Publications by authors named "Bart L Staker"

65 Publications

Identification of P218 as a potent inhibitor of DHFR.

RSC Med Chem 2021 Jan 22;12(1):103-109. Epub 2020 Oct 22.

Center for Infectious Disease Research, Seattle Children's Research Institute Seattle Washington 98109 USA

is the causative agent of Buruli ulcer, a debilitating chronic disease that mainly affects the skin. Current treatments for Buruli ulcer are efficacious, but rely on the use of antibiotics with severe side effects. The enzyme dihydrofolate reductase (DHFR) plays a critical role in the biosynthesis of folate species and is a validated target for several antimicrobials. Here we describe the biochemical and structural characterization of DHFR and identified P218, a safe antifolate compound in clinical evaluation for malaria, as a potent inhibitor of this enzyme. We expect our results to advance DHFR as a target for future structure-based drug discovery campaigns.
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http://dx.doi.org/10.1039/d0md00303dDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8130613PMC
January 2021

Crystal structure of acetoacetyl-CoA reductase from Rickettsia felis.

Acta Crystallogr F Struct Biol Commun 2021 Feb 16;77(Pt 2):54-60. Epub 2021 Feb 16.

Department of Chemistry, Vassar College, 124 Raymond Avenue, Poughkeepsie, New York, USA.

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.
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http://dx.doi.org/10.1107/S2053230X21001497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7900926PMC
February 2021

Structural analysis of CACHE domain of the McpA chemoreceptor from Leptospira interrogans.

Biochem Biophys Res Commun 2020 12 21;533(4):1323-1329. Epub 2020 Oct 21.

Laboratório de Desenvolvimento de Vacinas, Instituto Butantan, Avenida Vital Brasil, 1500, 05503-900, São Paulo, SP, Brazil.

Leptospira is a genus of spirochete bacteria highly motile that includes pathogenic species responsible to cause leptospirosis disease. Chemotaxis and motility are required for Leptospira infectivity, pathogenesis, and invasion of bacteria into the host. In prokaryotes, the most common chemoreceptors are methyl-accepting chemotaxis proteins that have a role play to detect the chemical signals and move to a favorable environment for its survival. Here, we report the first crystal structure of CACHE domain of the methyl-accepting chemotaxis protein (McpA) of L. interrogans. The structural analysis showed that McpA adopts similar α/β architecture of several other bacteria chemoreceptors. We also found a typical dimerization interface that appears to be functionally crucial for signal transmission and chemotaxis. In addition to McpA structural analyses, we have identified homologous proteins and conservative functional regions using bioinformatics techniques. These results improve our understanding the relationship between chemoreceptor structures and functions of Leptospira species.
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http://dx.doi.org/10.1016/j.bbrc.2020.10.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7744396PMC
December 2020

Identification of Selective Inhibitors of N-Myristoyltransferase by High-Throughput Screening.

J Med Chem 2020 01 8;63(2):591-600. Epub 2020 Jan 8.

Center for Infectious Disease Research , Seattle , Washington 98109 , United States.

New drugs that target species, the causative agents of malaria, are needed. The enzyme -myristoyltransferase (NMT) is an essential protein, which catalyzes the myristoylation of protein substrates, often to mediate membrane targeting. We screened ∼1.8 million small molecules for activity against () NMT. Hits were triaged based on potency and physicochemical properties and further tested against and () NMTs. We assessed the activity of hits against human NMT1 and NMT2 and discarded compounds with low selectivity indices. We identified 23 chemical classes specific for the inhibition of NMTs over human NMTs, including multiple novel scaffolds. Cocrystallization of NMT with one compound revealed peptide binding pocket binding. Other compounds show a range of potential modes of action. Our data provide insight into the activity of a collection of selective inhibitors of NMT and serve as a starting point for subsequent medicinal chemistry efforts.
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http://dx.doi.org/10.1021/acs.jmedchem.9b01343DOI Listing
January 2020

Structure-Guided Identification of Resistance Breaking Antimalarial N‑Myristoyltransferase Inhibitors.

Cell Chem Biol 2019 07 9;26(7):991-1000.e7. Epub 2019 May 9.

Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK. Electronic address:

The attachment of myristate to the N-terminal glycine of certain proteins is largely a co-translational modification catalyzed by N-myristoyltransferase (NMT), and involved in protein membrane-localization. Pathogen NMT is a validated therapeutic target in numerous infectious diseases including malaria. In Plasmodium falciparum, NMT substrates are important in essential processes including parasite gliding motility and host cell invasion. Here, we generated parasites resistant to a particular NMT inhibitor series and show that resistance in an in vitro parasite growth assay is mediated by a single amino acid substitution in the NMT substrate-binding pocket. The basis of resistance was validated and analyzed with a structure-guided approach using crystallography, in combination with enzyme activity, stability, and surface plasmon resonance assays, allowing identification of another inhibitor series unaffected by this substitution. We suggest that resistance studies incorporated early in the drug development process help selection of drug combinations to impede rapid evolution of parasite resistance.
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http://dx.doi.org/10.1016/j.chembiol.2019.03.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6658617PMC
July 2019

Structure and analysis of nucleoside diphosphate kinase from Borrelia burgdorferi prepared in a transition-state complex with ADP and vanadate moieties.

Acta Crystallogr F Struct Biol Commun 2018 06 31;74(Pt 6):373-384. Epub 2018 May 31.

Department of Allergy and Infectious Disease, University of Washington, Seattle, Washington, USA.

Nucleoside diphosphate kinases (NDKs) are implicated in a wide variety of cellular functions owing to their enzymatic conversion of NDP to NTP. NDK from Borrelia burgdorferi (BbNDK) was selected for functional and structural analysis to determine whether its activity is required for infection and to assess its potential for therapeutic inhibition. The Seattle Structural Genomics Center for Infectious Diseases (SSGCID) expressed recombinant BbNDK protein. The protein was crystallized and structures were solved of both the apoenzyme and a liganded form with ADP and vanadate ligands. This provided two structures and allowed the elucidation of changes between the apo and ligand-bound enzymes. Infectivity studies with ndk transposon mutants demonstrated that NDK function was important for establishing a robust infection in mice, and provided a rationale for therapeutic targeting of BbNDK. The protein structure was compared with other NDK structures found in the Protein Data Bank and was found to have similar primary, secondary, tertiary and quaternary structures, with conserved residues acting as the catalytic pocket, primarily using His132 as the phosphohistidine-transfer residue. Vanadate and ADP complexes model the transition state of this phosphoryl-transfer reaction, demonstrating that the pocket closes when bound to ADP, while allowing the addition or removal of a γ-phosphate. This analysis provides a framework for the design of potential therapeutics targeting BbNDK inhibition.
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http://dx.doi.org/10.1107/S2053230X18007392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5987747PMC
June 2018

The identification of inhibitory compounds of Rickettsia prowazekii methionine aminopeptidase for antibacterial applications.

Bioorg Med Chem Lett 2018 05 15;28(8):1376-1380. Epub 2018 Mar 15.

Department of Chemistry and Biochemistry, Northern Illinois University, 1425 W. Lincoln Hwy, DeKalb, IL 60115, USA. Electronic address:

Methionine aminopeptidase (MetAP) is a dinuclear metalloprotease responsible for the cleavage of methionine initiator residues from nascent proteins. MetAP activity is necessary for bacterial proliferation and is therefore a projected novel antibacterial target. A compound library consisting of 294 members containing metal-binding functional groups was screened against Rickettsia prowazekii MetAP to determine potential inhibitory motifs. The compounds were first screened against the target at a concentration of 10 µM and potential hits were determined to be those exhibiting greater than 50% inhibition of enzymatic activity. These hit compounds were then rescreened against the target in 8-point dose-response curves and 11 compounds were found to inhibit enzymatic activity with IC values of less than 10 µM. Finally, compounds (1-5) were docked against RpMetAP with AutoDock to determine potential binding mechanisms and the results were compared with crystal structures deposited within the PDB.
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http://dx.doi.org/10.1016/j.bmcl.2018.03.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5908248PMC
May 2018

Mycobacterium tuberculosis Rv3651 is a triple sensor-domain protein.

Protein Sci 2018 02 5;27(2):568-572. Epub 2017 Dec 5.

Center for Infectious Disease Research (formerly Seattle Biomedical Research Institute), Seattle, Washington.

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.
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http://dx.doi.org/10.1002/pro.3343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5775179PMC
February 2018

Ligand co-crystallization of aminoacyl-tRNA synthetases from infectious disease organisms.

Sci Rep 2017 03 16;7(1):223. Epub 2017 Mar 16.

Seattle Structural Genomics Center for Infectious Disease (SSGCID), Bethesda, MD, USA.

Aminoacyl-tRNA synthetases (aaRSs) charge tRNAs with their cognate amino acid, an essential precursor step to loading of charged tRNAs onto the ribosome and addition of the amino acid to the growing polypeptide chain during protein synthesis. Because of this important biological function, aminoacyl-tRNA synthetases have been the focus of anti-infective drug development efforts and two aaRS inhibitors have been approved as drugs. Several researchers in the scientific community requested aminoacyl-tRNA synthetases to be targeted in the Seattle Structural Genomics Center for Infectious Disease (SSGCID) structure determination pipeline. Here we investigate thirty-one aminoacyl-tRNA synthetases from infectious disease organisms by co-crystallization in the presence of their cognate amino acid, ATP, and/or inhibitors. Crystal structures were determined for a CysRS from Borrelia burgdorferi bound to AMP, GluRS from Borrelia burgdorferi and Burkholderia thailandensis bound to glutamic acid, a TrpRS from the eukaryotic pathogen Encephalitozoon cuniculi bound to tryptophan, a HisRS from Burkholderia thailandensis bound to histidine, and a LysRS from Burkholderia thailandensis bound to lysine. Thus, the presence of ligands may promote aaRS crystallization and structure determination. Comparison with homologous structures shows conformational flexibility that appears to be a recurring theme with this enzyme class.
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http://dx.doi.org/10.1038/s41598-017-00367-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5428304PMC
March 2017

Mycofactocin-associated mycobacterial dehydrogenases with non-exchangeable NAD cofactors.

Sci Rep 2017 01 25;7:41074. Epub 2017 Jan 25.

Seattle Structural Genomics Center for Infectious Disease (SSGCID), Seattle, WA 98109, USA.

During human infection, Mycobacterium tuberculosis (Mtb) survives the normally bacteriocidal phagosome of macrophages. Mtb and related species may be able to combat this harsh acidic environment which contains reactive oxygen species due to the mycobacterial genomes encoding a large number of dehydrogenases. Typically, dehydrogenase cofactor binding sites are open to solvent, which allows NAD/NADH exchange to support multiple turnover. Interestingly, mycobacterial short chain dehydrogenases/reductases (SDRs) within family TIGR03971 contain an insertion at the NAD binding site. Here we present crystal structures of 9 mycobacterial SDRs in which the insertion buries the NAD cofactor except for a small portion of the nicotinamide ring. Line broadening and STD-NMR experiments did not show NAD or NADH exchange on the NMR timescale. STD-NMR demonstrated binding of the potential substrate carveol, the potential product carvone, the inhibitor tricyclazol, and an external redox partner 2,6-dichloroindophenol (DCIP). Therefore, these SDRs appear to contain a non-exchangeable NAD cofactor and may rely on an external redox partner, rather than cofactor exchange, for multiple turnover. Incidentally, these genes always appear in conjunction with the mftA gene, which encodes the short peptide MftA, and with other genes proposed to convert MftA into the external redox partner mycofactocin.
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http://dx.doi.org/10.1038/srep41074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5264612PMC
January 2017

Rickettsia prowazekii methionine aminopeptidase as a promising target for the development of antibacterial agents.

Bioorg Med Chem 2017 02 10;25(3):813-824. Epub 2016 Nov 10.

Department of Chemistry and Biochemistry, Northern Illinois University, 1425 W. Lincoln Hwy, DeKalb, IL 60115, USA. Electronic address:

Methionine aminopeptidase (MetAP) is a class of ubiquitous enzymes essential for the survival of numerous bacterial species. These enzymes are responsible for the cleavage of N-terminal formyl-methionine initiators from nascent proteins to initiate post-translational modifications that are often essential to proper protein function. Thus, inhibition of MetAP activity has been implicated as a novel antibacterial target. We tested this idea in the present study by targeting the MetAP enzyme in the obligate intracellular pathogen Rickettsia prowazekii. We first identified potent RpMetAP inhibitory species by employing an in vitro enzymatic activity assay. The molecular docking program AutoDock was then utilized to compare published crystal structures of inhibited MetAP species to docked poses of RpMetAP. Based on these in silico and in vitro screens, a subset of 17 compounds was tested for inhibition of R. prowazekii growth in a pulmonary vascular endothelial cell (EC) culture infection model system. All compounds were tested over concentration ranges that were determined to be non-toxic to the ECs and 8 of the 17 compounds displayed substantial inhibition of R. prowazekii growth. These data highlight the therapeutic potential for inhibiting RpMetAP as a novel antimicrobial strategy and set the stage for future studies in pre-clinical animal models of infection.
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http://dx.doi.org/10.1016/j.bmc.2016.11.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5319851PMC
February 2017

The crystal structure of dihydrodipicolinate reductase from the human-pathogenic bacterium Bartonella henselae strain Houston-1 at 2.3 Å resolution.

Acta Crystallogr F Struct Biol Commun 2016 12 25;72(Pt 12):885-891. Epub 2016 Nov 25.

Thomas H. Gosnell School of Life Sciences, Rochester Institute of Technology, 85 Lomb Memorial Drive, Rochester, NY 14623-5603, USA.

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 P422, with unit-cell parameters a = 109.38, b = 109.38, c = 176.95 Å. R was 0.11, R was 0.177 and R 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.
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http://dx.doi.org/10.1107/S2053230X16018525DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5137465PMC
December 2016

Membrane skeletal association and post-translational allosteric regulation of Toxoplasma gondii GAPDH1.

Mol Microbiol 2017 02 23;103(4):618-634. Epub 2016 Dec 23.

Department of Biology, Boston College, MA, 02467, USA.

When Toxoplasma gondii egresses from the host cell, glyceraldehyde-3-phosphate dehydrogenase 1 (GAPDH1), which is primary a glycolysis enzyme but actually a quintessential multifunctional protein, translocates to the unique cortical membrane skeleton. Here, we report the 2.25 Å resolution crystal structure of the GAPDH1 holoenzyme in a quaternary complex providing the basis for the molecular dissection of GAPDH1 structure-function relationships Knockdown of GAPDH1 expression and catalytic site disruption validate the essentiality of GAPDH1 in intracellular replication but we confirmed that glycolysis is not strictly essential. We identify, for the first time, S-loop phosphorylation as a novel, critical regulator of enzymatic activity that is consistent with the notion that the S-loop is critical for cofactor binding, allosteric activation and oligomerization. We show that neither enzymatic activity nor phosphorylation state correlate with the ability to translocate to the cortex. However, we demonstrate that association of GAPDH1 with the cortex is mediated by the N-terminus, likely palmitoylation. Overall, glycolysis and cortical translocation are functionally decoupled by post-translational modifications.
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http://dx.doi.org/10.1111/mmi.13577DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5296235PMC
February 2017

Cloning, expression, purification, crystallization and X-ray diffraction analysis of dihydrodipicolinate synthase from the human pathogenic bacterium Bartonella henselae strain Houston-1 at 2.1 Å resolution.

Acta Crystallogr F Struct Biol Commun 2016 Jan 1;72(Pt 1):2-9. Epub 2016 Jan 1.

Thomas H. Gosnell School of Life Sciences, Rochester Institute of Technology, 85 Lomb Memorial Drive, Rochester, NY 14623-5603, USA.

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.
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http://dx.doi.org/10.1107/S2053230X15023213DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4708043PMC
January 2016

Structural Insight into How Bacteria Prevent Interference between Multiple Divergent Type IV Secretion Systems.

mBio 2015 Dec 8;6(6):e01867-15. Epub 2015 Dec 8.

Department of Biosciences, University of Helsinki, Helsinki, Finland Institute of Biomedicine, University of Turku, Turku, Finland

Unlabelled: Prokaryotes use type IV secretion systems (T4SSs) to translocate substrates (e.g., nucleoprotein, DNA, and protein) and/or elaborate surface structures (i.e., pili or adhesins). Bacterial genomes may encode multiple T4SSs, e.g., there are three functionally divergent T4SSs in some Bartonella species (vir, vbh, and trw). In a unique case, most rickettsial species encode a T4SS (rvh) enriched with gene duplication. Within single genomes, the evolutionary and functional implications of cross-system interchangeability of analogous T4SS protein components remains poorly understood. To lend insight into cross-system interchangeability, we analyzed the VirB8 family of T4SS channel proteins. Crystal structures of three VirB8 and two TrwG Bartonella proteins revealed highly conserved C-terminal periplasmic domain folds and dimerization interfaces, despite tremendous sequence divergence. This implies remarkable structural constraints for VirB8 components in the assembly of a functional T4SS. VirB8/TrwG heterodimers, determined via bacterial two-hybrid assays and molecular modeling, indicate that differential expression of trw and vir systems is the likely barrier to VirB8-TrwG interchangeability. We also determined the crystal structure of Rickettsia typhi RvhB8-II and modeled its coexpressed divergent paralog RvhB8-I. Remarkably, while RvhB8-I dimerizes and is structurally similar to other VirB8 proteins, the RvhB8-II dimer interface deviates substantially from other VirB8 structures, potentially preventing RvhB8-I/RvhB8-II heterodimerization. For the rvh T4SS, the evolution of divergent VirB8 paralogs implies a functional diversification that is unknown in other T4SSs. Collectively, our data identify two different constraints (spatiotemporal for Bartonella trw and vir T4SSs and structural for rvh T4SSs) that mediate the functionality of multiple divergent T4SSs within a single bacterium.

Importance: Assembly of multiprotein complexes at the right time and at the right cellular location is a fundamentally important task for any organism. In this respect, bacteria that express multiple analogous type IV secretion systems (T4SSs), each composed of around 12 different components, face an overwhelming complexity. Our work here presents the first structural investigation on factors regulating the maintenance of multiple T4SSs within a single bacterium. The structural data imply that the T4SS-expressing bacteria rely on two strategies to prevent cross-system interchangeability: (i) tight temporal regulation of expression or (ii) rapid diversification of the T4SS components. T4SSs are ideal drug targets provided that no analogous counterparts are known from eukaryotes. Drugs targeting the barriers to cross-system interchangeability (i.e., regulators) could dysregulate the structural and functional independence of discrete systems, potentially creating interference that prevents their efficient coordination throughout bacterial infection.
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http://dx.doi.org/10.1128/mBio.01867-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4676284PMC
December 2015

Recent contributions of structure-based drug design to the development of antibacterial compounds.

Curr Opin Microbiol 2015 Oct;27:133-8

Seattle Structural Genomics Center for Infectious Disease, United States; Center for Infectious Disease Research (formerly Seattle Biomedical Research Institute), 307 Westlake Ave N, Suite 500, Seattle, WA 98109, United States; Department of Global Health, University of Washington, Seattle, WA 98195, United States; Department of Biomedical Informatics and Health Education, University of Washington, Seattle, WA 98195, United States.

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.
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http://dx.doi.org/10.1016/j.mib.2015.09.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4659754PMC
October 2015

Advances in Bacterial Methionine Aminopeptidase Inhibition.

Curr Top Med Chem 2016 ;16(4):397-414

Northern Illinois University, Department of Chemistry and Biochemistry, DeKalb, IL 60115 USA.

Methionine aminopeptidases (MetAPs) are metalloenzymes that cleave the N-terminal methionine from newly synthesized peptides and proteins. These MetAP enzymes are present in bacteria, and knockout experiments have shown that MetAP activity is essential for cell life, suggesting that MetAPs are good antibacterial drug targets. MetAP enzymes are also present in the human host and selectivity is essential. There have been significant structural biology efforts and over 65 protein crystal structures of bacterial MetAPs are deposited into the PDB. This review highlights the available crystallographic data for bacterial MetAPs. Structural comparison of bacterial MetAPs with human MetAPs highlights differences that can lead to selectivity. In addition, this review includes the chemical diversity of molecules that bind and inhibit the bacterial MetAP enzymes. Analysis of the structural biology and chemical space of known bacterial MetAP inhibitors leads to a greater understanding of this antibacterial target and the likely development of potential antibacterial agents.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5009474PMC
http://dx.doi.org/10.2174/1568026615666150813145410DOI Listing
July 2016

Structures of prostaglandin F synthase from the protozoa Leishmania major and Trypanosoma cruzi with NADP.

Acta Crystallogr F Struct Biol Commun 2015 May 21;71(Pt 5):609-14. Epub 2015 Apr 21.

Seattle Structural Genomics Center for Infectious Disease, USA.

The crystal structures of prostaglandin F synthase (PGF) from both Leishmania major and Trypanosoma cruzi with and without their cofactor NADP have been determined to resolutions of 2.6 Å for T. cruzi PGF, 1.25 Å for T. cruzi PGF with NADP, 1.6 Å for L. major PGF and 1.8 Å for L. major PGF with NADP. These structures were determined by molecular replacement to a final R factor of less than 18.6% (Rfree of less than 22.9%). PGF in the infectious protozoa L. major and T. cruzi is a potential therapeutic target.
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http://dx.doi.org/10.1107/S2053230X15006883DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4427172PMC
May 2015

Structure of an ADP-ribosylation factor, ARF1, from Entamoeba histolytica bound to Mg(2+)-GDP.

Acta Crystallogr F Struct Biol Commun 2015 May 21;71(Pt 5):594-9. Epub 2015 Apr 21.

Seattle Structural Genomics Center for Infectious Disease (SSGCID), USA.

Entamoeba histolytica is the etiological agent of amebiasis, a diarrheal disease which causes amoebic liver abscesses and amoebic colitis. Approximately 50 million people are infected worldwide with E. histolytica. With only 10% of infected people developing symptomatic amebiasis, there are still an estimated 100,000 deaths each year. Because of the emergence of resistant strains of the parasite, it is necessary to find a treatment which would be a proper response to this challenge. ADP-ribosylation factor (ARF) is a member of the ARF family of GTP-binding proteins. These proteins are ubiquitous in eukaryotic cells; they generally associate with cell membranes and regulate vesicular traffic and intracellular signalling. The crystal structure of ARF1 from E. histolytica has been determined bound to magnesium and GDP at 1.8 Å resolution. Comparison with other structures of eukaryotic ARF proteins shows a highly conserved structure and supports the interswitch toggle mechanism of communicating the conformational state to partner proteins.
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http://dx.doi.org/10.1107/S2053230X15004677DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4427170PMC
May 2015

Structures of a histidine triad family protein from Entamoeba histolytica bound to sulfate, AMP and GMP.

Acta Crystallogr F Struct Biol Commun 2015 May 21;71(Pt 5):572-6. Epub 2015 Apr 21.

Seattle Structural Genomics Center for Infectious Disease, USA.

Three structures of the histidine triad family protein from Entamoeba histolytica, the causative agent of amoebic dysentery, were solved at high resolution within the Seattle Structural Genomics Center for Infectious Disease (SSGCID). The structures have sulfate (PDB entry 3oj7), AMP (PDB entry 3omf) or GMP (PDB entry 3oxk) bound in the active site, with sulfate occupying the same space as the α-phosphate of the two nucleotides. The C(α) backbones of the three structures are nearly superimposable, with pairwise r.m.s.d.s ranging from 0.06 to 0.13 Å.
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http://dx.doi.org/10.1107/S2053230X1500237XDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4427167PMC
May 2015

Structures of aspartate aminotransferases from Trypanosoma brucei, Leishmania major and Giardia lamblia.

Acta Crystallogr F Struct Biol Commun 2015 May 21;71(Pt 5):566-71. Epub 2015 Apr 21.

Seattle Structural Genomics Center for Infectious Disease, http://www.ssgcid.org, USA.

The structures of three aspartate aminotransferases (AATs) from eukaryotic pathogens were solved within the Seattle Structural Genomics Center for Infectious Disease (SSGCID). Both the open and closed conformations of AAT were observed. Pyridoxal phosphate was bound to the active site via a Schiff base to a conserved lysine. An active-site mutant showed that Trypanosoma brucei AAT still binds pyridoxal phosphate even in the absence of the tethering lysine. The structures highlight the challenges for the structure-based design of inhibitors targeting the active site, while showing options for inhibitor design targeting the N-terminal arm.
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http://dx.doi.org/10.1107/S2053230X15001831DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4427166PMC
May 2015

Structure of uridine diphosphate N-acetylglucosamine pyrophosphorylase from Entamoeba histolytica.

Acta Crystallogr F Struct Biol Commun 2015 May 21;71(Pt 5):560-5. Epub 2015 Apr 21.

Seattle Structural Genomics Center for Infectious Disease, USA.

Uridine diphosphate N-acetylglucosamine pyrophosphorylase (UAP) catalyzes the final step in the synthesis of UDP-GlcNAc, which is involved in cell-wall biogenesis in plants and fungi and in protein glycosylation. Small-molecule inhibitors have been developed against UAP from Trypanosoma brucei that target an allosteric pocket to provide selectivity over the human enzyme. A 1.8 Å resolution crystal structure was determined of UAP from Entamoeba histolytica, an anaerobic parasitic protozoan that causes amoebic dysentery. Although E. histolytica UAP exhibits the same three-domain global architecture as other UAPs, it appears to lack three α-helices at the N-terminus and contains two amino acids in the allosteric pocket that make it appear more like the enzyme from the human host than that from the other parasite T. brucei. Thus, allosteric inhibitors of T. brucei UAP are unlikely to target Entamoeba UAPs.
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http://dx.doi.org/10.1107/S2053230X1500179XDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4427165PMC
May 2015

Structure of a CutA1 divalent-cation tolerance protein from Cryptosporidium parvum, the protozoal parasite responsible for cryptosporidiosis.

Acta Crystallogr F Struct Biol Commun 2015 May 18;71(Pt 5):522-30. Epub 2015 Apr 18.

Seattle Structural Genomics Center for Infectious Disease, USA.

Cryptosporidiosis is an infectious disease caused by protozoan parasites of the Cryptosporidium genus. Infection is associated with mild to severe diarrhea that usually resolves spontaneously in healthy human adults, but may lead to severe complications in young children and in immunocompromised patients. The genome of C. parvum contains a gene, CUTA_CRYPI, that may play a role in regulating the intracellular concentration of copper, which is a toxic element in excess. Here, the crystal structure of this CutA1 protein, Cp-CutA1, is reported at 2.0 Å resolution. As observed for other CutA1 structures, the 117-residue protein is a trimer with a core ferrodoxin-like fold. Circular dichroism spectroscopy shows little, in any, unfolding of Cp-CutA1 up to 353 K. This robustness is corroborated by (1)H-(15)N HSQC spectra at 333 K, which are characteristic of a folded protein, suggesting that NMR spectroscopy may be a useful tool to further probe the function of the CutA1 proteins. While robust, Cp-CutA1 is not as stable as the homologous protein from a hyperthermophile, perhaps owing to a wide β-bulge in β2 that protrudes Pro48 and Ser49 outside the β-sheet.
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http://dx.doi.org/10.1107/S2053230X14028210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4427160PMC
May 2015

Crystal structures of Mycobacterial MeaB and MMAA-like GTPases.

J Struct Funct Genomics 2015 Jun 2;16(2):91-9. Epub 2015 Apr 2.

Beryllium, Seattle Structural Genomics Center for Infectious Disease (SSGCID), Bainbridge Island, WA, 98110, USA,

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.
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http://dx.doi.org/10.1007/s10969-015-9197-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4631608PMC
June 2015

Increasing the structural coverage of tuberculosis drug targets.

Tuberculosis (Edinb) 2015 Mar 19;95(2):142-8. Epub 2014 Dec 19.

Seattle Structural Genomics Center for Infectious Disease, United States; Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352, United States.

High-resolution three-dimensional structures of essential Mycobacterium tuberculosis (Mtb) proteins provide templates for TB drug design, but are available for only a small fraction of the Mtb proteome. Here we evaluate an intra-genus "homolog-rescue" strategy to increase the structural information available for TB drug discovery by using mycobacterial homologs with conserved active sites. Of 179 potential TB drug targets selected for x-ray structure determination, only 16 yielded a crystal structure. By adding 1675 homologs from nine other mycobacterial species to the pipeline, structures representing an additional 52 otherwise intractable targets were solved. To determine whether these homolog structures would be useful surrogates in TB drug design, we compared the active sites of 106 pairs of Mtb and non-TB mycobacterial (NTM) enzyme homologs with experimentally determined structures, using three metrics of active site similarity, including superposition of continuous pharmacophoric property distributions. Pair-wise structural comparisons revealed that 19/22 pairs with >55% overall sequence identity had active site Cα RMSD <1 Å, >85% side chain identity, and ≥80% PSAPF (similarity based on pharmacophoric properties) indicating highly conserved active site shape and chemistry. Applying these results to the 52 NTM structures described above, 41 shared >55% sequence identity with the Mtb target, thus increasing the effective structural coverage of the 179 Mtb targets over three-fold (from 9% to 32%). The utility of these structures in TB drug design can be tested by designing inhibitors using the homolog structure and assaying the cognate Mtb enzyme; a promising test case, Mtb cytidylate kinase, is described. The homolog-rescue strategy evaluated here for TB is also generalizable to drug targets for other diseases.
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http://dx.doi.org/10.1016/j.tube.2014.12.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4361283PMC
March 2015

Structural analysis of H1N1 and H7N9 influenza A virus PA in the absence of PB1.

Sci Rep 2014 Aug 4;4:5944. Epub 2014 Aug 4.

1] Seattle Structural Genomics Center for Infectious Disease (SSGCID) [2] Beryllium, 7869 NE Day Road West, Bainbridge Island, WA 98110, USA.

Influenza A viruses cause the respiratory illness influenza, which can be mild to fatal depending on the strain and host immune response. The flu polymerase acidic (PA), polymerase basic 1 (PB1), and polymerase basic 2 (PB2) proteins comprise the RNA-dependent RNA polymerase complex responsible for viral genome replication. The first crystal structures of the C-terminal domain of PA (PA-CTD) in the absence of PB1-derived peptides show a number of structural changes relative to the previously reported PB1-peptide bound structures. The human A/WSN/1933 (H1N1) and avian A/Anhui1/2013 (H7N9) strain PA-CTD proteins exhibit the same global topology as other strains in the absence of PB1, but differ extensively in the PB1 binding pocket including a widening of the binding groove and the unfolding of a β-turn. Both PA-CTD proteins exhibited a significant increase in thermal stability in the presence of either a PB1-derived peptide or a previously reported inhibitor in differential scanning fluorimetry assays. These structural changes demonstrate plasticity in the PA-PB1 binding interface which may be exploited in the development of novel therapeutics.
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http://dx.doi.org/10.1038/srep05944DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4123200PMC
August 2014

Crystal structure and putative substrate identification for the Entamoeba histolytica low molecular weight tyrosine phosphatase.

Mol Biochem Parasitol 2014 Jan 15;193(1):33-44. Epub 2014 Feb 15.

Division of Infectious Diseases and International Health, University of Virginia Health System, Charlottesville, VA 22908, USA; Department of Medicine, University of Virginia, Charlottesville, VA 22908, USA; Department of Pathology, University of Virginia, Charlottesville, VA 22908, USA. Electronic address:

Entamoeba histolytica is a eukaryotic intestinal parasite of humans, and is endemic in developing countries. We have characterized the E. histolytica putative low molecular weight protein tyrosine phosphatase (LMW-PTP). The structure for this amebic tyrosine phosphatase was solved, showing the ligand-induced conformational changes necessary for binding of substrate. In amebae, it was expressed at low but detectable levels as detected by immunoprecipitation followed by immunoblotting. A mutant LMW-PTP protein in which the catalytic cysteine in the active site was replaced with a serine lacked phosphatase activity, and was used to identify a number of trapped putative substrate proteins via mass spectrometry analysis. Seven of these putative substrate protein genes were cloned with an epitope tag and overexpressed in amebae. Five of these seven putative substrate proteins were demonstrated to interact specifically with the mutant LMW-PTP. This is the first biochemical study of a small tyrosine phosphatase in Entamoeba, and sets the stage for understanding its role in amebic biology and pathogenesis.
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http://dx.doi.org/10.1016/j.molbiopara.2014.01.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4022148PMC
January 2014

A structural biology approach enables the development of antimicrobials targeting bacterial immunophilins.

Antimicrob Agents Chemother 2014 23;58(3):1458-67. Epub 2013 Dec 23.

Emerald Bio, Bainbridge Island, Washington, USA.

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.
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http://dx.doi.org/10.1128/AAC.01875-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3957879PMC
October 2014

Mycobacterium tuberculosis Rv2179c protein establishes a new exoribonuclease family with broad phylogenetic distribution.

J Biol Chem 2014 Jan 4;289(4):2139-47. Epub 2013 Dec 4.

From Emerald Bio, Bainbridge Island, Washington 98110.

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.
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http://dx.doi.org/10.1074/jbc.M113.525683DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3900960PMC
January 2014

Cytidine derivatives as IspF inhibitors of Burkolderia pseudomallei.

Bioorg Med Chem Lett 2013 Dec 8;23(24):6860-3. Epub 2013 Oct 8.

Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL, USA.

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.
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http://dx.doi.org/10.1016/j.bmcl.2013.09.101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3874807PMC
December 2013
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