Seattle Structural Genomics Center for Infectious Disease

Seattle, United States

Seattle Structural Genomics Center for Infectious Disease

Seattle, United States
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Varela-Ramirez A.,University of Texas at El Paso | Abendroth J.,Seattle Structural Genomics Center for Infectious Disease | Abendroth J.,Beryllium | Mejia A.A.,University of Texas at El Paso | And 7 more authors.
Nucleic Acids Research | Year: 2017

Deoxyribonuclease II (DNase II) is also known as acid deoxyribonuclease because it has optimal activity at the low pH environment of lysosomes where it is typically found in higher eukaryotes. Interestingly, DNase II has also been identified in a few genera of bacteria and is believed to have arisen via horizontal transfer. Here, we demonstrate that recombinant Burkholderia thailandensis DNase II is highly active at low pH in the absence of divalent metal ions, similar to eukaryotic DNase II. The crystal structure of B. thailandensis DNase II shows a dimeric quaternary structure which appears capable of binding doublestranded DNA. Each monomer of B. thailandensis DNase II exhibits a similar overall fold as phospholipase D (PLD), phosphatidylserine synthase (PSS) and tyrosyl-DNA phosphodiesterase (TDP), and conserved catalytic residues imply a similarmechanism. The structural and biochemical data presented here provide insights into the atomic structure and catalytic mechanism of DNase II. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.


Helgren T.R.,Northern Illinois University | Wangtrakuldee P.,Northern Illinois University | Staker B.L.,Seattle Structural Genomics Center for Infectious Disease | Hagen T.J.,Northern Illinois University
Current Topics in Medicinal Chemistry | Year: 2016

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. © 2016 Bentham Science Publishers.


Staker B.L.,Seattle Structural Genomics Center for Infectious Disease | Staker B.L.,Seattle Biomedical Research Institute | Buchko G.W.,Seattle Structural Genomics Center for Infectious Disease | Buchko G.W.,Pacific Northwest National Laboratory | And 3 more authors.
Current Opinion in Microbiology | Year: 2015

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. © 2015.


PubMed | University of Washington, Australian National University, Seattle Structural Genomics Center for Infectious Disease, Beryllium and 2 more.
Type: Journal Article | Journal: ACS infectious diseases | Year: 2016

Plasmodium falciparum (Pf) prolyl-tRNA synthetase (ProRS) is one of the few chemical-genetically validated drug targets for malaria, yet highly selective inhibitors have not been described. In this paper, approximately 40,000 compounds were screened to identify compounds that selectively inhibit PfProRS enzyme activity versus Homo sapiens (Hs) ProRS. X-ray crystallography structures were solved for apo, as well as substrate- and inhibitor-bound forms of PfProRS. We identified two new inhibitors of PfProRS that bind outside the active site. These two allosteric inhibitors showed >100 times specificity for PfProRS compared to HsProRS, demonstrating this class of compounds could overcome the toxicity related to HsProRS inhibition by halofuginone and its analogues. Initial medicinal chemistry was performed on one of the two compounds, guided by the cocrystallography of the compound with PfProRS, and the results can instruct future medicinal chemistry work to optimize these promising new leads for drug development against malaria.


PubMed | University of Basel, Seattle Structural Genomics Center for Infectious Disease and Beryllium
Type: Journal Article | Journal: Structure (London, England : 1993) | Year: 2016

The BID (Bep intracellular delivery) domain functions as secretion signal in a subfamily of protein substrates of bacterial type IV secretion (T4S) systems. It mediates transfer of (1) relaxases and the attached DNA during bacterial conjugation, and (2) numerous Bartonella effector proteins (Beps) during protein transfer into host cells infected by pathogenic Bartonella species. Furthermore, BID domains of Beps have often evolved secondary effector functions within host cells. Here, we provide crystal structures for three representative BID domains and describe a novel conserved fold characterized by a compact, antiparallel four-helix bundle topped with a hook. The conserved hydrophobic core provides a rigid scaffold to a surface that, despite a few conserved exposed residues and similarities in charge distribution, displays significant variability. We propose that the genuine function of BID domains as T4S signal may primarily depend on their rigid structure, while the plasticity of their surface may facilitate adaptation to secondary effector functions.


PubMed | Rochester Institute of Technology, Seattle Structural Genomics Center for Infectious Disease, University of Canterbury and Beryllium
Type: Journal Article | Journal: Acta crystallographica. Section F, Structural biology communications | Year: 2016

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, 200mM sodium acetate, 100mM sodium citrate tribasic pH 5.5 and were shown to diffract to 2.3 resolution. They belonged to space group P4


PubMed | Northwestern University, Boston College, Seattle Structural Genomics Center for Infectious Disease and Stanford University
Type: | Journal: Molecular microbiology | Year: 2016

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 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. This article is protected by copyright. All rights reserved.


PubMed | Seattle Structural Genomics Center for Infectious Disease and U.S. National Institutes of Health
Type: | Journal: Scientific reports | Year: 2017

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.


PubMed | Rochester Institute of Technology, Seattle Structural Genomics Center for Infectious Disease, Lawrence Berkeley National Laboratory and University of Canterbury
Type: Journal Article | Journal: Acta crystallographica. Section F, Structural biology communications | Year: 2016

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, 100mM sodium citrate tribasic pH 5.5 and were shown to diffract to 2.10 resolution. They belonged to space group P212121, with unit-cell parameters a = 79.96, b = 106.33, c = 136.25. The final R values were Rr.i.m. = 0.098, Rwork = 0.183, Rfree = 0.233.

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