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Brucella ovis is an etiologic agent of ovine epididymitis and brucellosis that causes global devastation in sheep, rams, goats, small ruminants and deer. There are no cost-effective methods for the worldwide eradication of ovine brucellosis. B. ovis and other protein targets from various Brucella species are currently in the pipeline for high-throughput structural analysis at the Seattle Structural Genomics Center for Infectious Disease (SSGCID), with the aim of identifying new therapeutic targets. Furthermore, the wealth of structures generated are effective tools for teaching scientific communication, structural science and biochemistry. One of these structures, B. ovis leucine-, isoleucine-, valine-, threonine- and alanine-binding protein (BoLBP), is a putative periplasmic amino acid-binding protein. BoLBP shares less than 29% sequence identity with any other structure in the Protein Data Bank. The production, crystallization and high-resolution structures of BoLBP are reported. BoLBP is a prototypical bacterial periplasmic amino acid-binding protein with the characteristic Venus flytrap topology of two globular domains encapsulating a large central cavity containing the peptide-binding region. The central cavity contains small molecules usurped from the crystallization milieu. The reported structures reveal the conformational flexibility of the central cavity in the absence of bound peptides. The structural similarity to other LBPs can be exploited to accelerate drug repurposing., (open access.)

Background: The macrophage infectivity potentiator (Mip) protein, which belongs to the immunophilin superfamily, is a peptidyl-prolyl cis/trans isomerase (PPIase) enzyme. Mip has been shown to be important for virulence in a wide range of pathogenic microorganisms. It has previously been demonstrated that small-molecule compounds designed to target Mip from the Gram-negative bacterium Burkholderia pseudomallei bind at the site of enzymatic activity of the protein, inhibiting the in vitro activity of Mip., Objectives: In this study, co-crystallography experiments with recombinant B. pseudomallei Mip (BpMip) protein and Mip inhibitors, biochemical analysis and computational modelling were used to predict the efficacy of lead compounds for broad-spectrum activity against other pathogens., Methods: Binding activity of three lead compounds targeting BpMip was verified using surface plasmon resonance spectroscopy. The determination of crystal structures of BpMip in complex with these compounds, together with molecular modelling and in vitro assays, was used to determine whether the compounds have broad-spectrum antimicrobial activity against pathogens., Results: Of the three lead small-molecule compounds, two were effective in inhibiting the PPIase activity of Mip proteins from Neisseria meningitidis, Klebsiella pneumoniae and Leishmania major. The compounds also reduced the intracellular burden of these pathogens using in vitro cell infection assays., Conclusions: These results indicate that Mip is a novel antivirulence target that can be inhibited using small-molecule compounds that prove to be promising broad-spectrum drug candidates in vitro. Further optimization of compounds is required for in vivo evaluation and future clinical applications., (© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

Burkholderia pseudomallei infection causes melioidosis, which is often fatal if untreated. There is a need to develop new and more effective treatments for melioidosis. This study reports apo and cofactor-bound crystal structures of the potential drug target betaine aldehyde dehydrogenase (BADH) from B. pseudomallei. A structural comparison identified similarities to BADH from Pseudomonas aeruginosa which is inhibited by the drug disulfiram. This preliminary analysis could facilitate drug-repurposing studies for B. pseudomallei., (open access.)

Rickettsia felis, a Gram-negative bacterium that causes spotted fever, is of increasing interest as an emerging human pathogen. R. felis and several other Rickettsia strains are classed as National Institute of Allergy and Infectious Diseases priority pathogens. In recent years, R. felis has been shown to be adaptable to a wide range of hosts, and many fevers of unknown origin are now being attributed to this infectious agent. Here, the structure of acetoacetyl-CoA reductase from R. felis is reported at a resolution of 2.0 Å. While R. felis acetoacetyl-CoA reductase shares less than 50% sequence identity with its closest homologs, it adopts a fold common to other short-chain dehydrogenase/reductase (SDR) family members, such as the fatty-acid synthesis II enzyme FabG from the prominent pathogens Staphylococcus aureus and Bacillus anthracis. Continued characterization of the Rickettsia proteome may prove to be an effective means of finding new avenues of treatment through comparative structural studies.

A protein superfamily with a "Domain of Unknown Function,", DUF3349 (PF11829), is present predominately in Mycobacterium and Rhodococcus bacterial species suggesting that these proteins may have a biological function unique to these bacteria. We previously reported the inaugural structure of a DUF3349 superfamily member, Mycobacterium tuberculosis Rv0543c. Here, we report the structures determined for three additional DUF3349 proteins: Mycobacterium smegmatis MSMEG_1063 and MSMEG_1066 and Mycobacterium abscessus MAB_3403c. Like Rv0543c, the NMR solution structure of MSMEG_1063 revealed a monomeric five α-helix bundle with a similar overall topology. Conversely, the crystal structure of MSMEG_1066 revealed a five α-helix protein with a strikingly different topology and a tetrameric quaternary structure that was confirmed by size exclusion chromatography. The NMR solution structure of a fourth member of the DUF3349 superfamily, MAB_3403c, with 18 residues missing at the N-terminus, revealed a monomeric α-helical protein with a folding topology similar to the three C-terminal helices in the protomer of the MSMEG_1066 tetramer. These structures, together with a GREMLIN-based bioinformatics analysis of the DUF3349 primary amino acid sequences, suggest two subfamilies within the DUF3349 family. The division of the DUF3349 into two distinct subfamilies would have been lost if structure solution had stopped with the first structure in the DUF3349 family, highlighting the insights generated by solving multiple structures within a protein superfamily. Future studies will determine if the structural diversity at the tertiary and quaternary levels in the DUF3349 protein superfamily have functional roles in Mycobacteria and Rhodococcus species with potential implications for structure-based drug discovery., (© 2019 The Protein Society.)

Crystal structure determination requires solving the phase problem. This can be accomplished using ab initio direct methods for small molecules and macromolecules at resolutions higher than 1.2 Å, whereas macromolecular structure determination at lower resolution requires either molecular replacement using a homologous structure or experimental phases using a derivative such as covalent labeling (for example selenomethionine or mercury derivatization) or heavy-atom soaking (for example iodide ions). Here, a case is presented in which crystals were obtained from a 30.8 kDa protein sample and yielded a 1.6 Å resolution data set with a unit cell that could accommodate approximately 8 kDa of protein. Thus, it was unclear what had been crystallized. Molecular replacement with pieces of homologous proteins and attempts at iodide ion soaking failed to yield a solution. The crystals could not be reproduced. Sequence-independent molecular replacement using the structures available in the Protein Data Bank also failed to yield a solution. Ultimately, ab initio structure solution proved successful using the program ARCIMBOLDO, which identified two α-helical elements and yielded interpretable maps. The structure was the C-terminal dimerization domain of the intended target from Mycobacterium smegmatis. This structure is presented as a user-friendly test case in which an unknown protein fragment could be determined using ARCIMBOLDO.

The genome of the human pathogen Mycobacterium tuberculosis (Mtb) encodes ∼4,400 proteins, but one third of them have unknown functions. We solved the crystal structure of Rv3651, a hypothetical protein with no discernible similarity to proteins with known function. Rv3651 has a three-domain architecture that combines one cGMP-specific phosphodiesterases, adenylyl cyclases and FhlA (GAF) domain and two Per-ARNT-Sim (PAS) domains. GAF and PAS domains are sensor domains that are typically linked to signaling effector molecules. Unlike these sensor-effector proteins, Rv3651 is an unusual sensor domain-only protein with highly divergent sequence. The structure suggests that Rv3651 integrates multiple different signals and serves as a scaffold to facilitate signal transfer., (© 2017 The Protein Society.)

Thioredoxins are small ubiquitous proteins that participate in a diverse variety of redox reactions via the reversible oxidation of two cysteine thiol groups in a structurally conserved active site. Here, the NMR solution structures of a reduced and oxidized thioredoxin from Ehrlichia chaffeensis (Ec-Trx, ECH_0218), the etiological agent responsible for human monocytic ehrlichiosis, are described. The overall topology of the calculated structures is similar in both redox states and is similar to those of other thioredoxins: a five-stranded, mixed β-sheet (β1-β3-β2-β4-β5) surrounded by four α-helices. Unlike other thioredoxins studied by NMR in both redox states, the 1 H- 15 N HSQC spectrum of reduced Ec-Trx was missing eight additional amide cross peaks relative to the spectrum of oxidized Ec-Trx. These missing amides correspond to residues Cys35-Glu39 in the active-site-containing helix (α2) and Ser72-Ile75 in a loop near the active site, and suggest a change in backbone dynamics on the millisecond-to-microsecond timescale associated with the breakage of an intramolecular Cys32-Cys35 disulfide bond in a thioredoxin. A consequence of the missing amide resonances is the absence of observable or unambiguous NOEs to provide the distance restraints necessary to define the N-terminal end of the α-helix containing the CPGC active site in the reduced state. This region adopts a well defined α-helical structure in other reported reduced thioredoxin structures, is mostly helical in oxidized Ec-Trx and CD studies of Ec-Trx in both redox states suggests there is no significant difference in the secondary structure of the protein. The NMR solution structure of reduced Ec-Trx illustrates that the absence of canonical structure in a region of a protein may be owing to unfavorable dynamics prohibiting NOE observations or unambiguous NOE assignments.

Mycobacterium tuberculosis protein Rv0577 is a prominent antigen in tuberculosis patients, the component responsible for neutral red staining of virulent strains of M. tuberculosis, a putative component in a methylglyoxal detoxification pathway, and an agonist of toll-like receptor 2. It also has an amino acid sequence that is 36% identical to that of Streptomyces coelicolor AfsK-binding protein A (KbpA), a component in the complex secondary metabolite pathways in the Streptomyces genus. To gain insight into the biological function of Rv0577 and the family of KpbA kinase regulators, the crystal structure for Rv0577 was determined to a resolution of 1.75 Å, binding properties with neutral red and deoxyadenosine were surveyed, backbone dynamics were measured, and thermal stability was assayed by circular dichroism spectroscopy. The protein is composed of four approximate repeats with a βαβββ topology arranged radially in consecutive pairs to form two continuous eight-strand β-sheets capped on both ends with an α-helix. The two β-sheets intersect in the center at roughly a right angle and form two asymmetric deep "saddles" that may serve to bind ligands. Nuclear magnetic resonance chemical shift perturbation experiments show that neutral red and deoxyadenosine bind to Rv0577. Binding to deoxyadenosine is weaker with an estimated dissociation constants of 4.1 ± 0.3 mM for saddle 1. Heteronuclear steady-state { 1 H}- 15 N nuclear Overhauser effect, T 1 , and T 2 values were generally uniform throughout the sequence with only a few modest pockets of differences. Circular dichroism spectroscopy characterization of the thermal stability of Rv0577 indicated irreversible unfolding upon heating with an estimated melting temperature of 56 °C.

In bacteria, the second committed step in the diaminopimelate/lysine anabolic pathways is catalyzed by the enzyme dihydrodipicolinate reductase (DapB). DapB catalyzes the reduction of dihydrodipicolinate to yield tetrahydrodipicolinate. Here, the cloning, expression, purification, crystallization and X-ray diffraction analysis of DapB from the human-pathogenic bacterium Bartonella henselae, the causative bacterium of cat-scratch disease, are reported. Protein crystals were grown in conditions consisting of 5%(w/v) PEG 4000, 200 mM sodium acetate, 100 mM sodium citrate tribasic pH 5.5 and were shown to diffract to ∼2.3 Å resolution. They belonged to space group P4 3 22, with unit-cell parameters a = 109.38, b = 109.38, c = 176.95 Å. R r.i.m. was 0.11, R work was 0.177 and R free was 0.208. The three-dimensional structural features of the enzymes show that DapB from B. henselae is a tetramer consisting of four identical polypeptides. In addition, the substrate NADP + was found to be bound to one monomer, which resulted in a closed conformational change in the N-terminal domain.

The enzyme dihydrodipicolinate synthase catalyzes the committed step in the synthesis of diaminopimelate and lysine to facilitate peptidoglycan and protein synthesis. Dihydrodipicolinate synthase catalyzes the condensation of L-aspartate 4-semialdehyde and pyruvate to synthesize L-2,3-dihydrodipicolinate. Here, the cloning, expression, purification, crystallization and X-ray diffraction analysis of dihydrodipicolinate synthase from the pathogenic bacterium Bartonella henselae, the causative bacterium of cat-scratch disease, are presented. Protein crystals were grown in conditions consisting of 20%(w/v) PEG 4000, 100 mM sodium citrate tribasic pH 5.5 and were shown to diffract to ∼2.10 Å resolution. They belonged to space group P212121, with unit-cell parameters a = 79.96, b = 106.33, c = 136.25 Å. The final R values were Rr.i.m. = 0.098, Rwork = 0.183, Rfree = 0.233.

According to a Pew Research study published in February 2015, there are 37 antibacterial programs currently in clinical trials in the United States. Protein structure-based methods for guiding small molecule design were used in at least 34 of these programs. Typically, this occurred at an early stage (drug discovery and/or lead optimization) prior to an Investigational New Drug (IND) application, although sometimes in retrospective studies to rationalize biological activity. Recognizing that structure-based methods are resource-intensive and often require specialized equipment and training, the NIAID has funded two Structural Genomics Centers to determine structures of infectious disease species proteins with the aim of supporting individual investigators' research programs with structural biology methods., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The methylmalonyl Co-A mutase-associated GTPase MeaB from Methylobacterium extorquens is involved in glyoxylate regulation and required for growth. In humans, mutations in the homolog methylmalonic aciduria associated protein (MMAA) cause methylmalonic aciduria, which is often fatal. The central role of MeaB from bacteria to humans suggests that MeaB is also important in other, pathogenic bacteria such as Mycobacterium tuberculosis. However, the identity of the mycobacterial MeaB homolog is presently unclear. Here, we identify the M. tuberculosis protein Rv1496 and its homologs in M. smegmatis and M. thermoresistibile as MeaB. The crystal structures of all three homologs are highly similar to MeaB and MMAA structures and reveal a characteristic three-domain homodimer with GDP bound in the G domain active site. A structure of Rv1496 obtained from a crystal grown in the presence of GTP exhibited electron density for GDP, suggesting GTPase activity. These structures identify the mycobacterial MeaB and provide a structural framework for therapeutic targeting of M. tuberculosis MeaB.

Ribonucleases (RNases) maintain the cellular RNA pool by RNA processing and degradation. In many bacteria, including the human pathogen Mycobacterium tuberculosis (Mtb), the enzymes mediating several central RNA processing functions are still unknown. Here, we identify the hypothetical Mtb protein Rv2179c as a highly divergent exoribonuclease. Although the primary sequence of Rv2179c has no detectable similarity to any known RNase, the Rv2179c crystal structure reveals an RNase fold. Active site residues are equivalent to those in the DEDD family of RNases, and Rv2179c has close structural homology to Escherichia coli RNase T. Consistent with the DEDD fold, Rv2179c has exoribonuclease activity, cleaving the 3' single-strand overhangs of duplex RNA. Functional orthologs of Rv2179c are prevalent in actinobacteria and found in bacteria as phylogenetically distant as proteobacteria. Thus, Rv2179c is the founding member of a new, large RNase family with hundreds of members across the bacterial kingdom.

Macrophage infectivity potentiators (Mips) are immunophilin proteins and essential virulence factors for a range of pathogenic organisms. We applied a structural biology approach to characterize a Mip from Burkholderia pseudomallei (BpML1), the causative agent of melioidosis. Crystal structure and nuclear magnetic resonance analyses of BpML1 in complex with known macrocyclics and other derivatives led to the identification of a key chemical scaffold. This scaffold possesses inhibitory potency for BpML1 without the immunosuppressive components of related macrocyclic agents. Biophysical characterization of a compound series with this scaffold allowed binding site specificity in solution and potency determinations for rank ordering the set. The best compounds in this series possessed a low-micromolar affinity for BpML1, bound at the site of enzymatic activity, and inhibited a panel of homologous Mip proteins from other pathogenic bacteria, without demonstrating toxicity in human macrophages. Importantly, the in vitro activity of BpML1 was reduced by these compounds, leading to decreased macrophage infectivity and intracellular growth of Burkholderia pseudomallei. These compounds offer the potential for activity against a new class of antimicrobial targets and present the utility of a structure-based approach for novel antimicrobial drug discovery.

Published biological data suggest that the methyl erythritol phosphate (MEP) pathway, a non-mevalonate isoprenoid biosynthetic pathway, is essential for certain bacteria and other infectious disease organisms. One highly conserved enzyme in the MEP pathway is 2C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (IspF). Fragment-bound complexes of IspF from Burkholderia pseudomallei were used to design and synthesize a series of molecules linking the cytidine moiety to different zinc pocket fragment binders. Testing by surface plasmon resonance (SPR) found one molecule in the series to possess binding affinity equal to that of cytidine diphosphate, despite lacking any metal-coordinating phosphate groups. Close inspection of the SPR data suggest different binding stoichiometries between IspF and test compounds. Crystallographic analysis shows important variations between the binding mode of one synthesized compound and the pose of the bound fragment from which it was designed. The binding modes of these molecules add to our structural knowledge base for IspF and suggest future refinements in this compound series., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The genus Mycobacterium comprises major human pathogens such as the causative agent of tuberculosis, Mycobacterium tuberculosis (Mtb), and many environmental species. Tuberculosis claims ~1.5 million lives every year, and drug resistant strains of Mtb are rapidly emerging. To aid the development of new tuberculosis drugs, major efforts are currently under way to determine crystal structures of Mtb drug targets and proteins involved in pathogenicity. However, a major obstacle to obtaining crystal structures is the generation of well-diffracting crystals. Proteins from thermophiles can have better crystallization and diffraction properties than proteins from mesophiles, but their sequences and structures are often divergent. Here, we establish a thermophilic mycobacterial model organism, Mycobacterium thermoresistibile (Mth), for the study of Mtb proteins. Mth tolerates higher temperatures than Mtb or other environmental mycobacteria such as M. smegmatis. Mth proteins are on average more soluble than Mtb proteins, and comparison of the crystal structures of two pairs of orthologous proteins reveals nearly identical folds, indicating that Mth structures provide good surrogates for Mtb structures. This study introduces a thermophile as a source of protein for the study of a closely related human pathogen and marks a new approach to solving challenging mycobacterial protein structures., (Copyright © 2012 The Protein Society.)

Approximately one-third of mankind has been exposed to Mycobacterium tuberculosis, the etiological agent responsible for tuberculosis (TB). As part of an effort to develop a new generation of anti-TB agents, the chemical shifts for the 261-residue, virulence-associated protein Rv0577 from M. tuberculosis has been extensively assigned.

Brucella melitensis is the etiological agent responsible for brucellosis. Present in the B. melitensis genome is a 116-residue protein related to arsenate reductases (Bm-YffB; BR0369). Arsenate reductases (ArsC) convert arsenate ion (H(2)AsO(4)(-)), a compound that is toxic to bacteria, to arsenite ion (AsO(2)(-)), a product that may be efficiently exported out of the cell. Consequently, Bm-YffB is a potential drug target because if arsenate reduction is the protein's major biological function then disabling the cell's ability to reduce arsenate would make these cells more sensitive to the deleterious effects of arsenate. Size-exclusion chromatography and NMR spectroscopy indicate that Bm-YffB is a monomer in solution. The solution structure of Bm-YffB (PDB entry 2kok) shows that the protein consists of two domains: a four-stranded mixed β-sheet flanked by two α-helices on one side and an α-helical bundle. The α/β domain is characteristic of the fold of thioredoxin-like proteins and the overall structure is generally similar to those of known arsenate reductases despite the marginal sequence similarity. Chemical shift perturbation studies with (15)N-labeled Bm-YffB show that the protein binds reduced glutathione at a site adjacent to a region similar to the HX(3)CX(3)R catalytic sequence motif that is important for arsenic detoxification activity in the classical arsenate-reductase family of proteins. The latter observation supports the hypothesis that the ArsC-YffB family of proteins may function as glutathione-dependent thiol reductases. However, comparison of the structure of Bm-YffB with the structures of proteins from the classical ArsC family suggest that the mechanism and possibly the function of Bm-YffB and other related proteins (ArsC-YffB) may differ from those of the ArsC family of proteins.

Glutaredoxin proteins (GLXRs) are essential components of the glutathione system that reductively detoxify substances such as arsenic and peroxides and are important in the synthesis of DNA via ribonucleotide reductases. NMR solution structures of glutaredoxin domains from two Gram-negative opportunistic pathogens, Brucella melitensis and Bartonella henselae, are presented. These domains lack the N-terminal helix that is frequently present in eukaryotic GLXRs. The conserved active-site cysteines adopt canonical proline/tyrosine-stabilized geometries. A difference in the angle of α-helix 2 relative to the β-sheet surface and the presence of an extended loop in the human sequence suggests potential regulatory regions and/or protein-protein interaction motifs. This observation is consistent with mutations in this region that suppress defects in GLXR-ribonucleotide reductase interactions. These differences between the human and bacterial forms are adjacent to the dithiol active site and may permit species-selective drug design.

The Seattle Structural Genomics Center for Infectious Disease (SSGCID) focuses on the structure elucidation of potential drug targets from class A, B, and C infectious disease organisms. Many SSGCID targets are selected because they have homologs in other organisms that are validated drug targets with known structures. Thus, many SSGCID targets are expected to be solved by molecular replacement (MR), and reflective of this, all proteins are expressed in native form. However, many community request targets do not have homologs with known structures and not all internally selected targets readily solve by MR, necessitating experimental phase determination. We have adopted the use of iodide ion soaks and single wavelength anomalous dispersion (SAD) experiments as our primary method for de novo phasing. This method uses existing native crystals and in house data collection, resulting in rapid, low cost structure determination. Iodide ions are non-toxic and soluble at molar concentrations, facilitating binding at numerous hydrophobic or positively charged sites. We have used this technique across a wide range of crystallization conditions with successful structure determination in 16 of 17 cases within the first year of use (94% success rate). Here we present a general overview of this method as well as several examples including SAD phasing of proteins with novel folds and the combined use of SAD and MR for targets with weak MR solutions. These cases highlight the straightforward and powerful method of iodide ion SAD phasing in a high-throughput structural genomics environment.

As part of the Seattle Structural Genomics Center for Infectious Disease, we seek to enhance structural genomics with ligand-bound structure data which can serve as a blueprint for structure-based drug design. We have adapted fragment-based screening methods to our structural genomics pipeline to generate multiple ligand-bound structures of high priority drug targets from pathogenic organisms. In this study, we report fragment screening methods and structure determination results for 2C-methyl-D-erythritol-2,4-cyclo-diphosphate (MECP) synthase from Burkholderia pseudomallei, the gram-negative bacterium which causes melioidosis. Screening by nuclear magnetic resonance spectroscopy as well as crystal soaking followed by X-ray diffraction led to the identification of several small molecules which bind this enzyme in a critical metabolic pathway. A series of complex structures obtained with screening hits reveal distinct binding pockets and a range of small molecules which form complexes with the target. Additional soaks with these compounds further demonstrate a subset of fragments to only bind the protein when present in specific combinations. This ensemble of fragment-bound complexes illuminates several characteristics of MECP synthase, including a previously unknown binding surface external to the catalytic active site. These ligand-bound structures now serve to guide medicinal chemists and structural biologists in rational design of novel inhibitors for this enzyme.

The first structure for a member of the DUF3349 (PF11829) family of proteins, Rv0543c from Mycobacterium tuberculosis, has been determined using NMR-based methods and some of its biophysical properties characterized. Rv0543c is a 100 residue, 11.3 kDa protein that both size exclusion chromatography and NMR spectroscopy show to be a monomer in solution. The structure of the protein consists of a bundle of five α-helices, α1 (M1-Y16), α2 (P21-C33), α3 (S37-G52), α4 (G58-H65) and α5 (S72-G87), held together by a largely conserved group of hydrophobic amino acid side chains. Heteronuclear steady-state {¹H}-¹⁵N NOE, T₁, and T₂ values are similar through-out the sequence indicating that the backbones of the five helices are in a single motional regime. The thermal stability of Rv0543c, characterized by circular dichroism spectroscopy, indicates that Rv0543c irreversibly unfolds upon heating with an estimated melting temperature of 62.5 °C. While the biological function of Rv0543c is still unknown, the presence of DUF3349 proteins predominantly in Mycobacterium and Rhodococcus bacterial species suggests that Rv0543 may have a biological function unique to these bacteria, and consequently, may prove to be an attractive drug target to combat tuberculosis., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The Mycobacterium tuberculosis protein Rv2377c (71 residues, MW=8.4kDa) has been characterized using nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. Rv2377c was the first identified member of the MbtH-like family of proteins. MbtH-like proteins have been implicated in siderophore biosynthesis, however, their precise biochemical function remain unknown. Size exclusion chromatography and NMR spectroscopy show that Rv2377c is a monomer in solution. Circular dichroism spectroscopy indicates that Rv2377c unfolds upon heating and will reversibly fold into its native conformation upon cooling. Using NMR-based methods the solution structure of Rv2377c was determined and some of the dynamic properties of the protein studied. The protein contains a three-strand, anti-parallel beta-sheet (beta3:beta1:beta2) nestled against one C-terminal alpha-helix (S44-N55). Weak or absent amide cross peaks in the (1)H-(15)N HSQC spectrum for many of the beta1 and beta2 residues suggest intermediate motion on the ms to mus time scale at the beta1:beta2 interface. Amide cross peaks in the (1)H-(15)N HSQC spectrum are absent for all but one residue at the C-terminus (W56-D71), a region that includes a highly conserved sequence WXDXR, suggesting this region is intrinsically disordered. The latter observation differs with the crystal structure of another MbtH-like protein, PA2412 from Pseudomonas aeruginosa, where a second ordered alpha-helix was observed at the extreme C-terminus.

Burkholderia pseudomallei is a NIAID Category B microorganism responsible for melioidosis. Here we report backbone and side chain NMR assignments for the 139-residue, homodimeric, organic hydroperoxide resistance protein (Ohr) from this organism.

In the following work, we test a generalized approach to integrating, transforming and learning data from disparate data sources for the classification of bacterial proteins involved in pathogenesis. We rely on the implicit inter-linkages between biological databases to draw relevant records, and leverage statistical learning methods to infer classification based on abundant, albeit noisy, data. Results suggest that types of public biological information have varying degrees of effectiveness in predictive data mining.

Immunization with an Anaplasma marginale surface protein complex containing two polypeptides (Am105U and Am105L), each having a molecular weight of 105,000, protected cattle against challenge with virulent organisms. These polypeptides were immunoprecipitated together from detergent extracts of A. marginale by a neutralizing monoclonal antibody. After surface radioiodination of intact parasites, both Am105U and Am105L contained the radiolabel. To define the structural and antigenic relationships between Am105U and Am105L and to determine individual efficacies as protective immunogens, we cloned and expressed A. marginale DNA in Escherichia coli. We identified recombinant bacteria which expressed a novel protein of 105,000 molecular weight as a major cellular component. The recombinant protein was structurally and antigenically homologous to Am105L. There were multiple, partially homologous copies of the cloned DNA sequence in the rickettsial genome.