Jennifer L. Martin

The structure of the caspase recruitment domain of BinCARD reveals that all three cysteines can be oxidized

Publisher: International Union of Crystallography (IUCr)

Date: 11-04-2013

DOI: 10.1107/S0907444913001558

Molecular Recognition of Sub-micromolar Inhibitors by the Epinephrine-Synthesizing Enzyme Phenylethanolamine N-Methyltransferase

Publisher: American Chemical Society (ACS)

Date: 09-12-2003

DOI: 10.1021/JM0205752

Abstract: The crystal structures of human phenylethanolamine N-methyltransferase in complex with S-adenosyl-l-homocysteine (7, AdoHcy) and either 7-iodo-1,2,3,4-tetrahydroisoquinoline (2) or 8,9-dichloro-2,3,4,5-tetrahydro-1H-2-benzazepine (3, LY134046) were determined and compared with the structure of the enzyme complex with 7 and 7-aminosulfonyl-1,2,3,4-tetrahydroisoquinoline (1, SK&F 29661). The enzyme is able to accommodate a variety of chemically disparate functional groups on the aromatic ring of the inhibitors through adaptation of the binding pocket for this substituent and by subtle adjustments of the orientation of the inhibitors within the relatively planar binding site. In addition, the interactions formed by the amine nitrogen of all three inhibitors reinforce the hypothesis that this functional group mimics the beta-hydroxyl of norepinephrine rather than the amine. These studies provide further clues for the development of improved inhibitors for use as pharmacological probes.

Protein disulfide isomerase: the structure of oxidative folding.

Publisher: Elsevier BV

Date: 08-2006

DOI: 10.1016/J.TIBS.2006.06.001

Abstract: Cellular functions hinge on the ability of proteins to adopt their correct folds, and misfolded proteins can lead to disease. Here, we focus on the proteins that catalyze disulfide bond formation, a step in the oxidative folding pathway that takes place in specialized cellular compartments. In the endoplasmic reticulum of eukaryotes, disulfide formation is catalyzed by protein disulfide isomerase (PDI) by contrast, prokaryotes produce a family of disulfide bond (Dsb) proteins, which together achieve an equivalent outcome in the bacterial periplasm. The recent crystal structure of yeast PDI has increased our understanding of the function and mechanism of PDI. Comparison of the structure of yeast PDI with those of bacterial DsbC and DsbG reveals some similarities but also striking differences that suggest directions for future research aimed at unraveling the catalytic mechanism of disulfide bond formation in the cell.

DisarmingBurkholderia pseudomallei: Structural and Functional Characterization of a Disulfide Oxidoreductase (DsbA) Required for VirulenceIn Vivo

Publisher: Mary Ann Liebert Inc

Date: 02-2014

DOI: 10.1089/ARS.2013.5375

Crystal Structure of the Dithiol Oxidase DsbA Enzyme from Proteus Mirabilis Bound Non-covalently to an Active Site Peptide Ligand

Publisher: Elsevier BV

Date: 07-2014

DOI: 10.1074/JBC.M114.552380

The mammalian DUF59 protein Fam96a forms two distinct types of domain-swapped dimer

Publisher: International Union of Crystallography (IUCr)

Date: 17-05-2012

DOI: 10.1107/S0907444912006592

The Weak Complex between RhoGAP Protein ARHGAP22 and Signal Regulatory Protein 14-3-3 Has 1:2 Stoichiometry and a Single Peptide Binding Mode

Publisher: Public Library of Science (PLoS)

Date: 28-08-2012

DOI: 10.1371/JOURNAL.PONE.0041731

Milligram Quantities of Homogeneous Recombinant Full-Length Mouse Munc18c from Escherichia coli Cultures

Publisher: Public Library of Science (PLoS)

Date: 31-12-2013

DOI: 10.1371/JOURNAL.PONE.0083499

Comparison of three commercial sparse-matrix crystallization screens

Publisher: International Union of Crystallography (IUCr)

Date: 25-03-2003

DOI: 10.1107/S0907444903002919

Abstract: Sparse-matrix s ling using commercially available crystallization screen kits has become the most popular way of determining the preliminary crystallization conditions for macromolecules. In this study, the efficiency of three commercial screening kits, Crystal Screen and Crystal Screen 2 (H ton Research), Wizard Screens I and II (Emerald BioStructures) and Personal Structure Screens 1 and 2 (Molecular Dimensions), has been compared using a set of 19 erse proteins. 18 proteins yielded crystals using at least one crystallization screen. Surprisingly, Crystal Screens and Personal Structure Screens showed dramatically different results, although most of the crystallization formulations are identical as listed by the manufacturers. Higher molecular weight polyethylene glycols and mixed precipitants were found to be the most effective precipitants in this study.

The nature of the Syntaxin4 C-terminus affects Munc18c-supported SNARE assembly

Publisher: Public Library of Science (PLoS)

Date: 25-08-2017

DOI: 10.1371/JOURNAL.PONE.0183366

Structure of West Nile Virus NS3 Protease: Ligand Stabilization of the Catalytic Conformation

Publisher: Elsevier BV

Date: 02-2009

DOI: 10.1016/J.JMB.2008.11.026

Abstract: Over the last decade, West Nile virus has spread rapidly via mosquito transmission from infected migratory birds to humans. One potential therapeutic approach to treating infection is to inhibit the virally encoded serine protease that is essential for viral replication. Here we report the crystal structure of the viral NS3 protease tethered to its essential NS2B cofactor and bound to a potent substrate-based tripeptide inhibitor, 2-naphthoyl-Lys-Lys-Arg-H (K(i)=41 nM), capped at the N-terminus by 2-naphthoyl and capped at the C-terminus by aldehyde. An important and unexpected feature of this structure is the presence of two conformations of the catalytic histidine suggesting a role for ligand stabilization of the catalytically competent His conformation. Analysis of other West Nile virus NS3 protease structures and related serine proteases supports this hypothesis, suggesting that the common catalytic mechanism involves an induced-fit mechanism.

Expression and crystallization of DsbA fromStaphylococcus aureus

Publisher: International Union of Crystallography (IUCr)

Date: 24-10-2007

DOI: 10.1107/S174430910704821X

Comparative studies of Munc18c and Munc18-1 reveal conserved and divergent mechanisms of Sec1/Munc18 proteins

Publisher: Proceedings of the National Academy of Sciences

Date: 05-08-2013

DOI: 10.1073/PNAS.1311232110

Abstract: Sec1/Munc18 (SM) proteins are essential for every vesicle fusion pathway, but their molecular mechanisms remain poorly understood. Our comparative studies of two functionally distinct SM proteins, Munc18c and Munc18-1, suggest that one conserved function of SM proteins is to recognize their cognate trans -SNARE complexes and accelerate fusion kinetics. The “closed” syntaxin binding mode of Munc18-1, however, is not conserved in Munc18c. Unexpectedly, we discovered that the architecture of the SNARE/SM complex differs across fusion pathways. Together, these findings reveal conserved as well as ergent functions of SM proteins in vesicle fusion.

Backbone and side chain H-1, N-15 and C-13 assignments for the oxidised and reduced forms of the oxidoreductase protein DsbA from Staphylococcus aureus

Publisher: Springer Science and Business Media LLC

Date: 20-11-2010

DOI: 10.1007/S12104-009-9199-8

Abstract: The function and dynamics of the thiol-disulfide oxidoreductase DsbA in the low-GC gram positive bacterium, Staphylococcus aureus, are yet to be elucidated. Here we report 13C, 15N and 1H assignments for the oxidised and reduced forms of SaDsbA as a prelude to further studies on the enzyme.

Reducing Loss of Resin Flowing in Porous Fibrous Media in Simulation of Composites Fabrication

Publisher: Wiley

Date: 2010

DOI: 10.1002/PC.20790

Disulfide-linked dimers of human adrenaline synthesizing enzyme PNMT are catalytically active

Publisher: Elsevier BV

Date: 06-2005

DOI: 10.1016/J.BBAPAP.2005.03.006

Abstract: The crystal structure of human phenylethanolamine N-methyltransferase (hPNMT) reveals a disulfide-linked dimer, despite the presence of reducing agent in the crystallisation conditions. By removing the reducing agent, hPNMT crystals grow more rapidly and at lower protein concentrations. However, it was unclear whether the disulfide bonds are only present in the crystal form or whether these affect enzyme activity. The solution oligomeric state of hPNMT was investigated using biochemical techniques and activity assays. We found that in the absence of reducing agent, hPNMT forms dimers in solution. Furthermore, the solution dimer of hPNMT incorporates disulfide bonds, since this form is sensitive to reducing agent. The C48A and C139A mutants of hPNMT, which are incapable of forming the disulfide bond observed in the crystal structure, have a decreased propensity to form dimer in solution. Those dimers that do form are also sensitive to reducing agent. Further, the C48A/C139A double mutant shows only monomeric behaviour. Both dimeric and monomeric hPNMT, as well as mutants have wildtype enzyme activity. These results show that a variety of disulfides, including those observed in the crystal structure, can form in solution. In addition, disulfide-linked dimers are as active as the monomeric enzyme indicating that the crystal structure of the protein is a valid target for inhibitor design.

Crystal structures of the DsbG disulfide isomerase reveal an unstable disulfide

Publisher: Proceedings of the National Academy of Sciences

Date: 07-06-2004

DOI: 10.1073/PNAS.0402769101

Abstract: Dsb proteins control the formation and rearrangement of disulfide bonds during the folding of secreted and membrane proteins in bacteria. DsbG, a member of this family, has disulfide bond isomerase and chaperone activity. Here, we present two crystal structures of DsbG at 1.7and 2.0-Å resolution that are meant to represent the reduced and oxidized forms, respectively. The oxidized structure, however, reveals a mixture of both redox forms, suggesting that oxidized DsbG is less stable than the reduced form. This trait would contribute to DsbG isomerase activity, which requires that the active-site Cys residues are kept reduced, regardless of the highly oxidative environment of the periplasm. We propose that a Thr residue that is conserved in the cis -Pro loop of DsbG and DsbC but not found in other Dsb proteins could play a role in this process. Also, the structure of DsbG reveals an unanticipated and surprising feature that may help define its specific role in oxidative protein folding. Thus, the dimensions and surface features of DsbG show a very large and charged binding surface that is consistent with interaction with globular protein substrates having charged surfaces. This finding suggests that, rather than catalyzing disulfide rearrangement in unfolded substrates, DsbG may preferentially act later in the folding process to catalyze disulfide rearrangement in folded or partially folded proteins.

Crystal structures of free, IMP-, and GMP-bound Escherichia coli hypoxanthine phosphoribosyltransferase

Publisher: Wiley

Date: 07-2002

DOI: 10.1110/PS.0201002

Abstract: Crystal structures have been determined for free Escherichia coli hypoxanthine phosphoribosyltransferase (HPRT) (2.9 A resolution) and for the enzyme in complex with the reaction products, inosine 5'-monophosphate (IMP) and guanosine 5'-monophosphate (GMP) (2.8 A resolution). Of the known 6-oxopurine phosphoribosyltransferase (PRTase) structures, E. coli HPRT is most similar in structure to that of Tritrichomonas foetus HGXPRT, with a rmsd for 150 Calpha atoms of 1.0 A. Comparison of the free and product bound structures shows that the side chain of Phe156 and the polypeptide backbone in this vicinity move to bind IMP or GMP. A nonproline cis peptide bond, also found in some other 6-oxopurine PRTases, is observed between Leu46 and Arg47 in both the free and complexed structures. For catalysis to occur, the 6-oxopurine PRTases have a requirement for alent metal ion, usually Mg(2+) in vivo. In the free structure, a Mg(2+) is coordinated to the side chains of Glu103 and Asp104. This interaction may be important for stabilization of the enzyme before catalysis. E. coli HPRT is unique among the known 6-oxopurine PRTases in that it exhibits a marked preference for hypoxanthine as substrate over both xanthine and guanine. The structures suggest that its substrate specificity is due to the modes of binding of the bases. In E. coli HPRT, the carbonyl oxygen of Asp163 would likely form a hydrogen bond with the 2-exocyclic nitrogen of guanine (in the HPRT-guanine-PRib-PP-Mg(2+) complex). However, hypoxanthine does not have a 2-exocyclic atom and the HPRT-IMP structure suggests that hypoxanthine is likely to occupy a different position in the purine-binding pocket.

Comparison of the Binding of 3-Fluoromethyl-7-sulfonyl-1,2,3,4-tetrahydroisoquinolines with Their Isosteric Sulfonamides to the Active Site of Phenylethanolamine N-Methyltransferase

Publisher: American Chemical Society (ACS)

Date: 11-08-2006

DOI: 10.1021/JM060466D

Abstract: 3-Fluoromethyl-7-(N-substituted aminosulfonyl)-1,2,3,4-tetrahydroisoquinolines (14, 16, and 18-22) are highly potent and selective inhibitors of phenylethanolamine N-methyltransferase (PNMT). Molecular modeling studies with 3-fluoromethyl-7-(N-alkyl aminosulfonyl)-1,2,3,4-tetrahydroisoquinolines, such as 16, suggested that the sulfonamide -NH- could form a hydrogen bond with the side chain of Lys57. However, SAR studies and analysis of the crystal structure of human PNMT (hPNMT) in complex with 7 indicated that the sulfonamide oxygens, and not the sulfonamide -NH-, formed favorable interactions with the enzyme. Thus, we hypothesized that replacement of the sulfonamide -NH- with a methylene group could result in compounds that would retain potency at PNMT and that would have increased lipophilicity, thus increasing the likelihood they will cross the blood brain barrier. A series of 3-fluoromethyl-7-sulfonyl-1,2,3,4-tetrahydroisoquinolines (23-30) were synthesized and evaluated for their PNMT inhibitory potency and affinity for the alpha2-adrenoceptor. A comparison of these compounds with their isosteric sulfonamides (14, 16, and 18-22) showed that the sulfones were more lipophilic but less potent than their corresponding sulfonamides. Sulfone 24 (hPNMT Ki = 1.3 microM) is the most potent compound in this series and is quite selective for PNMT versus the alpha2-adrenoceptor, but 24 is less potent than the corresponding sulfonamide, 16 (hPNMT Ki = 0.13 microM). We also report the crystal structure of hPNMT in complex with sulfonamide 15, from which a potential hydrogen bond acceptor within the hPNMT active site has been identified, the main chain carbonyl oxygen of Asn39. The interaction of this residue with the sulfonamide -NH- is likely responsible for much of the enhanced inhibitory potency of the sulfonamides versus the sulfones.

Enzyme adaptation to inhibitor binding: A cryptic binding site in phenylethanolamine N-methyltransferase

Publisher: American Chemical Society (ACS)

Date: 11-09-2007

DOI: 10.1021/JM0703385

Abstract: Shape complementarity is a fundamental principle of inhibitor design. Here we show that an enzyme for which the crystal structure has been determined (phenylethanolamine N-methyltransferase, PNMT) conceals a cryptic binding site. This site is revealed upon binding of inhibitors that are double the size of the physiological substrate. These large inhibitors are not predicted to bind in that they protrude through the accessible surface calculated from a PNMT/7-aminosulfonyl-1,2,3,4-tetrahydroisoquinoline (SK&F 29661) crystal structure, yet they are potent inhibitors of PNMT. We determined structures of the enzyme complexed with large inhibitors and found that the volume of the active site increases by 140 A3 upon binding. Changes in active site size and shape are brought about by unfavorable side chain conformations and rigid body helix motions. The energetic cost is modest, estimated at 2-3 kcal/mol from mutational analyses. Our findings further underline the importance of protein flexibility in structure-based inhibitor design studies.

Structure of a human carcinogen-converting enzyme, SULT1A1 - Structural and kinetic implications of substrate inhibition

Publisher: Elsevier BV

Date: 02-2003

DOI: 10.1074/JBC.M207246200

Expression, purification and preliminary X-ray diffraction studies of VERNALIZATION1(208-341) from Arabidopsis thaliana

Publisher: International Union of Crystallography (IUCr)

Date: 26-02-2009

DOI: 10.1107/S1744309109004217

Structure and Function of DsbA, a Key Bacterial Oxidative Folding Catalyst

Publisher: Mary Ann Liebert Inc

Date: 05-2011

DOI: 10.1089/ARS.2010.3344

Abstract: Since its discovery in 1991, the bacterial periplasmic oxidative folding catalyst DsbA has been the focus of intense research. Early studies addressed why it is so oxidizing and how it is maintained in its less stable oxidized state. The crystal structure of Escherichia coli DsbA (EcDsbA) revealed that the oxidizing periplasmic enzyme is a distant evolutionary cousin of the reducing cytoplasmic enzyme thioredoxin. Recent significant developments have deepened our understanding of DsbA function, mechanism, and interactions: the structure of the partner membrane protein EcDsbB, including its complex with EcDsbA, proved a landmark in the field. Studies of DsbA machineries from bacteria other than E. coli K-12 have highlighted dramatic differences from the model organism, including a striking ergence in redox parameters and surface features. Several DsbA structures have provided the first clues to its interaction with substrates, and finally, evidence for a central role of DsbA in bacterial virulence has been demonstrated in a range of organisms. Here, we review current knowledge on DsbA, a bacterial periplasmic protein that introduces disulfide bonds into erse substrate proteins and which may one day be the target of a new class of anti-virulence drugs to treat bacterial infection.

The 1.2 A resolution crystal structure of TcpG, the Vibrio cholerae DsbA disulfide-forming protein required for pilus and cholera-toxin production

Publisher: International Union of Crystallography (IUCr)

Date: 13-09-2012

DOI: 10.1107/S0907444912026388

Crystallization of the C-terminal domain of the mouse brain cytosolic long-chain acyl-CoA thioesterase

Publisher: International Union of Crystallography (IUCr)

Date: 27-01-2006

DOI: 10.1107/S1744309106000030

Engineered variants provide new insight into the structural properties important for activity of the highly dynamic, trimeric protein disulfide isomerase ScsC from Proteus mirabilis

Publisher: International Union of Crystallography (IUCr)

Date: 26-02-2019

DOI: 10.1107/S2059798319000081

Abstract: Suppressor of copper sensitivity protein C from Proteus mirabilis (PmScsC) is a homotrimeric disulfide isomerase that plays a role in copper tolerance, which is a key virulence trait of this uropathogen. Each protomer of the enzyme has an N-terminal trimerization stem (59 residues) containing a flexible linker (11 residues) connected to a thioredoxin-fold-containing catalytic domain (163 residues). Here, two PmScsC variants, PmScsCΔN and PmScsCΔLinker, are characterized. PmScsCΔN is an N-terminally truncated form of the protomer with two helices of the trimerization stem removed, generating a protein with dithiol oxidase rather than disulfide isomerase activity. The crystal structure of PmScsCΔN reported here reveals, as expected, a monomer that is structurally similar to the catalytic domain of native PmScsC. The second variant, PmScsCΔLinker, was designed to remove the 11-amino-acid linker, and it is shown that it generates a protein that has neither disulfide isomerase nor dithiol oxidase activity. The crystal structure of PmScsCΔLinker reveals a trimeric arrangement, with the catalytic domains packed together very closely. Small-angle X-ray scattering analysis found that native PmScsC is predominantly trimeric in solution even at low concentrations, whereas PmScsCΔLinker exists as an equilibrium between monomeric, dimeric and trimeric states, with the monomeric form dominating at low concentrations. These findings increase the understanding of disulfide isomerase activity, showing how (i) oligomerization, (ii) the spacing between and (iii) the dynamic motion of catalytic domains in PmScsC all contribute to its native function.

A shape-shifting redox foldase contributes to Proteus mirabilis copper resistance

Publisher: Springer Science and Business Media LLC

Date: 19-07-2017

DOI: 10.1038/NCOMMS16065

Abstract: Copper resistance is a key virulence trait of the uropathogen Proteus mirabilis . Here we show that P. mirabilis ScsC (PmScsC) contributes to this defence mechanism by enabling swarming in the presence of copper. We also demonstrate that PmScsC is a thioredoxin-like disulfide isomerase but, unlike other characterized proteins in this family, it is trimeric. PmScsC trimerization and its active site cysteine are required for wild-type swarming activity in the presence of copper. Moreover, PmScsC exhibits unprecedented motion as a consequence of a shape-shifting motif linking the catalytic and trimerization domains. The linker accesses strand, loop and helical conformations enabling the s ling of an enormous folding landscape by the catalytic domains. Mutation of the shape-shifting motif abolishes disulfide isomerase activity, as does removal of the trimerization domain, showing that both features are essential to foldase function. More broadly, the shape-shifter peptide has the potential for ‘plug and play’ application in protein engineering.

Reconciling the regulatory role of Munc18 proteins in SNARE-complex assembly

Publisher: International Union of Crystallography (IUCr)

Date: 21-10-2014

DOI: 10.1107/S2052252514020727

Abstract: Membrane fusion is essential for human health, playing a vital role in processes as erse as neurotransmission and blood glucose control. Two protein families are key: (1) the Sec1p/Munc18 (SM) and (2) the soluble N -ethylmaleimide-sensitive attachment protein receptor (SNARE) proteins. Whilst the essential nature of these proteins is irrefutable, their exact regulatory roles in membrane fusion remain controversial. In particular, whether SM proteins promote and/or inhibit the SNARE-complex formation required for membrane fusion is not resolved. Crystal structures of SM proteins alone and in complex with their cognate SNARE proteins have provided some insight, however, these structures lack the transmembrane spanning regions of the SNARE proteins and may not accurately reflect the native state. Here, we review the literature surrounding the regulatory role of mammalian Munc18 SM proteins required for exocytosis in eukaryotes. Our analysis suggests that the conflicting roles reported for these SM proteins may reflect differences in experimental design. SNARE proteins appear to require C-terminal immobilization or anchoring, for ex le through a transmembrane domain, to form a functional fusion complex in the presence of Munc18 proteins.

United we stand: combining structural methods

Publisher: Elsevier BV

Date: 10-2008

DOI: 10.1016/J.SBI.2008.07.004

Abstract: High-resolution techniques are the mainstay of structural biologists however, to address challenging biological systems many are now turning to hybrid approaches that use complementary structural data. In this review we outline the types of structural problems that benefit from combining results of many methods, we summarise the types of data that can be generated by complementary approaches, and we highlight the application of combined methods in structural biology with recent structural studies of membrane proteins, mega-complexes and inherently flexible proteins.

Responses to`Atomic resolution': a badly abused term in structural biology

Publisher: International Union of Crystallography (IUCr)

Date: 31-03-2017

DOI: 10.1107/S205979831700417X

Life inside and out: making and breaking protein disulfide bonds inChlamydia

Publisher: Informa UK Limited

Date: 02-01-2019

DOI: 10.1080/1040841X.2018.1538933

Virtual Screening of Peptide and Peptidomimetic Fragments Targeted to Inhibit Bacterial Dithiol Oxidase DsbA

Publisher: Public Library of Science (PLoS)

Date: 30-07-2015

DOI: 10.1371/JOURNAL.PONE.0133805

Disulfide isomerase activity of the dynamic, trimeric ScsC protein is primed by the tandem immunoglobulin-fold domain of ScsB

Publisher: Elsevier BV

Date: 04-2018

DOI: 10.1074/JBC.RA118.001860

Sent packing: protein engineering generates a new crystal form ofPseudomonas aeruginosaDsbA1 with increased catalytic surface accessibility

Publisher: International Union of Crystallography (IUCr)

Date: 26-11-2015

DOI: 10.1107/S1399004715018519

Abstract: Pseudomonas aeruginosa is an opportunistic human pathogen for which new antimicrobial drug options are urgently sought. P. aeruginosa disulfide-bond protein A1 (PaDsbA1) plays a pivotal role in catalyzing the oxidative folding of multiple virulence proteins and as such holds great promise as a drug target. As part of a fragment-based lead discovery approach to PaDsbA1 inhibitor development, the identification of a crystal form of PaDsbA1 that was more suitable for fragment-soaking experiments was sought. A previously identified crystallization condition for this protein was unsuitable, as in this crystal form of PaDsbA1 the active-site surface loops are engaged in the crystal packing, occluding access to the target site. A single residue involved in crystal-packing interactions was substituted with an amino acid commonly found at this position in closely related enzymes, and this variant was successfully used to generate a new crystal form of PaDsbA1 in which the active-site surface is more accessible for soaking experiments. The PaDsbA1 variant displays identical redox character and in vitro activity to wild-type PaDsbA1 and is structurally highly similar. Two crystal structures of the PaDsbA1 variant were determined in complex with small molecules bound to the protein active site. These small molecules (MES, glycerol and ethylene glycol) were derived from the crystallization or cryoprotectant solutions and provide a proof of principle that the reported crystal form will be amenable to co-crystallization and soaking with small molecules designed to target the protein active-site surface.

The atypical thiol–disulfide exchange protein α-DsbA2 fromWolbachia pipientisis a homotrimeric disulfide isomerase

Publisher: International Union of Crystallography (IUCr)

Date: 26-02-2019

DOI: 10.1107/S2059798318018442

Abstract: Disulfide-bond-forming (DSB) oxidative folding enzymes are master regulators of virulence that are localized to the periplasm of many Gram-negative bacteria. The archetypal DSB machinery from Escherichia coli K-12 consists of a dithiol-oxidizing redox-relay pair (DsbA/B), a disulfide-isomerizing redox-relay pair (DsbC/D) and the specialist reducing enzymes DsbE and DsbG that also interact with DsbD. By contrast, the Gram-negative bacterium Wolbachia pipientis encodes just three DSB enzymes. Two of these, α-DsbA1 and α-DsbB, form a redox-relay pair analogous to DsbA/B from E. coli . The third enzyme, α-DsbA2, incorporates a DsbA-like sequence but does not interact with α-DsbB. In comparison to other DsbA enzymes, α-DsbA2 has ∼50 extra N-terminal residues (excluding the signal peptide). The crystal structure of α-DsbA2ΔN, an N-terminally truncated form in which these ∼50 residues are removed, confirms the DsbA-like nature of this domain. However, α-DsbA2 does not have DsbA-like activity: it is structurally and functionally different as a consequence of its N-terminal residues. Firstly, α-DsbA2 is a powerful disulfide isomerase and a poor dithiol oxidase: i.e. its role is to shuffle rather than to introduce disulfide bonds. Moreover, small-angle X-ray scattering (SAXS) of α-DsbA2 reveals a homotrimeric arrangement that differs from those of the other characterized bacterial disulfide isomerases DsbC from Escherichia coli (homodimeric) and ScsC from Proteus mirabilis (PmScsC homotrimeric with a shape-shifter peptide). α-DsbA2 lacks the shape-shifter motif and SAXS data suggest that it is less flexible than PmScsC. These results allow conclusions to be drawn about the factors that are required for functionally equivalent disulfide isomerase enzymatic activity across structurally erse protein architectures.

Structural Insights into the Role of the Cyclic Backbone in a Squash Trypsin Inhibitor

Publisher: Elsevier BV

Date: 12-2013

DOI: 10.1074/JBC.M113.528240

Fragment-based screening by X-ray crystallography, MS and isothermal titration calorimetry to identify PNMT (phenylethanolamine N-methyltransferase) inhibitors

Publisher: Portland Press Ltd.

Date: 14-09-2010

DOI: 10.1042/BJ20100651

Abstract: CNS (central nervous system) adrenaline (epinephrine) is implicated in a wide range of physiological and pathological conditions. PNMT (phenylethanolamine N-methyltransferase) catalyses the final step in the biosynthesis of adrenaline, the conversion of noradrenaline (norepinephrine) to adrenaline by methylation. To help elucidate the role of CNS adrenaline, and to develop potential drug leads, potent, selective and CNS-active inhibitors are required. The fragment screening approach has advantages over other lead discovery methods including high hit rates, more efficient hits and the ability to s le chemical ersity more easily. In the present study we applied fragment-based screening approaches to the enzyme PNMT. We used crystallography as the primary screen and identified 12 hits from a small commercial library of 384 drug-like fragments. The hits include nine chemicals with two fused rings and three single-ring chemical systems. Eight of the hits come from three chemical classes: benzimidazoles (a known class of PNMT inhibitor), purines and quinolines. Nine of the hits have measurable binding affinities (~5–700 μM) as determined by isothermal titration calorimetry and all nine have ligand efficiencies of 0.39 kcal/mol per heavy atom or better (1 kcal≈4.184 kJ). We synthesized five elaborated benzimidazole compounds and characterized their binding to PNMT, showing for the first time how this class of inhibitors interact with the noradrenaline-binding site. Finally, we performed a pilot study with PNMT for fragment-based screening by MS showing that this approach could be used as a fast and efficient first-pass screening method prior to characterization of binding mode and affinity of hits.

Comparative Sequence, Structure and Redox Analyses of Klebsiella pneumoniae DsbA Show That Anti-Virulence Target DsbA Enzymes Fall into Distinct Classes

Publisher: Public Library of Science (PLoS)

Date: 14-11-2013

DOI: 10.1371/JOURNAL.PONE.0080210

Molecular dissection of the Munc18c/syntaxin4 interaction: Implications for regulation of membrane trafficking

Publisher: Wiley

Date: 10-08-2006

DOI: 10.1111/J.1600-0854.2006.00474.X

Abstract: Sec1p/Munc18 (SM) proteins are believed to play an integral role in vesicle transport through their interaction with SNAREs. Different SM proteins have been shown to interact with SNAREs via different mechanisms, leading to the conclusion that their function has erged. To further explore this notion, in this study, we have examined the molecular interactions between Munc18c and its cognate SNAREs as these molecules are ubiquitously expressed in mammals and likely regulate a universal plasma membrane trafficking step. Thus, Munc18c binds to monomeric syntaxin4 and the N-terminal 29 amino acids of syntaxin4 are necessary for this interaction. We identified key residues in Munc18c and syntaxin4 that determine the N-terminal interaction and that are consistent with the N-terminal binding mode of yeast proteins Sly1p and Sed5p. In addition, Munc18c binds to the syntaxin4/SNAP23/VAMP2 SNARE complex. Pre-assembly of the syntaxin4/Munc18c dimer accelerates the formation of SNARE complex compared to assembly with syntaxin4 alone. These data suggest that Munc18c interacts with its cognate SNAREs in a manner that resembles the yeast proteins Sly1p and Sed5p rather than the mammalian neuronal proteins Munc18a and syntaxin1a. The Munc18c-SNARE interactions described here imply that Munc18c could play a positive regulatory role in SNARE assembly.

Properties of the Thioredoxin Fold Superfamily Are Modulated by a Single Amino Acid Residue

Publisher: Elsevier BV

Date: 04-2009

DOI: 10.1074/JBC.M809509200

Structure of the Munc18c/Syntaxin4 N-peptide complex defines universal features of the N-peptide binding mode of Sec1/Munc18 proteins

Publisher: Proceedings of the National Academy of Sciences

Date: 22-05-2007

DOI: 10.1073/PNAS.0701124104

Abstract: Sec1/Munc18 proteins (SM proteins) bind to soluble NSF attachment protein receptors (SNAREs) and play an essential role in membrane fusion. Divergent modes of regulation have been proposed for different SM proteins indicating that they can either promote or inhibit SNARE assembly. This is in part because of discrete modes of binding that have been described for various SM/SNARE complexes. One mode suggests that SM proteins bind only to Syntaxins (Stx) preventing SNARE assembly, whereas in another they facilitate SNARE assembly and bind to SNARE complexes. The mammalian cell surface SM protein Munc18c binds to an N-peptide in Stx4, and this is compatible with its interaction with SNARE complexes. Here we describe the crystal structure of Munc18c in complex with the Stx4 N-peptide. This structure shows remarkable similarity with a yeast complex indicating that the mode of binding, which can accommodate SNARE complexes, is highly conserved throughout evolution. Modeling reveals the presence of the N-peptide binding mode in most but not all yeast and mammalian SM/Stx pairs, suggesting that it has coevolved to fulfill a specific regulatory function. It is unlikely that the N-peptide interaction alone accounts for the specificity in SM/SNARE binding, implicating other contact surfaces in this function. Together with other data, our results support a sequential two-state model for SM/SNARE binding involving an initial interaction via the Stx N-peptide, which somehow facilitates a second, more comprehensive interaction comprising other contact surfaces in both proteins.

Structural, mutagenic, and kinetic analysis of the binding of substrates and inhibitors of human phenylethanolamine N-methyltransferase

Publisher: American Chemical Society (ACS)

Date: 14-10-2005

DOI: 10.1021/JM050568O

Abstract: The X-ray structure of human phenylethanolamine N-methyltransferase (hPNMT) complexed with its product, S-adenosyl-L-homocysteine (4), and the most potent inhibitor reported to date, SK&F 64139 (7), was used to identify the residues involved in inhibitor binding. Four of these residues, Val53, Lys57, Glu219 and Asp267, were replaced, in turn, with alanine. All variants had increased Km values for phenylethanolamine (10), but only D267A showed a noteworthy (20-fold) decrease in its kcat value. Both WT hPNMT and D267A had similar kcat values for a rigid analogue, anti-9-amino-6-(trifluoromethyl)benzonorbornene (12), suggesting that Asp267 plays an important role in positioning the substrate but does not participate directly in catalysis. The Ki values for the binding of inhibitors such as 7 to the E219A and D267A variants increased by 2-3 orders of magnitude. Further, the inhibitors were shown to bind up to 50-fold more tightly in the presence of S-adenosyl-L-methionine (3), suggesting that the binding of the latter brings about a conformational change in the enzyme.

Studying Munc18:Syntaxin Interactions Using Small-Angle Scattering

Publisher: Springer New York

Date: 14-10-2018

DOI: 10.1007/978-1-4939-8760-3_7

Abstract: The interaction between the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein syntaxin (Sx) and regulatory partner Sec/Munc18 (SM) protein is a critical step in vesicle fusion. The exact role played by SM proteins, whether positive or negative, has been the topic of much debate. High-resolution structures of the SM:Sx complex have shown that SM proteins can bind syntaxin in a closed fusion incompetent state. However, in vitro and in vivo experiments also point to a positive regulatory role for SM proteins that is inconsistent with binding syntaxin in a closed conformation. Here we present protocols we used for the expression and purification of the SM proteins Munc18a and Munc18c and syntaxins 1 and 4 along with procedures used for small-angle X-ray and neutron scattering that showed that syntaxins can bind in an open conformation to SM proteins. We also describe methods for chemical cross-linking experiments and detail how this information can be combined with scattering data to obtain low-resolution structural models for SM:Sx protein complexes.

Crystal Structures of Highly Constrained Substrate and Hydrolysis Products Bound to HIV-1 Protease. Implications for the Catalytic Mechanism

Publisher: American Chemical Society (ACS)

Date: 03-2008

DOI: 10.1021/BI7023157

Abstract: HIV-1 protease is a key target in treating HIV infection and AIDS, with 10 inhibitors used clinically. Here we used an unusual hexapeptide substrate, containing two macrocyclic tripeptides constrained to mimic a beta strand conformation, linked by a scissile peptide bond, to probe the structural mechanism of proteolysis. The substrate has been cocrystallized with catalytically active synthetic HIV-1 protease and an inactive isosteric (D25N) mutant, and three-dimensional structures were determined (1.60 A). The structure of the inactive HIVPR(D25N)/substrate complex shows an intact substrate molecule in a single orientation that perfectly mimics the binding of conventional peptide ligands of HIVPR. The structure of the active HIVPR roduct complex shows two monocyclic hydrolysis products trapped in the active site, revealing two molecules of the N-terminal monocyclic product bound adjacent to one another, one molecule occupying the nonprime site, as expected, and the other monocycle binding in the prime site in the reverse orientation. The results suggest that both hydrolysis products are released from the active site upon cleavage and then rebind to the enzyme. These structures reveal that N-terminal binding of ligands is preferred, that the C-terminal site is more flexible, and that HIVPR can recognize substrate shape rather than just sequence alone. The product complex reveals three carboxylic acids in an almost planar orientation, indicating an unusual hexagonal homodromic complex between three carboxylic acids. The data presented herein regarding orientation of catalytic aspartates support the cleavage mechanism proposed by Northrop. The results imply strategies for design of inhibitors targeting the N-terminal side of the cleavage site or taking advantage of the flexibility in the protease domain that accommodates substrate/inhibitor segments C-terminal to the cleavage site.

In vivo oxidative protein folding can be facilitated by oxidation-reduction cycling

Publisher: Wiley

Date: 2010

DOI: 10.1111/J.1365-2958.2009.06952.X

Abstract: Current dogma dictates that bacterial proteins with misoxidized disulfide bonds are shuffled into correctly oxidized states by DsbC. There are two proposed mechanisms for DsbC activity. The first involves a DsbC-only model of substrate disulfide rearrangement. The second invokes cycles of reduction and oxidation of substrate disulfide bonds by DsbC and DsbA respectively. Here, we addressed whether the second mechanism is important in vivo by identifying whether a periplasmic reductase could complement DsbC. We screened for naturally occurring periplasmic reductases in Bacteroides fragilis, a bacterium chosen because we predicted it encodes reductases and has a reducing periplasm. We found that the B. fragilis periplasmic protein TrxP has a thioredoxin fold with an extended N-terminal region that it is a very active reductase but a poor isomerase and that it fully complements dsbC. These results provide direct in vivo evidence that correctly folded protein is achievable via cycles of oxidation and reduction.

Recombinant expression of Munc18c in a baculovirus system and interaction with syntaxin

Publisher: Elsevier BV

Date: 10-2003

DOI: 10.1016/S1046-5928(03)00197-9

Abstract: Two protein families that are critical for vesicle transport are the Syntaxin and Munc18/Sec1 families of proteins. These two molecules form a high affinity complex and play an essential role in vesicle docking and fusion. Munc18c was expressed as an N-terminally His-tagged fusion protein from recombinant baculovirus in Sf9 insect cells. His-tagged Munc18c was purified to homogeneity using both cobalt-chelating affinity chromatography and gel filtration chromatography. With this simple two-step protocol, 3.5 mg of purified Munc18c was obtained from a 1L culture. Further, the N-terminal His-tag could be removed by thrombin cleavage while the tagged protein was bound to metal affinity resin. Recombinant Munc18c produced in this way is functional, in that it forms a stable complex with the SNARE interacting partner, syntaxin4. Thus we have developed a method for producing and purifying large amounts of functional Munc18c--both tagged and detagged--from a baculovirus expression system. We have also developed a method to purify the Munc18c:syntaxin4 complex. These methods will be employed for future functional and structural studies.

Interaction between plate make and protein in protein crystallisation screening

Publisher: Public Library of Science (PLoS)

Date: 16-11-2009

DOI: 10.1371/JOURNAL.PONE.0007851

Laboratory evolution of one disulfide isomerase to resemble another

Publisher: Proceedings of the National Academy of Sciences

Date: 10-07-2007

DOI: 10.1073/PNAS.0704692104

Abstract: It is often difficult to determine which of the sequence and structural differences between ergent members of multigene families are functionally important. Here we use a laboratory evolution approach to determine functionally important structural differences between two distantly related disulfide isomerases, DsbC and DsbG from Escherichia coli . Surprisingly, we found single amino acid substitutions in DsbG that were able to complement dsbC in vivo and have more DsbC-like isomerase activity in vitro . Crystal structures of the three strongest point mutants, DsbG K113E, DsbG V216M, and DsbG T200M, reveal changes in highly surface-exposed regions that cause DsbG to more closely resemble the distantly related DsbC. In this case, laboratory evolution appears to have taken a direct route to allow one protein family member to complement another, with single substitutions apparently bypassing much of the need for multiple changes that took place over ≈0.5 billion years of evolution. Our findings suggest that, for these two proteins at least, regions important in determining functional differences may represent only a tiny fraction of the overall protein structure.

Countering Cooperative Effects in Protease Inhibitors Using Constrained β-Strand-Mimicking Templates in Focused Combinatorial Libraries

Publisher: American Chemical Society (ACS)

Date: 19-02-2004

DOI: 10.1021/JM030337M

Abstract: A major problem in de novo design of enzyme inhibitors is the unpredictability of the induced fit, with the shape of both ligand and enzyme changing cooperatively and unpredictably in response to subtle structural changes within a ligand. We have investigated the possibility of d ening the induced fit by using a constrained template as a replacement for adjoining segments of a ligand. The template preorganizes the ligand structure, thereby organizing the local enzyme environment. To test this approach, we used templates consisting of constrained cyclic tripeptides, formed through side chain to main chain linkages, as structural mimics of the protease-bound extended beta-strand conformation of three adjoining amino acid residues at the N- or C-terminal sides of the scissile bond of substrates. The macrocyclic templates were derivatized to a range of 30 structurally erse molecules via focused combinatorial variation of nonpeptidic appendages incorporating a hydroxyethylamine transition-state isostere. Most compounds in the library were potent inhibitors of the test protease (HIV-1 protease). Comparison of crystal structures for five protease-inhibitor complexes containing an N-terminal macrocycle and three protease-inhibitor complexes containing a C-terminal macrocycle establishes that the macrocycles fix their surrounding enzyme environment, thereby permitting independent variation of acyclic inhibitor components with only local disturbances to the protease. In this way, the location in the protease of various acyclic fragments on either side of the macrocyclic template can be accurately predicted. This type of templating strategy minimizes the problem of induced fit, reducing unpredictable cooperative effects in one inhibitor region caused by changes to adjacent enzyme-inhibitor interactions. This idea might be exploited in template-based approaches to inhibitors of other proteases, where a beta-strand mimetic is also required for recognition, and also other protein-binding ligands where different templates may be more appropriate.

Virulence of the Melioidosis Pathogen Burkholderia pseudomallei Requires the Oxidoreductase Membrane Protein DsbB

Publisher: American Society for Microbiology

Date: 05-2018

DOI: 10.1128/IAI.00938-17

Abstract: The naturally antibiotic-resistant bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a disease with stubbornly high mortality and a complex, protracted treatment regimen. The worldwide incidence of melioidosis is likely grossly underreported, though it is known to be highly endemic in northern Australia and Southeast Asia. Bacterial disulfide bond (DSB) proteins catalyze the oxidative folding and isomerization of disulfide bonds in substrate proteins. In the present study, we demonstrate that B. pseudomallei membrane protein disulfide bond protein B (BpsDsbB) forms a functional redox relay with the previously characterized virulence mediator B. pseudomallei disulfide bond protein A (BpsDsbA). Genomic analysis of erse B. pseudomallei clinical isolates demonstrated that dsbB is a highly conserved core gene. Critically, we show that DsbB is required for virulence in B. pseudomallei . A panel of B. pseudomallei dsbB deletion strains (K96243, 576, MSHR2511, MSHR0305b, and MSHR5858) were phenotypically erse according to the results of in vitro assays that assess hallmarks of virulence. Irrespective of their in vitro virulence phenotypes, two deletion strains were attenuated in a BALB/c mouse model of infection. A crystal structure of a DsbB-derived peptide complexed with BpsDsbA provides the first molecular characterization of their interaction. This work contributes to our broader understanding of DSB redox biology and will support the design of antimicrobial drugs active against this important family of bacterial virulence targets.

Prediction of Burkholderia pseudomallei DsbA substrates identifies potential virulence factors and vaccine targets

Publisher: Public Library of Science (PLoS)

Date: 20-11-2020

DOI: 10.1371/JOURNAL.PONE.0241306

Abstract: Identification of bacterial virulence factors is critical for understanding disease pathogenesis, drug discovery and vaccine development. In this study we used two approaches to predict virulence factors of Burkholderia pseudomallei , the Gram-negative bacterium that causes melioidosis. B . pseudomallei is naturally antibiotic resistant and there are no clinically available melioidosis vaccines. To identify B . pseudomallei protein targets for drug discovery and vaccine development, we chose to search for substrates of the B . pseudomallei periplasmic disulfide bond forming protein A (DsbA). DsbA introduces disulfide bonds into extra-cytoplasmic proteins and is essential for virulence in many Gram-negative organism, including B . pseudomallei . The first approach to identify B . pseudomallei DsbA virulence factor substrates was a large-scale genomic analysis of 511 unique B . pseudomallei disease-associated strains. This yielded 4,496 core gene products, of which we hypothesise 263 are DsbA substrates. Manual curation and database screening of the 263 mature proteins yielded 81 associated with disease pathogenesis or virulence. These were screened for structural homologues to predict potential B-cell epitopes. In the second approach, we searched the B . pseudomallei genome for homologues of the more than 90 known DsbA substrates in other bacteria. Using this approach, we identified 15 putative B . pseudomallei DsbA virulence factor substrates, with two of these previously identified in the genomic approach, bringing the total number of putative DsbA virulence factor substrates to 94. The two putative B . pseudomallei virulence factors identified by both methods are homologues of PenI family β-lactamase and a molecular chaperone. These two proteins could serve as high priority targets for future B . pseudomallei virulence factor characterization.

The Munc18-1 domain 3a loop is essential for neuroexocytosis but not for syntaxin-1A transport to the plasma membrane

Publisher: The Company of Biologists

Date: 06-2013

DOI: 10.1242/JCS.126813

Abstract: Munc18-1 plays a dual role in transporting syntaxin-1A (Sx1a) to the plasma membrane and regulating SNARE-mediated membrane fusion. As impairment of either function leads to a common exocytic defect, assigning specific roles for various Munc18-1 domains has proved difficult. Structural analyses predict that a loop region in Munc18-1 domain 3a could catalyse the conversion of Sx1a from a ‘closed’, fusion-incompetent to an ‘open’, fusion-competent conformation. As this conversion occurs at the plasma membrane, mutations in this loop could potentially separate the chaperone and exocytic functions of Munc18-1. Expression of a Munc18-1 deletion mutant lacking 17 residues of the domain 3a loop (Munc18-1Δ317–333) in PC12 cells deficient in endogenous Munc18 (DKD-PC12 cells) fully rescued transport of Sx1a to the plasma membrane, but not exocytic secretory granule fusion. In vitro binding of Munc18-1Δ317–333 to Sx1a was indistinguishable from that of full-length Munc18-1, consistent with the critical role of the closed conformation in Sx1a transport. However, in DKD-PC12 cells, Munc18-1Δ317–333 binding to Sx1a was greatly reduced compared to that of full-length Munc18-1, suggesting that closed conformation binding contributes little to the overall interaction at the cell surface. Furthermore, we found that Munc18-1Δ317–333 could bind SNARE complexes in vitro, suggesting that additional regulatory factors underpin the exocytic function of Munc18-1 in vivo. Together, these results point to a defined role for Munc18-1 in facilitating exocytosis linked to the loop region of domain 3a that is clearly distinct from its function in Sx1a transport.

Focusing in on structural genomics: The University of Queensland structural biology pipeline

Publisher: Elsevier BV

Date: 12-2006

DOI: 10.1016/J.BIOENG.2006.09.002

Abstract: The flood of new genomic sequence information together with technological innovations in protein structure determination have led to worldwide structural genomics (SG) initiatives. The goals of SG initiatives are to accelerate the process of protein structure determination, to fill in protein fold space and to provide information about the function of uncharacterized proteins. In the long-term, these outcomes are likely to impact on medical biotechnology and drug discovery, leading to a better understanding of disease as well as the development of new therapeutics. Here we describe the high throughput pipeline established at the University of Queensland in Australia. In this focused pipeline, the targets for structure determination are proteins that are expressed in mouse macrophage cells and that are inferred to have a role in innate immunity. The aim is to characterize the molecular structure and the biochemical and cellular function of these targets by using a parallel processing pipeline. The pipeline is designed to work with tens to hundreds of target gene products and comprises target selection, cloning, expression, purification, crystallization and structure determination. The structures from this pipeline will provide insights into the function of previously uncharacterized macrophage proteins and could lead to the validation of new drug targets for chronic obstructive pulmonary disease and arthritis.

The suppressor of copper sensitivity protein C from Caulobacter crescentus is a trimeric disulfide isomerase that binds copper(I) with subpicomolar affinity

Publisher: International Union of Crystallography (IUCr)

Date: 21-02-2022

DOI: 10.1107/S2059798322000729

Abstract: The introduction of disulfide bonds into periplasmic proteins is a critical process in many Gram-negative bacteria. The formation and regulation of protein disulfide bonds have been linked to the production of virulence factors. Understanding the different pathways involved in this process is important in the development of strategies to disarm pathogenic bacteria. The well characterized disulfide bond-forming (DSB) proteins play a key role by introducing or isomerizing disulfide bonds between cysteines in substrate proteins. Curiously, the suppressor of copper sensitivity C proteins (ScsCs), which are part of the bacterial copper-resistance response, share structural and functional similarities with DSB oxidase and isomerase proteins, including the presence of a catalytic thioredoxin domain. However, the oxidoreductase activity of ScsC varies with its oligomerization state, which depends on a poorly conserved N-terminal domain. Here, the structure and function of Caulobacter crescentus ScsC (CcScsC) have been characterized. It is shown that CcScsC binds copper in the copper(I) form with subpicomolar affinity and that its isomerase activity is comparable to that of Escherichia coli DsbC, the prototypical dimeric bacterial isomerase. It is also reported that CcScsC functionally complements trimeric Proteus mirabilis ScsC (PmScsC) in vivo , enabling the swarming of P. mirabilis in the presence of copper. Using mass photometry and small-angle X-ray scattering (SAXS) the protein is demonstrated to be trimeric in solution, like PmScsC, and not dimeric like EcDsbC. The crystal structure of CcScsC was also determined at a resolution of 2.6 Å, confirming the trimeric state and indicating that the trimerization results from interactions between the N-terminal α-helical domains of three CcScsC protomers. The SAXS data analysis suggested that the protomers are dynamic, like those of PmScsC, and are able to s le different conformations in solution.

Low-resolution solution structures of Munc18:Syntaxin protein complexes indicate an open binding mode driven by the Syntaxin N-peptide

Publisher: Proceedings of the National Academy of Sciences

Date: 05-06-2012

DOI: 10.1073/PNAS.1116975109

Abstract: When nerve cells communicate, vesicles from one neuron fuse with the presynaptic membrane releasing chemicals that signal to the next. Similarly, when insulin binds its receptor on adipocytes or muscle, glucose transporter-4 vesicles fuse with the cell membrane, allowing glucose to be imported. These essential processes require the interaction of SNARE proteins on vesicle and cell membranes, as well as the enigmatic protein Munc18 that binds the SNARE protein Syntaxin. Here, we show that in solution the neuronal protein Syntaxin1a interacts with Munc18-1 whether or not the Syntaxin1a N-peptide is present. Conversely, the adipocyte protein Syntaxin4 does not bind its partner Munc18c unless the N-peptide is present. Solution-scattering data for the Munc18-1:Syntaxin1a complex in the absence of the N-peptide indicates that this complex adopts the inhibitory closed binding mode, exemplified by a crystal structure of the complex. However, when the N-peptide is present, the solution-scattering data indicate both Syntaxin1a and Syntaxin4 adopt extended conformations in complexes with their respective Munc18 partners. The low-resolution solution structure of the open Munc18:Syntaxin binding mode was modeled using data from cross-linking/mass spectrometry, small-angle X-ray scattering, and small-angle neutron scattering with contrast variation, indicating significant differences in Munc18:Syntaxin interactions compared with the closed binding mode. Overall, our results indicate that the neuronal Munc18-1:Syntaxin1a proteins can adopt two alternate and functionally distinct binding modes, closed and open, depending on the presence of the N-peptide, whereas Munc18c:Syntaxin4 adopts only the open binding mode.

Active site mutations and substrate inhibition in human sulfotransferase 1A1 and 1A3

Publisher: Elsevier BV

Date: 04-2004

DOI: 10.1074/JBC.M312253200

Interrogating Fragments Using a Protein Thermal Shift Assay

Publisher: CSIRO Publishing

Date: 2013

DOI: 10.1071/CH13279

Abstract: Protein thermal shift is a relatively rapid and inexpensive technique for the identification of low molecular weight compound interactions with protein targets. An increase in the melting temperature of the target protein in the presence of a test ligand is indicative of a promising ligand–protein interaction. Due to its simplicity, protein thermal shift is an attractive method for screening libraries and validating hits in drug discovery programs. The methodology has been used successfully in high throughput screens of small molecule libraries, and its application has been extended to report on protein–drug-like-fragment interactions. Here, we review how protein thermal shift has been employed recently in fragment-based drug discovery (FBDD) efforts, and highlight its application to protein–protein interaction targets. Multiple validation of fragment hits by independent means is paramount to ensure efficient and economical progress in a FBDD c aign. We discuss the applicability of thermal shift assays in this light, and discuss more generally what one does when orthogonal approaches disagree.

Molecular recognition of physiological substrate noradrenaline by the adrenaline-synthesizing enzyme PNMT and factors influencing its methyltransferase activity

Publisher: Portland Press Ltd.

Date: 27-08-2009

DOI: 10.1042/BJ20090702

Abstract: Substrate specificity is critically important for enzyme catalysis. In the adrenaline-synthesizing enzyme PNMT (phenylethanolamine N-methyltransferase), minor changes in substituents can convert substrates into inhibitors. Here we report the crystal structures of six human PNMT complexes, including the first structure of the enzyme in complex with its physiological ligand R-noradrenaline. Determining this structure required rapid soak methods because of the tendency for noradrenaline to oxidize. Comparison of the PNMT–noradrenaline complex with the previously determined PNMT–p-octopamine complex demonstrates that these two substrates form almost equivalent interactions with the enzyme and show that p-octopamine is a valid model substrate for PNMT. The crystal structures illustrate the adaptability of the PNMT substrate binding site in accepting multi-fused ring systems, such as substituted norbornene, as well as noradrenochrome, the oxidation product of noradrenaline. These results explain why only a subset of ligands recognized by PNMT are methylated by the enzyme bulky substituents dictate the binding orientation of the ligand and can thereby place the acceptor amine too far from the donor methyl group for methylation to occur. We also show how the critical Glu185 catalytic residue can be replaced by aspartic acid with a loss of only 10-fold in catalytic efficiency. This is because protein backbone movements place the Asp185 carboxylate almost coincident with the carboxylate of Glu185. Conversely, replacement of Glu185 by glutamine reduces catalytic efficiency almost 300-fold, not only because of the loss of charge, but also because the variant residue does not adopt the same conformation as Glu185.

Oxidoreductase disulfide bond proteins DsbA and DsbB form an active redox pair in Chlamydia trachomatis, a bacterium with disulfide dependent infection and development

Publisher: Public Library of Science (PLoS)

Date: 19-09-2019

DOI: 10.1371/JOURNAL.PONE.0222595

The Multidrug Resistance IncA/C Transferable Plasmid Encodes a Novel Domain-swapped Dimeric Protein-disulfide Isomerase

Publisher: Elsevier BV

Date: 2014

DOI: 10.1074/JBC.M113.516898

Human sulfotransferases and their role in chemical metabolism

Publisher: Oxford University Press (OUP)

Date: 12-2006

DOI: 10.1093/TOXSCI/KFJ061

Abstract: Sulfonation is an important reaction in the metabolism of numerous xenobiotics, drugs, and endogenous compounds. A supergene family of enzymes called sulfotransferases (SULTs) catalyze this reaction. In most cases, the addition of a sulfonate moiety to a compound increases its water solubility and decreases its biological activity. However, many of these enzymes are also capable of bioactivating procarcinogens to reactive electrophiles. In humans three SULT families, SULT1, SULT2, and SULT4, have been identified that contain at least thirteen distinct members. SULTs have a wide tissue distribution and act as a major detoxification enzyme system in adult and the developing human fetus. Nine crystal structures of human cytosolic SULTs have now been determined, and together with site-directed mutagenesis experiments and molecular modeling, we are now beginning to understand the factors that govern distinct but overlapping substrate specificities. These studies have also provided insight into the enzyme kinetics and inhibition characteristics of these enzymes. The regulation of human SULTs remains as one of the least explored areas of research in the field, though there have been some recent advances on the molecular transcription mechanism controlling the in idual SULT promoters. Interin idual variation in sulfonation capacity may be important in determining an in idual's response to xenobiotics, and recent studies have begun to suggest roles for SULT polymorphism in disease susceptibility. This review aims to provide a summary of our present understanding of the function of human cytosolic sulfotransferases.

Structural basis for recruitment of tandem hotdog domains in acyl-CoA thioesterase 7 and its role in inflammation

Publisher: Proceedings of the National Academy of Sciences

Date: 19-06-2007

DOI: 10.1073/PNAS.0700974104

Abstract: Acyl-CoA thioesterases (Acots) catalyze the hydrolysis of fatty acyl-CoA to free fatty acid and CoA and thereby regulate lipid metabolism and cellular signaling. We present a comprehensive structural and functional characterization of mouse acyl-CoA thioesterase 7 (Acot7). Whereas prokaryotic homologues possess a single thioesterase domain, mammalian Acot7 contains a pair of domains in tandem. We determined the crystal structures of both the N- and C-terminal domains of the mouse enzyme, and inferred the structure of the full-length enzyme using a combination of chemical cross-linking, mass spectrometry, and molecular modeling. The quaternary arrangement in Acot7 features a trimer of hotdog fold dimers. Both domains of Acot7 are required for activity, but only one of two possible active sites in the dimer is functional. Asn-24 and Asp-213 (from N- and C-domains, respectively) were identified as the catalytic residues through site-directed mutagenesis. An enzyme with higher activity than wild-type Acot7 was obtained by mutating the residues in the nonfunctional active site. Recombinant Acot7 was shown to have the highest activity toward arachidonoyl-CoA, suggesting a function in eicosanoid metabolism. In line with the proposal, Acot7 was shown to be highly expressed in macrophages and up-regulated by lipopolysaccharide. Overexpression of Acot7 in a macrophage cell line modified the production of prostaglandins D2 and E2. Together, the results link the molecular and cellular functions of Acot7 and identify the enzyme as a candidate drug target in inflammatory disease.

Combined x-ray and NMR analysis of the stability of the cyclotide cystine knot fold that underpins its insecticidal activity and potential use as a drug scaffold

Publisher: Elsevier BV

Date: 04-2009

DOI: 10.1074/JBC.M900021200

Causes of death in a cohort treated for opioid dependence between 1985 and 2005

Publisher: Wiley

Date: 09-10-2013

DOI: 10.1111/ADD.12337

Abstract: To examine changes in causes of death in a cohort treated for opioid dependence, across time and age quantify years of potential life lost (YPLL) and identify avoidable causes of death. People in New South Wales (NSW) who registered for opioid substitution therapy (OST), 1985-2005, were linked to a register of all deaths in Australia. NSW, Australia. Crude mortality rates (CMRs), age-sex-standardized mortality rates (ASSRs) and standardized mortality ratios (SMRs) across time, sex and age. Years of potential life lost (YPLL) were calculated with reference to Australian life tables and by calculating years lost before the age of 65 years. There were 43 789 people in the cohort, with 412 216 person-years of follow-up. The proportion of the cohort aged 40+ years increased from 1% in 1985 to 39% in 2005. Accidental opioid overdoses, suicides, transport accidents and violent deaths declined with age deaths from cardiovascular disease, liver disease and cancer increased. Among men, 89% of deaths were potentially avoidable among women, 86% of deaths were avoidable. There were an estimated 160 555 YPLL in the cohort, an average of 44 YPLL per decedent and an average of 29 YPLL before age 65 years. Among a cohort of opioid-dependent people in New South Wales, 1985-2005, almost nine in 10 deaths in the cohort were avoidable. There is huge scope to improve mortality among opioid-dependent people.

The α-Proteobacteria Wolbachia pipientis Protein Disulfide Machinery Has a Regulatory Mechanism Absent in γ-Proteobacteria

Publisher: Public Library of Science (PLoS)

Date: 25-11-2013

DOI: 10.1371/JOURNAL.PONE.0081440

Evaluating protein:protein complex formation using synchrotron radiation circular dichroism spectroscopy

Publisher: Wiley

Date: 25-09-2007

DOI: 10.1002/PROT.21631

Abstract: Circular dichroism (CD) spectroscopy beamlines at synchrotrons produce dramatically higher light flux than conventional CD instruments. This property of synchrotron radiation circular dichroism (SRCD) results in improved signal-to-noise ratios and allows data collection to lower wavelengths, characteristics that have led to the development of novel SRCD applications. Here we describe the use of SRCD to study protein complex formation, specifically evaluating the complex formed between carboxypeptidase A and its protein inhibitor latexin. Crystal structure analyses of this complex and the in idual proteins reveal only minor changes in secondary structure of either protein upon complex formation (i.e., it involves only rigid body interactions). Conventional CD spectroscopy reports on changes in secondary structure and would therefore not be expected to be sensitive to such interactions. However, in this study we have shown that SRCD can identify differences in the vacuum ultraviolet CD spectra that are significant and attributable to complex formation.

Four structural subclasses of the antivirulence drug target disulfide oxidoreductase DsbA provide a platform for design of subclass-specific inhibitors

Publisher: Elsevier BV

Date: 08-2014

DOI: 10.1016/J.BBAPAP.2014.01.013

Abstract: By catalyzing oxidative protein folding, the bacterial disulfide bond protein A (DsbA) plays an essential role in the assembly of many virulence factors. Predictably, DsbA disruption affects multiple downstream effector molecules, resulting in pleiotropic effects on the virulence of important human pathogens. These findings mark DsbA as a master regulator of virulence, and identify the enzyme as a target for a new class of antivirulence agents that disarm pathogenic bacteria rather than killing them. The purpose of this article is to discuss and expand upon recent findings on DsbA and to provide additional novel insights into the druggability of this important disulfide oxidoreductase by comparing the structures and properties of 13 well-characterized DsbA enzymes. Our structural analysis involved comparison of the overall fold, the surface properties, the conformations of three loops contributing to the binding surface and the sequence identity of residues contributing to these loops. Two distinct structural classes were identified, classes I and II, which are differentiated by their central β-sheet arrangements and which roughly separate the DsbAs produced by Gram-negative from Gram-positive organisms. The classes can be further sub ided into a total of four subclasses on the basis of surface features. Class Ia is equivalent to the Enterobacteriaceae class that has been defined previously. Bioinformatic analyses support the classification of DsbAs into 3 of the 4 subclasses, but did not pick up the 4th subclass which is only apparent from analysis of DsbA electrostatic surface properties. In the context of inhibitor development, the discrete structural subclasses provide a platform for developing DsbA inhibitory scaffolds with a subclass-wide spectrum of activity. We expect that more DsbA classes are likely to be identified, as enzymes from other pathogens are explored, and we highlight the issues associated with structure-based inhibitor development targeting this pivotal mediator of bacterial virulence. This article is part of a Special Issue entitled: Thiol-Based Redox Processes.

Author Correction: A shape-shifting redox foldase contributes to Proteus mirabilis copper resistance.

Publisher: Springer Science and Business Media LLC

Date: 03-2019

DOI: 10.1038/S41467-019-08920-9

Abstract: This Article contains errors in Fig. 1, Table 1 and the Methods section. In panel c, the labels for PmScsC and EcDsbC in the upper two curves are interchanged. In Table 1 and the Methods section entitled ‘Extended structure’, the space group of the extended PmScsC structure is incorrectly referred to as H3 2 and should read H32. Correct versions of Fig. 1 and Table 1 are presented below the errors have not been corrected in the Article.

Cortactin adopts a globular conformation and bundles actin into sheets

Publisher: Elsevier BV

Date: 06-2008

DOI: 10.1074/JBC.M708917200

Staphylococcus aureus DsbA Does Not Have a Destabilizing Disulfide

Publisher: Elsevier BV

Date: 02-2008

DOI: 10.1074/JBC.M707838200

An Inflammatory Role for the Mammalian Carboxypeptidase Inhibitor Latexin: Relationship to Cystatins and the Tumor Suppressor TIG1

Publisher: Elsevier BV

Date: 02-2005

DOI: 10.1016/J.STR.2004.12.013

Abstract: Latexin, the only known mammalian carboxypeptidase inhibitor, has no detectable sequence similarity with plant and parasite inhibitors, but it is related to a human putative tumor suppressor protein, TIG1. Latexin is expressed in the developing brain, and we find that it plays a role in inflammation, as it is expressed at high levels and is inducible in macrophages in concert with other protease inhibitors and potential protease targets. The crystal structure of mouse latexin, solved at 1.83 A resolution, shows no structural relationship with other carboxypeptidase inhibitors. Furthermore, despite a lack of detectable sequence duplication, the structure incorporates two topologically analogous domains related by pseudo two-fold symmetry. Surprisingly, these domains share a cystatin fold architecture found in proteins that inhibit cysteine proteases, suggesting an evolutionary and possibly functional relationship. The structure of the tumor suppressor protein TIG1 was modeled, revealing its putative membrane binding surface.

Structural and Functional Characterization of the Oxidoreductase α-DsbA1 fromWolbachia pipientis

Publisher: Mary Ann Liebert Inc

Date: 07-2009

DOI: 10.1089/ARS.2008.2420

Abstract: The alpha-proteobacterium Wolbachia pipientis is a highly successful intracellular endosymbiont of invertebrates that manipulates its host's reproductive biology to facilitate its own maternal transmission. The fastidious nature of Wolbachia and the lack of genetic transformation have h ered analysis of the molecular basis of these manipulations. Structure determination of key Wolbachia proteins will enable the development of inhibitors for chemical genetics studies. Wolbachia encodes a homologue (alpha-DsbA1) of the Escherichia coli dithiol oxidase enzyme EcDsbA, essential for the oxidative folding of many exported proteins. We found that the active-site cysteine pair of Wolbachia alpha-DsbA1 has the most reducing redox potential of any characterized DsbA. In addition, Wolbachia alpha-DsbA1 possesses a second disulfide that is highly conserved in alpha-proteobacterial DsbAs but not in other DsbAs. The alpha-DsbA1 structure lacks the characteristic hydrophobic features of EcDsbA, and the protein neither complements EcDsbA deletion mutants in E. coli nor interacts with EcDsbB, the redox partner of EcDsbA. The surface characteristics and redox profile of alpha-DsbA1 indicate that it probably plays a specialized oxidative folding role with a narrow substrate specificity. This first report of a Wolbachia protein structure provides the basis for future chemical genetics studies.

Modification of recombinatorial cloning for small affinity tag fusion protein construct generation

Publisher: Elsevier BV

Date: 11-2005

DOI: 10.1016/J.AB.2005.06.016

Crystallization and preliminary diffraction analysis of a DsbA homologue from Wolbachia pipientis

Publisher: International Union of Crystallography (IUCr)

Date: 31-01-2007

DOI: 10.1107/S1744309108000055

Crystal structure of Toll-like receptor adaptor MAL/TIRAP reveals the molecular basis for signal transduction and disease protection

Publisher: Proceedings of the National Academy of Sciences

Date: 22-08-2011

DOI: 10.1073/PNAS.1104780108

Abstract: Initiation of the innate immune response requires agonist recognition by pathogen-recognition receptors such as the Toll-like receptors (TLRs). Toll/interleukin-1 receptor (TIR) domain-containing adaptors are critical in orchestrating the signal transduction pathways after TLR and interleukin-1 receptor activation. Myeloid differentiation primary response gene 88 (MyD88) adaptor-like (MAL)/TIR domain-containing adaptor protein (TIRAP) is involved in bridging MyD88 to TLR2 and TLR4 in response to bacterial infection. Genetic studies have associated a number of unique single-nucleotide polymorphisms in MAL with protection against invasive microbial infection, but a molecular understanding has been h ered by a lack of structural information. The present study describes the crystal structure of MAL TIR domain. Significant structural differences exist in the overall fold of MAL compared with other TIR domain structures: A sequence motif comprising a β-strand in other TIR domains instead corresponds to a long loop, placing the functionally important “BB loop” proline motif in a unique surface position in MAL. The structure suggests possible dimerization and MyD88-interacting interfaces, and we confirm the key interface residues by coimmunoprecipitation using site-directed mutants. Jointly, our results provide a molecular and structural basis for the role of MAL in TLR signaling and disease protection.

Cloning, expression, purification and characterization of a DsbA-like protein from Wolbachia pipientis

Publisher: Elsevier BV

Date: 06-2008

DOI: 10.1016/J.PEP.2008.02.008

Abstract: Wolbachia pipientis are obligate endosymbionts that infect a wide range of insect and other arthropod species. They act as reproductive parasites by manipulating the host reproduction machinery to enhance their own transmission. This unusual phenotype is thought to be a consequence of the actions of secreted Wolbachia proteins that are likely to contain disulfide bonds to stabilize the protein structure. In bacteria, the introduction or isomerization of disulfide bonds in proteins is catalyzed by Dsb proteins. The Wolbachia genome encodes two proteins, alpha-DsbA1 and alpha-DsbA2, that might catalyze these steps. In this work we focussed on the 234 residue protein alpha-DsbA1 the gene was cloned and expressed in Escherichia coli, the protein was purified and its identity confirmed by mass spectrometry. The sequence identity of alpha-DsbA1 for both dithiol oxidants (E. coli DsbA, 12%) and disulfide isomerases (E. coli DsbC, 14%) is similar. We therefore sought to establish whether alpha-DsbA1 is an oxidant or an isomerase based on functional activity. The purified alpha-DsbA1 was active in an oxidoreductase assay but had little isomerase activity, indicating that alpha-DsbA1 is DsbA-like rather than DsbC-like. This work represents the first successful ex le of the characterization of a recombinant Wolbachia protein. Purified alpha-DsbA1 will now be used in further functional studies to identify protein substrates that could help explain the molecular basis for the unusual Wolbachia phenotypes, and in structural studies to explore its relationship to other disulfide oxidoreductase proteins.

The structure of human SULT1A1 crystallized with estradiol - An insight into active site plasticity and substrate inhibition with multi-ring substrates

Publisher: Elsevier BV

Date: 12-2005

DOI: 10.1074/JBC.M508289200

Solving the α-conotoxin folding problem: Efficient selenium-directed on-resin generation of more potent and stable nicotinic acetylcholine receptor antaqonists

Publisher: American Chemical Society (ACS)

Date: 17-02-2010

DOI: 10.1021/JA910602H

Abstract: Alpha-conotoxins are tightly folded miniproteins that antagonize nicotinic acetylcholine receptors (nAChR) with high specificity for erse subtypes. Here we report the use of selenocysteine in a supported phase method to direct native folding and produce alpha-conotoxins efficiently with improved biophysical properties. By replacing complementary cysteine pairs with selenocysteine pairs on an hiphilic resin, we were able to chemically direct all five structural subclasses of alpha-conotoxins exclusively into their native folds. X-ray analysis at 1.4 A resolution of alpha-selenoconotoxin PnIA confirmed the isosteric character of the diselenide bond and the integrity of the alpha-conotoxin fold. The alpha-selenoconotoxins exhibited similar or improved potency at rat diaphragm muscle and alpha3beta4, alpha7, and alpha1beta1 deltagamma nAChRs expressed in Xenopus oocytes plus improved disulfide bond scrambling stability in plasma. Together, these results underpin the development of more stable and potent nicotinic antagonists suitable for new drug therapies, and highlight the application of selenocysteine technology more broadly to disulfide-bonded peptides and proteins.

Crystallography and protein–protein interactions: biological interfaces and crystal contacts

Publisher: Portland Press Ltd.

Date: 19-11-2008

DOI: 10.1042/BST0361438

Abstract: Crystallography is commonly used for studying the structures of protein–protein complexes. However, a crystal structure does not define a unique protein–protein interface, and distinguishing a ‘biological interface’ from ‘crystal contacts’ is often not straightforward. A number of computational approaches exist for distinguishing them, but their error rate is high, emphasizing the need to obtain further data on the biological interface using complementary structural and functional approaches. In addition to reviewing the computational and experimental approaches for addressing this problem, we highlight two relevant ex les. The first ex le from our laboratory involves the structure of acyl-CoA thioesterase 7, where each domain of this two-domain protein was crystallized separately, but both yielded a non-functional assembly. The structure of the full-length protein was uncovered using a combination of complementary approaches including chemical cross-linking, analytical ultracentrifugation and mutagenesis. The second ex le involves the platelet glycoprotein Ibα–thrombin complex. Two groups reported the crystal structures of this complex, but all the interacting interfaces differed between the two structures. Our computational analysis did not fully resolve the reasons for the discrepancies, but provided interesting insights into the system. This review highlights the need to complement crystallographic studies with complementary experimental and computational approaches.

Rv2969c, essential for optimal growth inMycobacterium tuberculosis, is a DsbA-like enzyme that interacts with VKOR-derived peptides and has atypical features of DsbA-like disulfide oxidases

Publisher: International Union of Crystallography (IUCr)

Date: 20-09-2013

DOI: 10.1107/S0907444913017800

Abstract: The bacterial disulfide machinery is an attractive molecular target for developing new antibacterials because it is required for the production of multiple virulence factors. The archetypal disulfide oxidase proteins in Escherichia coli (Ec) are DsbA and DsbB, which together form a functional unit: DsbA introduces disulfides into folding proteins and DsbB reoxidizes DsbA to maintain it in the active form. In Mycobacterium tuberculosis (Mtb), no DsbB homologue is encoded but a functionally similar but structurally ergent protein, MtbVKOR, has been identified. Here, the Mtb protein Rv2969c is investigated and it is shown that it is the DsbA-like partner protein of MtbVKOR. It is found that it has the characteristic redox features of a DsbA-like protein: a highly acidic catalytic cysteine, a highly oxidizing potential and a destabilizing active-site disulfide bond. Rv2969c also has peptide-oxidizing activity and recognizes peptide segments derived from the periplasmic loops of MtbVKOR. Unlike the archetypal EcDsbA enzyme, Rv2969c has little or no activity in disulfide-reducing and disulfide-isomerase assays. The crystal structure of Rv2969c reveals a canonical DsbA fold comprising a thioredoxin domain with an embedded helical domain. However, Rv2969c erges considerably from other DsbAs, including having an additional C-terminal helix (H8) that may restrain the mobility of the catalytic helix H1. The enzyme is also characterized by a very shallow hydrophobic binding surface and a negative electrostatic surface potential surrounding the catalytic cysteine. The structure of Rv2969c was also used to model the structure of a paralogous DsbA-like domain of the Ser/Thr protein kinase PknE. Together, these results show that Rv2969c is a DsbA-like protein with unique properties and a limited substrate-binding specificity.

Improved Success of Sparse Matrix Protein Crystallization Screening with Heterogeneous Nucleating Agents

Publisher: Public Library of Science (PLoS)

Date: 31-10-2007

DOI: 10.1371/JOURNAL.PONE.0001091

DSB proteins and bacterial pathogenicity

Publisher: Springer Science and Business Media LLC

Date: 09-02-2009

DOI: 10.1038/NRMICRO2087

Abstract: If DNA is the information of life, then proteins are the machines of life--but they must be assembled and correctly folded to function. A key step in the protein-folding pathway is the introduction of disulphide bonds between cysteine residues in a process called oxidative protein folding. Many bacteria use an oxidative protein-folding machinery to assemble proteins that are essential for cell integrity and to produce virulence factors. Although our current knowledge of this machinery stems largely from Escherichia coli K-12, this view must now be adjusted to encompass the wider range of disulphide catalytic systems present in bacteria.

Characterization of the DsbA Oxidative Folding Catalyst fromPseudomonas aeruginosaReveals a Highly Oxidizing Protein that Binds Small Molecules

Publisher: Mary Ann Liebert Inc

Date: 15-04-2010

DOI: 10.1089/ARS.2009.2736

Abstract: Bacterial antibiotic resistance is an emerging global crisis, and treatment of multidrug-resistant gram-negative infections, particularly those caused by the opportunistic human pathogen Pseudomonas aeruginosa, remains a major challenge. This problem is compounded by a lack of new antibiotics in the development pipeline: only two new classes have been developed since the 1960s, and both are indicated for multidrug-resistant gram-positive infections. A promising new approach to combat antibiotic resistance is by targeting bacterial virulence, rather than bacterial viability. The bacterial periplasmic protein DsbA represents a central point for antivirulence intervention because its oxidoreductase activity is essential for the folding and function of almost all exported virulence factors. Here we describe the three-dimensional structure of this DsbA target from P. aeruginosa, and we establish for the first time that a member of this enzyme family is capable of binding small molecules. We also describe biochemical assays that validate the redox activity of PaDsbA. Together, the structural and functional characterization of PaDsbA provides the basis for future studies aimed at designing a new class of antivirulence compounds to combat antibiotic-resistant P. aeruginosa infection.

Revisiting interaction specificity reveals neuronal and adipocyte Munc18 membrane fusion regulatory proteins differ in their binding interactions with partner SNARE Syntaxins

Publisher: Public Library of Science (PLoS)

Date: 31-10-2017

DOI: 10.1371/JOURNAL.PONE.0187302

Ten simple rules to achieve conference speaker gender balance.

Publisher: Public Library of Science (PLoS)

Date: 20-11-2014

DOI: 10.1371/JOURNAL.PCBI.1003903

Mode of binding of methyl acceptor substrates to the adrenaline-synthesizing enzyme phenylethanolamine N-methyltransferase: Implications for catalysis

Publisher: American Chemical Society (ACS)

Date: 12-2005

DOI: 10.1021/BI051636B

Abstract: Here we report three crystal structure complexes of human phenylethanolamine N-methyltransferase (PNMT), one bound with a substrate that incorporates a flexible ethanolamine side chain (p-octopamine), a second bound with a semirigid analogue substrate [cis-(1R,2S)-2-amino-1-tetralol, cis-(1R,2S)-AT], and a third with trans-(1S,2S)-2-amino-1-tetralol [trans-(1S,2S)-AT] that acts as an inhibitor of PNMT rather than a substrate. A water-mediated interaction between the critical beta-hydroxyl of the flexible ethanolamine group of p-octopamine and an acidic residue, Asp267, is likely to play a key role in positioning the side chain correctly for methylation to occur at the amine. A second interaction with Glu219 may play a lesser role. Catalysis likely occurs via deprotonation of the amine through the action of Glu185 mutation of this residue significantly reduced the kcat without affecting the Km. The mode of binding of cis-(1R,2S)-AT supports the notion that this substrate is a conformationally restrained analogue of flexible PNMT substrates, in that it forms interactions with the enzyme similar to those observed for p-octopamine. By contrast, trans-(1S,2S)-AT, an inhibitor rather than a substrate, binds in an orientation that is flipped by 180 degrees compared with cis-(1R,2S)-AT. A consequence of this flipped binding mode is that the interactions between the hydroxyl and Asp267 and Glu219 are lost. However, the amines of inhibitor trans-(1S,2S)-AT and substrate cis-(1R,2S)-AT are both within methyl transfer distance of the cofactor. These results suggest that PNMT catalyzes transfer of methyl to ligand amines only when "anchor" interactions, such as those identified for the beta-hydroxyls of p-octopamine and cis-AT, are present.

Human cytosolic sulfotransferase SULT1A1

Publisher: Elsevier BV

Date: 2007

DOI: 10.1016/J.BIOCEL.2006.10.002

Abstract: Sulfonation is an important conjugation reaction required for a range of biological processes including phase II metabolism, whereby sulfo-conjugation renders a compound more hydrophilic to aid its excretion. The major enzyme responsible for xenobiotic sulfonation is the widely expressed cytosolic sulfotransferase SULT1A1. The SULT1A1 crystal structure has provided insights into this enzyme's substrate specificity and catalytic function, including its role in the sulfonation of endogenous substrates such as oestrogens. Contrary to its metabolic role, SULT1A1 can also bioactivate compounds it is known to sulfonate pro-carcinogens such as hydroxymethyl polycyclic aromatic hydrocarbons leading to highly reactive intermediates capable of forming DNA adducts, potentially resulting in mutagenesis. Given the role of SULT1A1 in these erse functions and the discovery of allelic variants with differing catalytic activities, this enzyme has been the focus of numerous polymorphic studies investigating the link between inter-in idual SULT1A1 variance and the etiology of a variety of cancers.

Post-crystallization treatments for improving diffraction quality of protein crystals

Publisher: International Union of Crystallography (IUCr)

Date: 16-08-2005

DOI: 10.1107/S0907444905019451

Possible roles for Munc18-1 domain 3a and Syntaxin1 N-peptide and C-terminal anchor in SNARE complex formation

Publisher: Proceedings of the National Academy of Sciences

Date: 30-12-2011

DOI: 10.1073/PNAS.0914906108

Abstract: Munc18-1 and Syntaxin1 are essential proteins for SNARE-mediated neurotransmission. Munc18-1 participates in synaptic vesicle fusion via dual roles: as a docking/chaperone protein by binding closed Syntaxin1, and as a fusion protein that binds SNARE complexes in a Syntaxin1 N-peptide dependent manner. The two roles are associated with a closed–open Syntaxin1 conformational transition. Here, we show that Syntaxin N-peptide binding to Munc18-1 is not highly selective, suggesting that other parts of the SNARE complex are involved in binding to Munc18-1. We also find that Syntaxin1, with an N peptide and a physically anchored C terminus, binds to Munc18-1 and that this complex can participate in SNARE complex formation. We report a Munc18-1–N-peptide crystal structure that, together with other data, reveals how Munc18-1 might transit from a conformation that binds closed Syntaxin1 to one that may be compatible with binding open Syntaxin1 and SNARE complexes. Our results suggest the possibility that structural transitions occur in both Munc18-1 and Syntaxin1 during their binary interaction. We hypothesize that Munc18-1 domain 3a undergoes a conformational change that may allow coiled-coil interactions with SNARE complexes.

The Acidic Nature of the CcmG Redox-Active Center Is Important for Cytochrome c Maturation in Escherichia coli

Publisher: American Society for Microbiology

Date: 15-06-2004

DOI: 10.1128/JB.186.12.4030-4033.2004

Abstract: Cytochrome c biogenesis in Escherichia coli is a complex process requiring at least eight genes ( ccmABCDEFGH ). One of these genes, ccmG , encodes a thioredoxin-like protein with unusually specific redox activity. Here, we investigate the basis for CcmG function and demonstrate the importance of acidic residues surrounding the redox-active center.

Backbone resonance assignments of the monomeric DUF59 domain of human Fam96a

Publisher: Springer Science and Business Media LLC

Date: 22-05-2012

DOI: 10.1007/S12104-012-9390-1

Abstract: Proteins containing a domain of unknown function 59 (DUF59) appear to have a variety of physiological functions, ranging from iron-sulfur cluster assembly to DNA repair. DUF59 proteins have been found in bacteria, archaea and eukaryotes, however Fam96a and Fam96b are the only mammalian proteins predicted to contain a DUF59 domain. Fam96a is an 18 kDa protein comprised primarily of a DUF59 domain (residues 31-157) and an N-terminal signal peptide (residues 1-27). Interestingly, the DUF59 domain of Fam96a exists as monomeric and dimeric forms in solution, and X-ray crystallography studies of both forms unexpectedly revealed two different domain-swapped dimer structures. Here we report the backbone resonance assignments and secondary structure of the monomeric form of the 127 residue DUF59 domain of human Fam96a. This study provides the basis for further understanding the structural variability exhibited by Fam96a and the mechanism for domain swapping.

Crystallization and preliminary X-ray diffraction of the Munc18c-syntaxin41-29 complex

Publisher: International Union of Crystallography (IUCr)

Date: 18-05-2007

DOI: 10.1107/S1744309107022361

Towards selective lysophospholipid GPCR modulators

Publisher: Elsevier BV

Date: 05-2014

DOI: 10.1016/J.TIPS.2014.03.004

Abstract: G-protein-coupled receptors (GPCRs) that recognize the lysophospholipids (LPLs) are grouped into two phylogenetically distinct families: the endothelial differentiation gene (Edg) and non-Edg GPCRs. Owing to their more recent identification, and hindered by a lack of selective pharmacological tools, our understanding of the functions and signaling pathways of the non-Edg GPCRs is still in its infancy. Targeting the non-conserved allosteric binding sites of the LPL GPCRs shows particular promise for the development of selective modulators by structure-based drug design. However, only one Edg GPCR (S1PR1) structure has been determined to date, and it has low sequence identity with the non-Edg GPCRs (<20%). Thus, a representative structure of a non-Edg GPCR remains a pressing objective for selective structure-based drug design. Obtaining selective modulators targeting the non-Edg receptors would help to unravel the biology behind these novel GPCRs and potentially will support therapeutic treatment of diseases such as cancer, inflammation, and neuropsychiatric disorders.

An automatable screen for the rapid identification of proteins amenable to refolding

Publisher: Wiley

Date: 03-2006

DOI: 10.1002/PMIC.200500056

Abstract: Insoluble expression of heterologous proteins in Escherichia coli is a major bottleneck of many structural genomics and high-throughput protein biochemistry projects. Many of these proteins may be amenable to refolding, but their identification is h ered by a lack of high-throughput methods. We have developed a matrix-assisted refolding approach in which correctly folded proteins are distinguished from misfolded proteins by their elution from affinity resin under non-denaturing conditions. Misfolded proteins remain adhered to the resin, presumably via hydrophobic interactions. The assay can be applied to insoluble proteins on an in idual basis but is particularly well suited for high-throughput applications because it is rapid, automatable and has no rigorous s le preparation requirements. The efficacy of the screen is demonstrated on small-scale expression s les for 15 proteins. Refolding is then validated by large-scale expressions using SEC and circular dichroism.

A method for screening the temperature dependence of three-dimensional crystal formation

Publisher: International Union of Crystallography (IUCr)

Date: 19-04-2006

DOI: 10.1107/S0907444906006093

Incorporating a TEV cleavage site reduces the solubility of nine recombinant mouse proteins

Publisher: Elsevier BV

Date: 11-2006

DOI: 10.1016/J.PEP.2006.05.006

Abstract: Failure to express soluble proteins in bacteria is mainly attributed to the properties of the target protein itself, as well as the choice of the vector, the purification tag and the linker between the tag and protein, and codon usage. The expression of proteins with fusion tags to facilitate subsequent purification steps is a widely used procedure in the production of recombinant proteins. However, the additional residues can affect the properties of the protein therefore, it is often desirable to remove the tag after purification. This is usually done by engineering a cleavage site between the tag and the encoded protein that is recognised by a site-specific protease, such as the one from tobacco etch virus (TEV). In this study, we investigated the effect of four different tags on the bacterial expression and solubility of nine mouse proteins. Two of the four engineered constructs contained hexahistidine tags with either a long or short linker. The other two constructs contained a TEV cleavage site engineered into the linker region. Our data show that inclusion of the TEV recognition site directly downstream of the recombination site of the Invitrogen Gateway vector resulted in a loss of solubility of the nine mouse proteins. Our work suggests that one needs to be very careful when making modifications to expression vectors and combining different affinity and fusion tags and cleavage sites.

Identification and characterization of two drug-like fragments that bind to the same cryptic binding pocket of Burkholderia pseudomallei DsbA

Publisher: International Union of Crystallography (IUCr)

Date: 2022

DOI: 10.1107/S2059798321011475

Abstract: Disulfide-bond-forming proteins (Dsbs) play a crucial role in the pathogenicity of many Gram-negative bacteria. Disulfide-bond-forming protein A (DsbA) catalyzes the formation of the disulfide bonds necessary for the activity and stability of multiple substrate proteins, including many virulence factors. Hence, DsbA is an attractive target for the development of new drugs to combat bacterial infections. Here, two fragments, bromophenoxy propanamide ( 1 ) and 4-methoxy- N -phenylbenzenesulfonamide ( 2 ), were identified that bind to DsbA from the pathogenic bacterium Burkholderia pseudomallei , the causative agent of melioidosis. The crystal structures of oxidized B. pseudomallei DsbA (termed BpsDsbA) co-crystallized with 1 or 2 show that both fragments bind to a hydrophobic pocket that is formed by a change in the side-chain orientation of Tyr110. This conformational change opens a `cryptic' pocket that is not evident in the apoprotein structure. This binding location was supported by 2D-NMR studies, which identified a chemical shift perturbation of the Tyr110 backbone amide resonance of more than 0.05 p.p.m. upon the addition of 2 m M fragment 1 and of more than 0.04 p.p.m. upon the addition of 1 m M fragment 2 . Although binding was detected by both X-ray crystallography and NMR, the binding affinity ( K d ) for both fragments was low (above 2 m M ), suggesting weak interactions with BpsDsbA. This conclusion is also supported by the crystal structure models, which ascribe partial occupancy to the ligands in the cryptic binding pocket. Small fragments such as 1 and 2 are not expected to have a high energetic binding affinity due to their relatively small surface area and the few functional groups that are available for intermolecular interactions. However, their simplicity makes them ideal for functionalization and optimization. The identification of the binding sites of 1 and 2 to BpsDsbA could provide a starting point for the development of more potent novel antimicrobial compounds that target DsbA and bacterial virulence.

University Of Oxford

Location: United Kingdom of Great Britain and Northern Ireland

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University Of Queensland

Location: Australia

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University Of Wollongong

Location: Australia

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Victorian College Of Pharmacy

Location: Australia

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Griffith University

Location: Australia

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London Business School

Location: United Kingdom of Great Britain and Northern Ireland

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Linkage Infrastructure, Equipment And Facilities - Grant ID: LE170100075

Start Date: 2017

End Date: 12-2017

Amount: $315,000.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP0347228

Start Date: 2003

End Date: 12-2005

Amount: $360,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0561041

Start Date: 04-2005

End Date: 04-2006

Amount: $347,358.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0664564

Start Date: 2006

End Date: 03-2009

Amount: $286,000.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP0560794

Start Date: 06-2005

End Date: 06-2008

Amount: $251,704.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0450641

Start Date: 2004

End Date: 12-2006

Amount: $600,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP190101613

Start Date: 03-2019

End Date: 03-2022

Amount: $550,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0345019

Start Date: 2003

End Date: 12-2006

Amount: $270,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE180100179

Start Date: 03-2018

End Date: 12-2019

Amount: $3,189,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0668521

Start Date: 2006

End Date: 12-2007

Amount: $300,000.00

Funder: Australian Research Council

Special Research Initiatives - Grant ID: SR0354775

Start Date: 01-2004

End Date: 12-2003

Amount: $10,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE120100128

Start Date: 03-2012

End Date: 12-2013

Amount: $237,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0770465

Start Date: 2007

End Date: 03-2012

Amount: $1,462,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP160101702

Start Date: 2016

End Date: 12-2019

Amount: $497,400.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0668382

Start Date: 2006

End Date: 12-2008

Amount: $1,000,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0347585

Start Date: 2003

End Date: 12-2004

Amount: $320,000.00

Funder: Australian Research Council

Australian Laureate Fellowships - Grant ID: FL0992138

Start Date: 12-2009

End Date: 12-2014

Amount: $3,100,000.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP0990166

Start Date: 03-2010

End Date: 12-2012

Amount: $600,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP130100576

Start Date: 2013

End Date: 12-2016

Amount: $480,000.00

Funder: Australian Research Council

Special Research Initiatives - Grant ID: SR0354691

Start Date: 01-2004

End Date: 11-2004

Amount: $30,000.00

Funder: Australian Research Council

Research Networks - Grant ID: RN0460256

Start Date: 02-2005

End Date: 02-2010

Amount: $1,500,000.00

Funder: Australian Research Council

Special Research Initiatives - Grant ID: SR0354892

Start Date: 2004

End Date: 12-2004

Amount: $40,000.00

Funder: Australian Research Council

Special Research Initiatives - Grant ID: SR120200004

Start Date: 07-2012

End Date: 12-2015

Amount: $30,000,000.00

Funder: Australian Research Council