Publications by authors named "Jason J Paxman"

17 Publications

  • Page 1 of 1

BcfH Is a Trimeric Thioredoxin-Like Bifunctional Enzyme with Both Thiol Oxidase and Disulfide Isomerase Activities.

Antioxid Redox Signal 2021 Apr 12. Epub 2021 Apr 12.

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Australia.

Thioredoxin (TRX)-fold proteins are ubiquitous in nature. This redox scaffold has evolved to enable a variety of functions, including redox regulation, protein folding, and oxidative stress defense. In bacteria, the TRX-like disulfide bond (Dsb) family mediates the oxidative folding of multiple proteins required for fitness and pathogenic potential. Conventionally, Dsb proteins have specific redox functions with monomeric and dimeric Dsbs exclusively catalyzing thiol oxidation and disulfide isomerization, respectively. This contrasts with the eukaryotic disulfide forming machinery where the modular TRX protein disulfide isomerase (PDI) mediates thiol oxidation and disulfide reshuffling. In this study, we identified and structurally and biochemically characterized a novel Dsb-like protein from termed bovine colonization factor protein H (BcfH) and defined its role in virulence. In the conserved bovine colonization factor () fimbrial operon, the Dsb-like enzyme BcfH forms a trimeric structure, exceptionally uncommon among the large and evolutionary conserved TRX superfamily. This protein also displays very unusual catalytic redox centers, including an unwound α-helix holding the redox active site and a proline instead of the conserved -proline active site loop. Remarkably, BcfH displays both thiol oxidase and disulfide isomerase activities contributing to fimbrial biogenesis. Typically, oligomerization of bacterial Dsb proteins modulates their redox function, with monomeric and dimeric Dsbs mediating thiol oxidation and disulfide isomerization, respectively. This study demonstrates a further structural and functional malleability in the TRX-fold protein family. BcfH trimeric architecture and unconventional catalytic sites permit multiple redox functions emulating in bacteria the eukaryotic PDI dual oxidoreductase activity.
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http://dx.doi.org/10.1089/ars.2020.8218DOI Listing
April 2021

A Molecular Chameleon for Mapping Subcellular Polarity in an Unfolded Proteome Environment.

Angew Chem Int Ed Engl 2020 06 8;59(25):10129-10135. Epub 2020 Jan 8.

Department of Chemistry and Physics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, 3086, Australia.

Environmental polarity is an important factor that drives biomolecular interactions to regulate cell function. Herein, a general method of using the fluorogenic probe NTPAN-MI is reported to quantify the subcellular polarity change in response to protein unfolding. NTPAN-MI fluorescence is selectively activated upon labeling unfolded proteins with exposed thiols, thereby reporting on the extent of proteostasis. NTPAN-MI also reveals the collapse of the host proteome caused by influenza A virus infection. The emission profile of NTPAN-MI contains information of the local polarity of the unfolded proteome, which can be resolved through spectral phasor analysis. Under stress conditions that disrupt different checkpoints of protein quality control, distinct patterns of dielectric constant distribution in the cytoplasm can be observed. However, in the nucleus, the unfolded proteome was found to experience a more hydrophilic environment across all the stress conditions, indicating the central role of nucleus in the stress response process.
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http://dx.doi.org/10.1002/anie.201914263DOI Listing
June 2020

Molecular and structural insights into an asymmetric proteolytic complex (ClpP1P2) from Mycobacterium smegmatis.

Sci Rep 2019 12 2;9(1):18019. Epub 2019 Dec 2.

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, 3086, Australia.

The ClpP protease is found in all kingdoms of life, from bacteria to humans. In general, this protease forms a homo-oligomeric complex composed of 14 identical subunits, which associates with its cognate ATPase in a symmetrical manner. Here we show that, in contrast to this general architecture, the Clp protease from Mycobacterium smegmatis (Msm) forms an asymmetric hetero-oligomeric complex ClpP1P2, which only associates with its cognate ATPase through the ClpP2 ring. Our structural and functional characterisation of this complex demonstrates that asymmetric docking of the ATPase component is controlled by both the composition of the ClpP1 hydrophobic pocket (Hp) and the presence of a unique C-terminal extension in ClpP1 that guards this Hp. Our structural analysis of ClpP1 also revealed openings in the side-walls of the inactive tetradecamer, which may represent sites for product egress.
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http://dx.doi.org/10.1038/s41598-019-53736-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6889138PMC
December 2019

The Scs disulfide reductase system cooperates with the metallochaperone CueP in copper resistance.

J Biol Chem 2019 11 23;294(44):15876-15888. Epub 2019 Aug 23.

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Kingsbury Drive, Bundoora, Victoria 3083, Australia

The human pathogen serovar Typhimurium ( Typhimurium) contains a complex disulfide bond (Dsb) catalytic machinery. This machinery encompasses multiple Dsb thiol-disulfide oxidoreductases that mediate oxidative protein folding and a less-characterized suppressor of copper sensitivity () gene cluster, associated with increased tolerance to copper. To better understand the function of the Scs system, here we characterized two of its key components, the membrane protein ScsB and the periplasmic protein ScsC. Our results revealed that these two proteins form a redox pair in which the electron transfer from the periplasmic domain of ScsB (n-ScsB) to ScsC is thermodynamically driven. We also demonstrate that the Scs reducing pathway remains separate from the Dsb oxidizing pathways and thereby avoids futile redox cycles. Additionally, we provide new insight into the molecular mechanism underlying Scs-mediated copper tolerance in We show that both ScsB and ScsC can bind toxic copper(I) with femtomolar affinities and transfer it to the periplasmic copper metallochaperone CueP. Our results indicate that the Scs machinery has evolved a dual mode of action, capable of transferring reducing power to the oxidizing periplasm and protecting against copper stress by cooperating with the regulon, a major copper resistance mechanism in Overall, these findings expand our understanding of the functional diversity of Dsb-like systems, ranging from those mediating oxidative folding of proteins required for infection to those contributing to defense mechanisms against oxidative stress and copper toxicity, critical traits for niche adaptation and survival.
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http://dx.doi.org/10.1074/jbc.RA119.010164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6827279PMC
November 2019

Unique structural features of a bacterial autotransporter adhesin suggest mechanisms for interaction with host macromolecules.

Nat Commun 2019 04 29;10(1):1967. Epub 2019 Apr 29.

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, 3086, VIC, Australia.

Autotransporters are the largest family of outer membrane and secreted proteins in Gram-negative bacteria. Most autotransporters are localised to the bacterial surface where they promote colonisation of host epithelial surfaces. Here we present the crystal structure of UpaB, an autotransporter that is known to contribute to uropathogenic E. coli (UPEC) colonisation of the urinary tract. We provide evidence that UpaB can interact with glycosaminoglycans and host fibronectin. Unique modifications to its core β-helical structure create a groove on one side of the protein for interaction with glycosaminoglycans, while the opposite face can bind fibronectin. Our findings reveal far greater diversity in the autotransporter β-helix than previously thought, and suggest that this domain can interact with host macromolecules. The relevance of these interactions during infection remains unclear.
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http://dx.doi.org/10.1038/s41467-019-09814-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6488583PMC
April 2019

Structural and biochemical insights into the disulfide reductase mechanism of DsbD, an essential enzyme for neisserial pathogens.

J Biol Chem 2018 10 4;293(43):16559-16571. Epub 2018 Sep 4.

From the Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne 3086, Victoria, Australia,

The worldwide incidence of neisserial infections, particularly gonococcal infections, is increasingly associated with antibiotic-resistant strains. In particular, extensively drug-resistant strains that are resistant to third-generation cephalosporins are a major public health concern. There is a pressing clinical need to identify new targets for the development of antibiotics effective against -specific processes. In this study, we report that the bacterial disulfide reductase DsbD is highly prevalent and conserved among spp. and that this enzyme is essential for survival of DsbD is a membrane-bound protein that consists of two periplasmic domains, n-DsbD and c-DsbD, which flank the transmembrane domain t-DsbD. In this work, we show that the two functionally essential periplasmic domains of DsbD catalyze electron transfer reactions through unidirectional interdomain interactions, from reduced c-DsbD to oxidized n-DsbD, and that this process is not dictated by their redox potentials. Structural characterization of the n- and c-DsbD domains in both redox states provides evidence that steric hindrance reduces interactions between the two periplasmic domains when n-DsbD is reduced, thereby preventing a futile redox cycle. Finally, we propose a conserved mechanism of electron transfer for DsbD and define the residues involved in domain-domain recognition. Inhibitors of the interaction of the two DsbD domains have the potential to be developed as anti-neisserial agents.
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http://dx.doi.org/10.1074/jbc.RA118.004847DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6204915PMC
October 2018

Production, biophysical characterization and initial crystallization studies of the N- and C-terminal domains of DsbD, an essential enzyme in Neisseria meningitidis.

Acta Crystallogr F Struct Biol Commun 2018 01 1;74(Pt 1):31-38. Epub 2018 Jan 1.

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria 3086, Australia.

The membrane protein DsbD is a reductase that acts as an electron hub, translocating reducing equivalents from cytoplasmic thioredoxin to a number of periplasmic substrates involved in oxidative protein folding, cytochrome c maturation and oxidative stress defence. DsbD is a multi-domain protein consisting of a transmembrane domain (t-DsbD) flanked by two periplasmic domains (n-DsbD and c-DsbD). Previous studies have shown that DsbD is required for the survival of the obligate human pathogen Neisseria meningitidis. To help understand the structural and functional aspects of N. meningitidis DsbD, the two periplasmic domains which are required for electron transfer are being studied. Here, the expression, purification and biophysical properties of n-NmDsbD and c-NmDsbD are described. The crystallization and crystallographic analysis of n-NmDsbD and c-NmDsbD are also described in both redox states, which differ only in the presence or absence of a disulfide bond but which crystallized in completely different conditions. Crystals of n-NmDsbD, n-NmDsbD, c-NmDsbD and c-NmDsbD diffracted to 2.3, 1.6, 2.3 and 1.7 Å resolution and belonged to space groups P23, P321, P4 and P121, respectively.
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http://dx.doi.org/10.1107/S2053230X17017800DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5947690PMC
January 2018

Recognition by host nuclear transport proteins drives disorder-to-order transition in Hendra virus V.

Sci Rep 2018 01 10;8(1):358. Epub 2018 Jan 10.

Infection & Immunity Program, Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.

Hendra virus (HeV) is a paramyxovirus that causes lethal disease in humans, for which no vaccine or antiviral agent is available. HeV V protein is central to pathogenesis through its ability to interact with cytoplasmic host proteins, playing key antiviral roles. Here we use immunoprecipitation, siRNA knockdown and confocal laser scanning microscopy to show that HeV V shuttles to and from the nucleus through specific host nuclear transporters. Spectroscopic and small angle X-ray scattering studies reveal HeV V undergoes a disorder-to-order transition upon binding to either importin α/β1 or exportin-1/Ran-GTP, dependent on the V N-terminus. Importantly, we show that specific inhibitors of nuclear transport prevent interaction with host transporters, and reduce HeV infection. These findings emphasize the critical role of host-virus interactions in HeV infection, and potential use of compounds targeting nuclear transport, such as the FDA-approved agent ivermectin, as anti-HeV agents.
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http://dx.doi.org/10.1038/s41598-017-18742-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5762688PMC
January 2018

Inhibition of Diverse DsbA Enzymes in Multi-DsbA Encoding Pathogens.

Antioxid Redox Signal 2018 09 1;29(7):653-666. Epub 2018 Feb 1.

2 Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University , Bundoora, Australia .

Aims: DsbA catalyzes disulfide bond formation in secreted and outer membrane proteins in bacteria. In pathogens, DsbA is a major facilitator of virulence constituting a target for antivirulence antimicrobial development. However, many pathogens encode multiple and diverse DsbA enzymes for virulence factor folding during infection. The aim of this study was to determine whether our recently identified inhibitors of Escherichia coli K-12 DsbA can inhibit the diverse DsbA enzymes found in two important human pathogens and attenuate their virulence.

Results: DsbA inhibitors from two chemical classes (phenylthiophene and phenoxyphenyl derivatives) inhibited the virulence of uropathogenic E. coli and Salmonella enterica serovar Typhimurium, encoding two and three diverse DsbA homologues, respectively. Inhibitors blocked the virulence of dsbA null mutants complemented with structurally diverse DsbL and SrgA, suggesting that they were not selective for prototypical DsbA. Structural characterization of DsbA-inhibitor complexes showed that compounds from each class bind in a similar region of the hydrophobic groove adjacent to the Cys30-Pro31-His32-Cys33 (CPHC) active site. Modeling of DsbL- and SrgA-inhibitor interactions showed that these accessory enzymes could accommodate the inhibitors in their different hydrophobic grooves, supporting our in vivo findings. Further, we identified highly conserved residues surrounding the active site for 20 diverse bacterial DsbA enzymes, which could be exploited in developing inhibitors with a broad spectrum of activity. Innovation and Conclusion: We have developed tools to analyze the specificity of DsbA inhibitors in bacterial pathogens encoding multiple DsbA enzymes. This work demonstrates that DsbA inhibitors can be developed to target diverse homologues found in bacteria. Antioxid. Redox Signal. 29, 653-666.
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http://dx.doi.org/10.1089/ars.2017.7104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6067686PMC
September 2018

Autotransporter Adhesins in Escherichia coli Pathogenesis.

Proteomics 2017 Dec 12;17(23-24). Epub 2017 Oct 12.

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia.

Most bacteria produce adhesion molecules to facilitate the interaction with host cells and establish successful infections. An important group of bacterial adhesins belong to the autotransporter (AT) superfamily, the largest group of secreted and outer membrane proteins in Gram-negative bacteria. AT adhesins possess diverse functions that facilitate bacterial colonisation, survival and persistence, and as such are often associated with increased bacterial fitness and pathogenic potential. In this review, we will describe AIDA-I type AT adhesins, which comprise the biggest and most diverse group in the AT family. We will focus on Escherichia coli proteins and define general aspects of their biogenesis, distribution, structural properties and key roles in infection.
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http://dx.doi.org/10.1002/pmic.201600431DOI Listing
December 2017

Bioinformatics Tools and Resources for Analyzing Protein Structures.

Methods Mol Biol 2017 ;1549:209-220

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Rm 521, LIMS1, Kingsbury Drive, Bundoora, Melbourne, VIC, 3086, Australia.

The dramatic increase in the number of protein sequences and structures deposited in biological databases has led to the development of many bioinformatics tools and programs to manage, validate, compare, and interpret this large volume of data. In addition, powerful tools are being developed to use this sequence and structural data to facilitate protein classification and infer biological function of newly identified proteins. This chapter covers freely available bioinformatics resources on the World Wide Web that are commonly used for protein structure analysis.
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http://dx.doi.org/10.1007/978-1-4939-6740-7_16DOI Listing
February 2018

Targeting Bacterial Dsb Proteins for the Development of Anti-Virulence Agents.

Molecules 2016 Jul 16;21(7). Epub 2016 Jul 16.

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Kingsbury Drive, Bundoora, Vic 3083, Australia.

Recent years have witnessed a dramatic increase in bacterial antimicrobial resistance and a decline in the development of novel antibiotics. New therapeutic strategies are urgently needed to combat the growing threat posed by multidrug resistant bacterial infections. The Dsb disulfide bond forming pathways are potential targets for the development of antimicrobial agents because they play a central role in bacterial pathogenesis. In particular, the DsbA/DsbB system catalyses disulfide bond formation in a wide array of virulence factors, which are essential for many pathogens to establish infections and cause disease. These redox enzymes are well placed as antimicrobial targets because they are taxonomically widespread, share low sequence identity with human proteins, and many years of basic research have provided a deep molecular understanding of these systems in bacteria. In this review, we discuss disulfide bond catalytic pathways in bacteria and their significance in pathogenesis. We also review the use of different approaches to develop inhibitors against Dsb proteins as potential anti-virulence agents, including fragment-based drug discovery, high-throughput screening and other structure-based drug discovery methods.
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http://dx.doi.org/10.3390/molecules21070811DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6273893PMC
July 2016

Structural Determinants Defining the Allosteric Inhibition of an Essential Antibiotic Target.

Structure 2016 08 14;24(8):1282-1291. Epub 2016 Jul 14.

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, VIC 3086, Australia. Electronic address:

Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step in the lysine biosynthesis pathway of bacteria. The pathway can be regulated by feedback inhibition of DHDPS through the allosteric binding of the end product, lysine. The current dogma states that DHDPS from Gram-negative bacteria are inhibited by lysine but orthologs from Gram-positive species are not. The 1.65-Å resolution structure of the Gram-negative Legionella pneumophila DHDPS and the 1.88-Å resolution structure of the Gram-positive Streptococcus pneumoniae DHDPS bound to lysine, together with comprehensive functional analyses, show that this dogma is incorrect. We subsequently employed our crystallographic data with bioinformatics, mutagenesis, enzyme kinetics, and microscale thermophoresis to reveal that lysine-mediated inhibition is not defined by Gram staining, but by the presence of a His or Glu at position 56 (Escherichia coli numbering). This study has unveiled the molecular determinants defining lysine-mediated allosteric inhibition of bacterial DHDPS.
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http://dx.doi.org/10.1016/j.str.2016.05.019DOI Listing
August 2016

Molecular and Structural Characterization of a Novel Escherichia coli Interleukin Receptor Mimic Protein.

mBio 2016 Mar 15;7(2):e02046. Epub 2016 Mar 15.

Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Australia

Unlabelled: Urinary tract infection (UTI) is a disease of extremely high incidence in both community and nosocomial settings. UTIs cause significant morbidity and mortality, with approximately 150 million cases globally per year. Uropathogenic Escherichia coli (UPEC) is the primary cause of UTI and is generally treated empirically. However, the rapidly increasing incidence of UTIs caused by multidrug-resistant UPEC strains has led to limited available treatment options and highlights the urgent need to develop alternative treatment and prevention strategies. In this study, we performed a comprehensive analysis to define the regulation, structure, function, and immunogenicity of recently identified UPEC vaccine candidate C1275 (here referred to as IrmA). We showed that the irmA gene is highly prevalent in UPEC, is cotranscribed with the biofilm-associated antigen 43 gene, and is regulated by the global oxidative stress response OxyR protein. Localization studies identified IrmA in the UPEC culture supernatant. We determined the structure of IrmA and showed that it adopts a unique domain-swapped dimer architecture. The dimeric structure of IrmA displays similarity to those of human cytokine receptors, including the interleukin-2 receptor (IL-2R), interleukin-4 receptor (IL-4R), and interleukin-10 receptor (IL-10R) binding domains, and we showed that purified IrmA can bind to their cognate cytokines. Finally, we showed that plasma from convalescent urosepsis patients contains high IrmA antibody titers, demonstrating the strong immunogenicity of IrmA. Taken together, our results indicate that IrmA may play an important role during UPEC infection.

Importance: Uropathogenic E. coli (UPEC) is the primary cause of urinary tract infection (UTI), a disease of major significance to human health. Globally, the incidence of UPEC-mediated UTI is strongly associated with increasing antibiotic resistance, making this extremely common infection a major public health concern. In this report, we describe the regulatory, structural, functional, and immunogenic properties of a candidate UPEC vaccine antigen, IrmA. We demonstrate that IrmA is a small UPEC protein that forms a unique domain-swapped dimer with structural mimicry to several human cytokine receptors. We also show that IrmA binds to IL-2, IL-4, and IL-10, is strongly immunogenic in urosepsis patients, and is coexpressed with factors associated with biofilm formation. Overall, this work suggests a potential novel contribution for IrmA in UPEC infection.
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http://dx.doi.org/10.1128/mBio.02046-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4807369PMC
March 2016

The antigen 43 structure reveals a molecular Velcro-like mechanism of autotransporter-mediated bacterial clumping.

Proc Natl Acad Sci U S A 2014 Jan 13;111(1):457-62. Epub 2013 Dec 13.

Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC 3086, Australia.

Aggregation and biofilm formation are critical mechanisms for bacterial resistance to host immune factors and antibiotics. Autotransporter (AT) proteins, which represent the largest group of outer-membrane and secreted proteins in Gram-negative bacteria, contribute significantly to these phenotypes. Despite their abundance and role in bacterial pathogenesis, most AT proteins have not been structurally characterized, and there is a paucity of detailed information with regard to their mode of action. Here we report the structure-function relationships of Antigen 43 (Ag43a), a prototypic self-associating AT protein from uropathogenic Escherichia coli. The functional domain of Ag43a displays a twisted L-shaped β-helical structure firmly stabilized by a 3D hydrogen-bonded scaffold. Notably, the distinctive Ag43a L shape facilitates self-association and cell aggregation. Combining all our data, we define a molecular "Velcro-like" mechanism of AT-mediated bacterial clumping, which can be tailored to fit different bacterial lifestyles such as the formation of biofilms.
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http://dx.doi.org/10.1073/pnas.1311592111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3890832PMC
January 2014

Cloning to crystallization of dihydrodipicolinate synthase from the intracellular pathogen Legionella pneumophila.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2013 Oct 30;69(Pt 10):1177-81. Epub 2013 Sep 30.

Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC 3086, Australia.

Dihydrodipicolinate synthase (DHDPS) catalyses the rate-limiting step in the biosynthesis of meso-diaminopimelate and lysine. Here, the cloning, expression, purification and crystallization of DHDPS from the intracellular pathogen Legionella pneumophila are described. Crystals grown in the presence of high-molecular-weight PEG precipitant and magnesium chloride were found to diffract beyond 1.65 Å resolution. The crystal lattice belonged to the hexagonal space group P6₁22, with unit-cell parameters a=b=89.31, c=290.18 Å, and contained two molecules in the asymmetric unit. The crystal structure was determined by molecular replacement using a single chain of Pseudomonas aeruginosa DHDPS as the search model.
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http://dx.doi.org/10.1107/S1744309113024639DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3792684PMC
October 2013

The structure of the bacterial oxidoreductase enzyme DsbA in complex with a peptide reveals a basis for substrate specificity in the catalytic cycle of DsbA enzymes.

J Biol Chem 2009 Jun 22;284(26):17835-45. Epub 2009 Apr 22.

Medicinal Chemistry and Drug Action, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville Campus, 381 Royal Parade, Parkville, Victoria 3052, Australia.

Oxidative protein folding in Gram-negative bacteria results in the formation of disulfide bonds between pairs of cysteine residues. This is a multistep process in which the dithiol-disulfide oxidoreductase enzyme, DsbA, plays a central role. The structure of DsbA comprises an all helical domain of unknown function and a thioredoxin domain, where active site cysteines shuttle between an oxidized, substrate-bound, reduced form and a DsbB-bound form, where DsbB is a membrane protein that reoxidizes DsbA. Most DsbA enzymes interact with a wide variety of reduced substrates and show little specificity. However, a number of DsbA enzymes have now been identified that have narrow substrate repertoires and appear to interact specifically with a smaller number of substrates. The transient nature of the DsbA-substrate complex has hampered our understanding of the factors that govern the interaction of DsbA enzymes with their substrates. Here we report the crystal structure of a complex between Escherichia coli DsbA and a peptide with a sequence derived from a substrate. The binding site identified in the DsbA-peptide complex was distinct from that observed for DsbB in the DsbA-DsbB complex. The structure revealed details of the DsbA-peptide interaction and suggested a mechanism by which DsbA can simultaneously show broad specificity for substrates yet exhibit specificity for DsbB. This mode of binding was supported by solution nuclear magnetic resonance data as well as functional data, which demonstrated that the substrate specificity of DsbA could be modified via changes at the binding interface identified in the structure of the complex.
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http://dx.doi.org/10.1074/jbc.M109.011502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2719422PMC
June 2009