 44 kDa fragment. The rate of Pi release is 0.0038 (+/- 0.0010) s-1 for hsc70 and 0.0051 (+/- 0.0006) s-1 for the 44 kDa fragment. For the 44 kDa fragment, Pi release is the slowest step in the ATPase cycle, while for hsc70, Pi release and chemical hydrolysis of MgATP have similar rates; in both cases, ADP release is a relatively rapid step in the ATPase cycle.

  View details for Web of Science ID A1994PV91100031

  View details for PubMedID 7981225

• MODEL FOR AN RNA TERTIARY INTERACTION FRONT THE STRUCTURE OF AN INTERMOLECULAR COMPLEX BETWEEN A GAAA TETRALOOP AND AN RNA HELIX NATURE PLEY, H. W., Flaherty, K. M., McKay, D. B. 1994; 372 (6501): 111-113

  Abstract

  In large structured RNAs, RNA hairpins in which the strands of the duplex stem are connected by a tetraloop of the consensus sequence 5'-GNRA (where N is any nucleotide, and R is either G or A) are unusually frequent. In group I introns there is a covariation in sequence between nucleotides in the third and fourth positions of the loop with specific distant base pairs in putative RNA duplex stems: GNAA loops correlate with successive 5'-C-C.G-C base pairs in stems, whereas GNGA loops correlate with 5'-C-U.G-A. This has led to the suggestion that GNRA tetraloops may be involved in specific long-range tertiary interactions, with each A in position 3 or 4 of the loop interacting with a C-G base pair in the duplex, and G in position 3 interacting with a U-A base pair. This idea is supported experimentally for the GAAA loop of the P5b extension of the group I intron of Tetrahymena thermophila and the L9 GUGA terminal loop of the td intron of bacteriophage T4 (ref. 4). NMR has revealed the overall structure of the tetraloop for 12-nucleotide hairpins with GCAA and GAAA loops and models have been proposed for the interaction of GNRA tetraloops with base pairs in the minor groove of A-form RNA. Here we describe the crystal structure of an intermolecular complex between a GAAA tetraloop and an RNA helix. The interactions we observe correlate with the specificity of GNRA tetraloops inferred from phylogenetic studies, suggesting that this complex is a legitimate model for intramolecular tertiary interactions mediated by GNRA tetraloops in large structured RNAs.

  View details for Web of Science ID A1994PQ34800085

  View details for PubMedID 7526219

• 3-DIMENSIONAL STRUCTURE OF A HAMMERHEAD RIBOZYME NATURE PLEY, H. W., Flaherty, K. M., McKay, D. B. 1994; 372 (6501): 68-74

  Abstract

  The hammerhead ribozyme is a small catalytic RNA motif made up of three base-paired stems and a core of highly conserved, non-complementary nucleotides essential for catalysis. The X-ray crystallographic structure of a hammerhead RNA-DNA ribozyme-inhibitor complex at 2.6 A resolution reveals that the base-paired stems are A-form helices and that the core has two structural domains. The first domain is formed by the sequence 5'-CUGA following stem I and is a sharp turn identical to the uridine turn of transfer RNA, whereas the second is a non-Watson-Crick three-base-pair duplex with a divalent-ion binding site. The phosphodiester backbone of the DNA inhibitor strand is splayed out at the phosphate 5' to the cleavage site. The structure indicates that the ribozyme may destabilize a substrate strand in order to facilitate twisting of the substrate to allow cleavage of the scissile bond.

  View details for Web of Science ID A1994PQ34800071

  View details for PubMedID 7969422

• STRUCTURAL BASIS OF THE 70-KILODALTON HEAT-SHOCK COGNATE PROTEIN ATP HYDROLYTIC ACTIVITY .2. STRUCTURE OF THE ACTIVE-SITE WITH ADP OR ATP BOUND TO WILD-TYPE AND MUTANT ATPASE FRAGMENT JOURNAL OF BIOLOGICAL CHEMISTRY Flaherty, K. M., Wilbanks, S. M., DELUCAFLAHERTY, C., McKay, D. B. 1994; 269 (17): 12899-12907

  Abstract

  The ATPase fragment of the bovine 70-kDa heat shock cognate protein is an attractive construct in which to study its mechanism of ATP hydrolysis. The three-dimensional structure suggests several residues that might participate in the ATPase reaction. Four acidic residues (Asp-10, Glu-175, Asp-199, and Asp-206) have been individually mutated to both the cognate amine (asparagine/glutamine) and to serine, and the effects of the mutations on the kinetics of the ATPase activity (Wilbanks, S. M., DeLuca-Flaherty, C., and McKay, D. B. (1994) J. Biol. Chem. 269, 12893-12898) and the structure of the mutant ATPase fragments have been determined, typically to approximately 2.4 A resolution. Additionally, the structures of the wild type protein complexed with MgADP and Pi, MgAMPPNP (5'-adenylyl-beta, gamma-imidodiphosphate) and CaAMPPNP have been refined to 2.1, 2.4, and 2.4 A, respectively. Combined, these structures provide models for the prehydrolysis, MgATP-bound state and the post-hydrolysis, MgADP-bound state of the ATPase fragment. These models suggest a pathway for the hydrolytic reaction in which 1) the gamma phosphate of bound ATP reorients to form a beta, gamma-bidentate phosphate complex with the Mg2+ ion, allowing 2) in-line nucleophilic attack on the gamma phosphate by a H2O molecule or OH- ion, with 3) subsequent release of inorganic phosphate.

  View details for Web of Science ID A1994NH71600081

  View details for PubMedID 8175707

• STRUCTURAL BASIS OF THE 70-KILODALTON HEAT-SHOCK COGNATE PROTEIN ATP HYDROLYTIC ACTIVITY .1. KINETIC ANALYSES OF ACTIVE-SITE MUTANTS JOURNAL OF BIOLOGICAL CHEMISTRY Wilbanks, S. M., DELUCAFLAHERTY, C., McKay, D. B. 1994; 269 (17): 12893-12898

  Abstract

  The 70-kDa heat shock cognate protein is a member of a highly conserved family of molecular chaperones in which the binding and release of target polypeptides are coupled to the chaperones' ATPase activity. The ATPase activity resides in the amino-terminal 44-kDa fragment of the protein. Four acidic residues of the ATPase fragment which might participate in catalysis (Asp-10 and Asp-199, which are Mg2+ ion ligands; Glu-175 and Asp-206, which are candidates for a role as catalytic base) have been individually mutated to both the cognate amide residue (aspartate to asparagine, glutamate to glutamine) and to serine, and the effects of the mutations on the kinetics (this manuscript) and structure (Flaherty, K.M., Wilbanks, S. M., DeLuca-Flaherty, C., and McKay, D. B. (1994) J. Biol. Chem. 269, 12899-12907) have been determined. Changes at Asp-10 and Asp-199 reduced kcat to approximately 1% of wild type; changes at Glu-175 and Asp-206 reduced kcat to approximately 10% of wild type. Changes to Asp-199 and Asp-206 had little effect on Km; changes to Asp-10 and Glu-175 increased Km 10-100-fold. These data suggest that the bound magnesium ion and its local environment are crucial to catalysis; they argue against a single residue acting as the sole essential general base catalyst in the hydrolytic reaction.

  View details for Web of Science ID A1994NH71600080

  View details for PubMedID 8175706

• DEFINITION AND SPATIAL LOCATION OF MOUSE INTERLEUKIN-2 RESIDUES THAT INTERACT WITH ITS HETEROTRIMERIC RECEPTOR EMBO JOURNAL Zurawski, S. M., Vega, F., Doyle, E. L., Huyghe, B., Flaherty, K., McKay, D. B., Zurawski, G. 1993; 12 (13): 5113-5119

  Abstract

  The high affinity receptor for interleukin-2 (IL-2) contains three subunits called IL-2R alpha, beta and gamma. A biological and receptor binding analysis based on 1393 different mutant mouse IL-2 (mIL-2) proteins was used to define the function of each of the 149 residues. By this genetic analysis, 44 residues were assigned important functions, 21 of which were structural. The remaining 23 residues consisted of 19 residues, from three separate regions, that were important for IL-2R alpha interaction; three residues, from two separate regions, that were important for IL-2R beta interaction; and a single residue important for IL-2R gamma interaction. We built a model mIL-2 structure based on the homologous human IL-2 (hIL-2) crystal structure. The roles of the 21 residues presumed to be important for structure were consistent with the model. Despite discontinuity in the primary sequence, the residues specific for each IL-2R subunit interaction were clustered and located to three disparate regions of the tertiary mIL-2 structure. The relative spatial locations of these three surfaces are different from the two receptor binding sites known for the structurally related human growth hormone and the significance of this observation is discussed.

  View details for Web of Science ID A1993MM12000023

  View details for PubMedID 8262055

  View details for PubMedCentralID PMC413773

• 3-DIMENSIONAL STRUCTURE OF RECOVERIN, A CALCIUM SENSOR IN VISION CELL Flaherty, K. M., Zozulya, S., Stryer, L., McKay, D. B. 1993; 75 (4): 709-716

  Abstract

  Recoverin, a recently discovered member of the EF hand superfamily, serves as a calcium sensor in vision. We report here the crystal structure of recombinant unmyristoylated recoverin at 1.9 A resolution. The four EF hands of the protein are arranged in a compact array that contrasts with the dumbbell shape of calmodulin and troponin C. A calcium ion is bound to EF hand 3, while EF hand 2 can bind samarium but not calcium in this crystal form. The other two EF hands have novel structural features that prevent or impair calcium binding. A concave hydrophobic surface formed by EF hands 1 and 2 may participate in the read out of calcium signals by recoverin and its homologs.

  View details for Web of Science ID A1993MH74900016

  View details for PubMedID 8242744

• THREONINE-204 OF THE CHAPERONE PROTEIN HSC70 INFLUENCES THE STRUCTURE OF THE ACTIVE-SITE, BUT IS NOT ESSENTIAL FOR ATP HYDROLYSIS JOURNAL OF BIOLOGICAL CHEMISTRY OBRIEN, M. C., McKay, D. B. 1993; 268 (32): 24323-24329

  Abstract

  The chaperone protein Hsc70 is an ATPase of unknown mechanism, although the crystal structure of the 44-kDa ATPase domain has been solved. This structure shows that the hydroxyl of threonine 204 is located close to the gamma-phosphate of ATP, in a position where it might be an intermediate phosphate acceptor in the hydrolysis reaction. We made two point mutations at residue 204 of Hsc70, threonine to valine (T204V) and threonine to glutamic acid (T204E). The wild-type ATPase domain had a Km for ATP of approximately 1 microM; the mutants had Km values of approximately 90 microM. The kcat values for the mutant proteins were also increased. After crystallization, the structures of the T204V and T204E proteins were solved and refined with data to 2.3- and 2.4-A resolution, respectively. The overall tertiary structure of the mutants showed little change from the wild type; however, significant changes were observed in the active site. Analysis of the structures suggested possible reasons for the changes in kinetic constants. Threonine 204 does not seem to be an obligatory intermediate phosphate acceptor in the hydrolysis reaction since the mutants retained appreciable ATPase activity.

  View details for Web of Science ID A1993MF29400091

  View details for PubMedID 8226982

• CRYSTALS OF A HAMMERHEAD RIBOZYME JOURNAL OF BIOLOGICAL CHEMISTRY PLEY, H. W., LINDES, D. S., DELUCAFLAHERTY, C., McKay, D. B. 1993; 268 (26): 19656-19658

  Abstract

  Hammerhead ribozyme-inhibitor complexes consisting of an RNA "enzyme" strand and a DNA "inhibitor" strand have been crystallized in four different crystal forms. The crystal form that is most attractive for structure determination diffracts to approximately 3.2 A resolution; it is trigonal, space group P3(1)21 or enantiomorph, a = 92.5 A, c = 185.0 A.

  View details for Web of Science ID A1993LW81900078

  View details for PubMedID 8366108

• 3-DIMENSIONAL STRUCTURE OF THE ALKALINE PROTEASE OF PSEUDOMONAS-AERUGINOSA - A 2-DOMAIN PROTEIN WITH A CALCIUM-BINDING PARALLEL-BETA ROLL MOTIF EMBO JOURNAL Baumann, U., Wu, S., Flaherty, K. M., McKay, D. B. 1993; 12 (9): 3357-3364

  Abstract

  The three-dimensional structure of the alkaline protease of Pseudomonas aeruginosa, a zinc metalloprotease, has been solved to a resolution of 1.64 A by multiple isomorphous replacement and non-crystallographic symmetry averaging between different crystal forms. The molecule is elongated with overall dimensions of 90 x 35 x 25 A; it has two distinct structural domains. The N-terminal domain is the proteolytic domain; it has an overall tertiary fold and active site zinc ligation similar to that of astacin, a metalloprotease isolated from a European freshwater crayfish. The C-terminal domain consists of a 21-strand beta sandwich. Within this domain is a novel 'parallel beta roll' structure in which successive beta strands are wound in a right-handed spiral, and in which Ca2+ ions are bound within the turns between strands by a repeated GGXGXD sequence motif, a motif that is found in a diverse group of proteins secreted by Gram-negative bacteria.

  View details for Web of Science ID A1993LU51300002

  View details for PubMedID 8253063

• STRUCTURE AND MECHANISM OF 70-KDA HEAT-SHOCK-RELATED PROTEINS ADVANCES IN PROTEIN CHEMISTRY, VOL 44 McKay, D. B. 1993; 44: 67-98

  View details for Web of Science ID A1993BZ69T00003

  View details for PubMedID 8317298

• STRUCTURAL COMPARISON SUGGESTS THAT THERMOLYSIN AND RELATED NEUTRAL PROTEASES UNDERGO HINGE-BENDING MOTION DURING CATALYSIS BIOCHEMISTRY Holland, D. R., Tronrud, D. E., PLEY, H. W., Flaherty, K. M., Stark, W., Jansonius, J. N., McKay, D. B., Matthews, B. W. 1992; 31 (46): 11310-11316

  Abstract

  Crystal structures are known for three members of the bacterial neutral protease family: thermolysin from Bacillus thermoproteolyticus (TLN), the neutral protease from Bacillus cereus (NEU), and the elastase of Pseudomonas aeruginosa (PAE), both in free and ligand-bound forms. Each enzyme consists of an N-terminal and C-terminal domain with the active site formed at the junction of the two domains. Comparison of the different molecules reveals that the structure within each domain is well conserved, but there are substantial hinge-bending displacements (up to 16 degrees) of one domain relative to the other. These domain motions can be correlated with the presence or absence of bound inhibitor, as was previously observed in the specific example of PAE [Thayer, M.M., Flaherty, K.M., & McKay, D.B. (1991) J. Biol. Chem. 266, 2864-2871]. The binding of inhibitor appears to be associated with a reduction of the domain hinge-bending angle by 6-14 degrees and a closure of the "jaws" of the active site cleft by about 2 A. Crystallographic refinement of the structure of thermolysin suggests that electron density seen in the active site of the enzyme in the original structure determination probably corresponds to a bound dipeptide. Thus, the crystal structure appears to correspond to an enzyme-inhibitor or enzyme-product complex, rather than the free enzyme, as has previously been assumed.

  View details for Web of Science ID A1992JZ52300008

  View details for PubMedID 1445869

• CLONING, EXPRESSION, AND CRYSTALLIZATION OF RECOVERIN, A CALCIUM SENSOR IN VISION PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ray, S., Zozulya, S., Niemi, G. A., Flaherty, K. M., BROLLEY, D., Dizhoor, A. M., McKay, D. B., Hurley, J., Stryer, L. 1992; 89 (13): 5705-5709

  Abstract

  Recoverin, a recently discovered 23-kDa calcium-binding protein, activates retinal rod guanylate cyclase when the calcium level is lowered in the submicromolar range. We report here the cloning and sequencing of a cDNA for recoverin from a bovine retinal expression library. The recoverin coding sequence was inserted into a pET-11a expression vector under control of the T7 phage promoter. A second expression system, in which the coding sequence was placed under control of the lambda phage PR promoter, gave 10-fold higher yields (10 mg of purified recoverin per liter of Escherichia coli culture). The finding that retinal recoverin is myristoylated at its amino terminus led us to coexpress the recombinant protein and N-myristoyltransferase (EC 2.3.1.97). Myristoylated recombinant recoverin formed in this way in E. coli is like retinal recoverin in exhibiting a large calcium-induced shift in its tryptophan fluorescence emission spectrum. The availability of abundant protein enabled us to crystallize unmyristoylated recombinant recoverin and initiate x-ray studies. The space group of tetragonal crystals obtained from 75% saturation ammonium sulfate is I4 with unit cell dimensions a = 85.1 A and c = 59.8 A. These crystals of the calcium-bound form of the protein diffracted to a resolution of 2.2 A. The expression systems described here open the door to high-resolution x-ray crystallographic and nuclear magnetic resonance studies of this new member of the EF-hand superfamily and to the elucidation of its precise mode of action as a calcium switch.

  View details for Web of Science ID A1992JC86800002

  View details for PubMedID 1385864

  View details for PubMedCentralID PMC49365

• Crystallographic structures of the elastase of Pseudomonas aeruginosa. Matrix (Stuttgart, Germany). Supplement McKay, D. B., Thayer, M. M., Flaherty, K. M., Pley, H., BENVEGNU, D. 1992; 1: 112-115

  Abstract

  The elastase protein of Pseudomonas aeruginosa is a zinc metalloprotease which has been shown to be a member of the bacterial neutral protease family. Its overall tertiary structure is similar to that of thermolysin. The x-ray crystallographic structure of the elastase has been solved to high resolution in three different crystal forms. Substantial conformational differences are observed in the protein in different crystal forms. In the absence of ligand, and independently in the presence of a covalent noncompetitive inhibitor, the elastase is observed to have a relatively "open" substrate binding cleft, while in the presence of tight-binding competitive inhibitors, the active site cleft is "closed".

  View details for PubMedID 1480011

• SIMILARITY OF THE 3-DIMENSIONAL STRUCTURES OF ACTIN AND THE ATPASE FRAGMENT OF A 70-KDA HEAT-SHOCK COGNATE PROTEIN PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Flaherty, K. M., McKay, D. B., Kabsch, W., Holmes, K. C. 1991; 88 (11): 5041-5045

  Abstract

  Although there is very little sequence identity between the two proteins, the structures of rabbit skeletal muscle actin (375-amino acid residues) and the 44-kDa ATPase fragment of the bovine 70-kDa heat shock cognate protein (HSC70; 386 residues) are very similar. The alpha-carbon positions of 241 pairs of amino acid residues that are structurally equivalent within the two proteins can be superimposed with a root-mean-square difference in distance of 2.3 A; of these, 39 residues are identical, and 56 are conservative substitutions. In addition, the conformations of ADP are very similar in both proteins. A local sequence "fingerprint," which may be diagnostic of the adenine nucleotide beta-phosphate-binding pocket, has been derived. The fingerprint identifies members of the glycerol kinase family as candidates likely to have a similar structure in their nucleotide-binding domains. The structural differences between the two molecules mainly occur in loop regions of actin known to be involved in interactions with other monomers in the actin filament or in the binding of myosin; the corresponding regions in heat shock proteins may have functions that are as yet undetermined. Placing the Ca2+ ATP of actin on the ATPase fragment structure suggests Asp-206 (corresponding to His-161 of actin) as a candidate proton acceptor for the ATPase reaction.

  View details for Web of Science ID A1991FP08700099

  View details for PubMedID 1828889

• 3-DIMENSIONAL STRUCTURE OF THE ELASTASE OF PSEUDOMONAS-AERUGINOSA AT 1.5-A RESOLUTION JOURNAL OF BIOLOGICAL CHEMISTRY Thayer, M. M., Flaherty, K. M., McKay, D. B. 1991; 266 (5): 2864-2871

  Abstract

  Pseudomonas aeruginosa elastase (PAE) is a zinc metalloprotease with 301 amino acids. We have crystallized and solved the three-dimensional structure of PAE, using data to 1.5-A resolution, and have refined the native molecular structure to R = 0.188. The overall tertiary structure of the PAE molecule is similar to that of thermolysin, with which it shares 28% amino acid sequence identity. Nearly all of the active site residues that might potentially interact with substrates are identical in the two proteins. However, the active site cleft is significantly more "open" in PAE than in thermolysin.

  View details for Web of Science ID A1991EX60000033

  View details for PubMedID 1899664

• STRUCTURE OF THE 70-KILODALTON HEAT-SHOCK-RELATED PROTEINS SPRINGER SEMINARS IN IMMUNOPATHOLOGY McKay, D. B. 1991; 13 (1): 1-9

  View details for Web of Science ID A1991GM12400001

  View details for PubMedID 1776119

• NUCLEOTIDE-SEQUENCE OF THE CDNA OF A BOVINE-70 KILODALTON HEAT-SHOCK COGNATE PROTEIN NUCLEIC ACIDS RESEARCH DELUCAFLAHERTY, C., McKay, D. B. 1990; 18 (18): 5569-5569

  View details for Web of Science ID A1990EA88200055

  View details for PubMedID 2216746

• UNCOATING PROTEIN (HSC70) BINDS A CONFORMATIONALLY LABILE DOMAIN OF CLATHRIN LIGHT CHAIN LCA TO STIMULATE ATP HYDROLYSIS CELL DELUCAFLAHERTY, C., McKay, D. B., Parham, P., Hill, B. L. 1990; 62 (5): 875-887

  Abstract

  Uncoating of clathrin-coated vesicles is mediated by the heat shock cognate protein, hsc70, and requires clathrin light chains (LCa and LCb) and ATP hydrolysis. We demonstrate that purified light chains and synthetic peptides derived from their sequences bind hsc70 to stimulate ATP hydrolysis. LCa is more effective than LCb in stimulating hsc70 ATPase and in inhibiting clathrin uncoating by hsc70. These differences correlate with high sequence divergence in the proline- and glycine-rich region (residues 47-71) that forms the hsc70 binding site. For LCa, but not LCb, this region undergoes reversible conformational changes upon perturbation of the ionic strength or the calcium ion concentration. Our results show that LCa is more important for interactions with hsc70 than is LCb and suggest a model in which the LCa conformation regulates coated vesicle uncoating.

  View details for Web of Science ID A1990DY10000007

  View details for PubMedID 1975516

• 3-DIMENSIONAL STRUCTURE OF THE ATPASE FRAGMENT OF A 70K HEAT-SHOCK COGNATE PROTEIN NATURE Flaherty, K. M., DELUCAFLAHERTY, C., McKay, D. B. 1990; 346 (6285): 623-628

  Abstract

  The three-dimensional structure of the amino-terminal 44K ATPase fragment of the 70K bovine heat-shock cognate protein has been solved to a resolution of 2.2 A. The ATPase fragment has two structural lobes with a deep cleft between them; ATP binds at the base of the cleft. Surprisingly, the nucleotide-binding 'core' of the ATPase fragment has a tertiary structure similar to that of hexokinase, although the remainder of the structures of the two proteins are completely dissimilar, suggesting that both the phosphotransferase mechanism and the substrate-induced conformational change intrinsic to the hexokinases may be used by the 70K heat shock-related proteins.

  View details for Web of Science ID A1990DU72800050

  View details for PubMedID 2143562

• STRUCTURE OF EXOTOXIN-A OF PSEUDOMONAS-AERUGINOSA AT 3.0-ANGSTROM RESOLUTION PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA ALLURED, V. S., Collier, R. J., Carroll, S. F., McKay, D. B. 1986; 83 (5): 1320-1324

  Abstract

  Exotoxin A of Pseudomonas aeruginosa is a secreted bacterial toxin capable of translocating a catalytic domain into mammalian cells and inhibiting protein synthesis by the ADP-ribosylation of cellular elongation factor 2. The protein is a single polypeptide chain of 613 amino acids. The x-ray crystallographic structure of exotoxin A, determined to 3.0-A resolution, shows the following: an amino-terminal domain, composed primarily of antiparallel beta-structure and comprising approximately half of the molecule; a middle domain composed of alpha-helices; and a carboxyl-terminal domain comprising approximately one-third of the molecule. The carboxyl-terminal domain is the ADP-ribosyltransferase of the toxin. The other two domains are presumably involved in cell receptor binding and membrane translocation.

  View details for Web of Science ID A1986A426100032

  View details for PubMedID 3006045

• X-RAY GRADE CRYSTALS OF DIPHTHERIA-TOXIN JOURNAL OF BIOLOGICAL CHEMISTRY Collier, R. J., Westbrook, E. M., McKay, D. B., Eisenberg, D. 1982; 257 (9): 5283-5285

  Abstract

  Diphtheria toxin, complexed with the endogenous dinucleotide ApUp, has been crystallized under novel conditions involving high concentrations of both polyethylene glycol and salt. Among four crystal forms obtained, one was found suitable for high resolution structural analysis by x-ray diffraction. This form is triclinic (space group P1) with 2 molecules per unit cell; unit cell parameters are a = 70.8 A, b = 70.7 A, c = 65.3 A, alpha = 95.2 degrees, beta = 91.3 degrees, gamma = 99.7 degrees.

  View details for Web of Science ID A1982NN57300097

  View details for PubMedID 7068685