Seattle Structural Genomics Center For Infectious Disease Research Articles

Crystal structure of N-terminally hexahistidine-tagged Onchocerca volvulus macrophage migration inhibitory factor-1.

Onchocerca volvulus causes blindness, onchocerciasis, skin infections and devastating neurological diseases such as nodding syndrome. New treatments are needed because the currently used drug, ivermectin, is contraindicated in pregnant women and those co-infected with Loa loa. The Seattle Structural Genomics Center for Infectious Disease (SSGCID) produced, crystallized and determined the apo structure of N-terminally hexahistidine-tagged O. volvulus macrophage migration inhibitory factor-1 (His-OvMIF-1). OvMIF-1 is a possible drug target. His-OvMIF-1 has a unique jellyfish-like structure with a prototypical macrophage migration inhibitory factor (MIF) trimer as the `head' and a unique C-terminal `tail'. Deleting the N-terminal tag reveals an OvMIF-1 structure with a larger cavity than that observed in human MIF that can be targeted for drug repurposing and discovery. Removal of the tag will be necessary to determine the actual biological oligomer of OvMIF-1 because size-exclusion chomatographic analysis of His-OvMIF-1 suggests a monomer, while PISA analysis suggests a hexamer stabilized by the unique C-terminal tails.

Ternary structure of Plasmodium vivaxN-myristoyltransferase with myristoyl-CoA and inhibitor IMP-0001173.

Plasmodium vivax is a major cause of malaria, which poses an increased health burden on approximately one third of the world's population due to climate change. Primaquine, the preferred treatment for P. vivax malaria, is contraindicated in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency, a common genetic cause of hemolytic anemia, that affects ∼2.5% of the world's population and ∼8% of the population in areas of the world where P. vivax malaria is endemic. The Seattle Structural Genomics Center for Infectious Disease (SSGCID) conducted a structure-function analysis of P.vivax N-myristoyltransferase (PvNMT) as part of efforts to develop alternative malaria drugs. PvNMT catalyzes the attachment of myristate to the N-terminal glycine of many proteins, and this critical post-translational modification is required for the survival of P. vivax. The first step is the formation of aPvNMT-myristoyl-CoA binary complex that can bind to peptides. Understanding how inhibitors prevent protein binding will facilitate the development of PvNMT as a viable drug target. NMTs are secreted in all life stages of malarial parasites, making them attractive targets, unlike current antimalarials that are only effective during the plasmodial erythrocytic stages. The 2.3 Å resolution crystal structure of the ternary complex of PvNMT with myristoyl-CoA and a novel inhibitor is reported. One asymmetric unit contains two monomers. The structure reveals notable differences between the PvNMT and human enzymes and similarities to other plasmodial NMTs that can be exploited to develop new antimalarials.

Structures of Brucella ovis leucine-, isoleucine-, valine-, threonine- and alanine-binding protein reveal a conformationally flexible peptide-binding cavity.

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.

Crystal structures of glutamyl-tRNA synthetase from Elizabethkingia anopheles and E. meningosepticum.

Elizabethkingia bacteria are globally emerging pathogens that cause opportunistic and nosocomial infections, with up to 40% mortality among the immunocompromised. Elizabethkingia species are in the pipeline of organisms for high-throughput structural analysis at the Seattle Structural Genomics Center for Infectious Disease (SSGCID). These efforts include the structure-function analysis of potential therapeutic targets. Glutamyl-tRNA synthetase (GluRS) is essential for tRNA aminoacylation and is under investigation as a bacterial drug target. The SSGCID produced, crystallized and determined high-resolution structures of GluRS from E. meningosepticum (EmGluRS) and E.anopheles (EaGluRS). EmGluRS was co-crystallized with glutamate, while EaGluRS is an apo structure. EmGluRS shares ∼97% sequence identity with EaGluRS but less than 39% sequence identity with any other structure in the Protein Data Bank. EmGluRS and EaGluRS have the prototypical bacterial GluRS topology. EmGluRS and EaGluRS have similar binding sites and tertiary structures to other bacterial GluRSs that are promising drug targets. These structural similarities can be exploited for drug discovery.

Crystal structures of FolM alternative dihydrofolate reductase 1 from Brucella suis and Brucella canis.

Members of the bacterial genus Brucella cause brucellosis, a zoonotic disease that affects both livestock and wildlife. Brucella are category B infectious agents that can be aerosolized for biological warfare. As part of the structural genomics studies at the Seattle Structural Genomics Center for Infectious Disease (SSGCID), FolM alternative dihydrofolate reductases 1 from Brucella suis and Brucella canis were produced and their structures are reported. The enzymes share ∼95% sequence identity but have less than 33% sequence identity to other homologues with known structure. The structures are prototypical NADPH-dependent short-chain reductases that share their highest tertiary-structural similarity with protozoan pteridine reductases, which are being investigated for rational therapeutic development.

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

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.

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

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.

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

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.

Structural Genomics Support for Infectious Disease Drug Design.

Structural Genomics Support for Infectious Disease Drug Design.

Drug Target Discovery Is Focus for ID Structural Genomics Centers

The Seattle Structural Genomics Center for Infectious Disease (SSGCID) and the Chicago-based Center for Structural Genomics of Infectious Diseases (CSGID) recently received renewal grants, worth $25 million over five years and dedicated to solving three-dimensional (3-D) structures of proteins or other macromolecules from microorganisms of biodefense and emerging infectious disease (ID) importance. Solving such structures is part of a broader effort toward discovery and early-stage drug development. Support for the centers comes from the National Institute of Allergy and Infectious Disease (NIAID), part of the National Institutes of Health.

Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States alone, an average of 41,400 deaths and 1.86 million hospitalizations are caused by influenza virus infection each year 1. Point mutations in the polymerase basic protein 2 subunit (PB2) have been linked to the adaptation of the viral infection in humans 2. Findings from such studies have revealed the biological significance of PB2 as a virulence factor, thus highlighting its potential as an antiviral drug target.The structural genomics program put forth by the National Institute of Allergy and Infectious Disease (NIAID) provides funding to Emerald Bio and three other Pacific Northwest institutions that together make up the Seattle Structural Genomics Center for Infectious Disease (SSGCID). The SSGCID is dedicated to providing the scientific community with three-dimensional protein structures of NIAID category A-C pathogens. Making such structural information available to the scientific community serves to accelerate structure-based drug design.Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for new lead discovery by targeting a pathway or an entire protein family. Emerald Bio has developed a high-throughput, multi-target parallel processing pipeline (MTPP) for gene-to-structure determination to support the consortium. Here we describe the protocols used to determine the structure of the PB2 subunit from four different influenza A strains.

Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States alone, an average of 41,400 deaths and 1.86 million hospitalizations are caused by influenza virus infection each year 1. Point mutations in the polymerase basic protein 2 subunit (PB2) have been linked to the adaptation of the viral infection in humans 2. Findings from such studies have revealed the biological significance of PB2 as a virulence factor, thus highlighting its potential as an antiviral drug target. The structural genomics program put forth by the National Institute of Allergy and Infectious Disease (NIAID) provides funding to Emerald Bio and three other Pacific Northwest institutions that together make up the Seattle Structural Genomics Center for Infectious Disease (SSGCID). The SSGCID is dedicated to providing the scientific community with three-dimensional protein structures of NIAID category A-C pathogens. Making such structural information available to the scientific community serves to accelerate structure-based drug design. Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for new lead discovery by targeting a pathway or an entire protein family. Emerald Bio has developed a high-throughput, multi-target parallel processing pipeline (MTPP) for gene-to-structure determination to support the consortium. Here we describe the protocols used to determine the structure of the PB2 subunit from four different influenza A strains.

Solution structure of small molecule-influenza RNA complex, Seattle Structural Genomics Center for Infectious Disease (SSGCID)

solution structure of small molecule-influenza RNA complex, Seattle Structural Genomics Center for Infectious Disease (SSGCID)

Behind Every Good Metabolite there is a Great Enzyme (and perhaps a structure)

Today, due to great technological advancements, it is possible to study everything at the same time. This ability has given birth to “totality” studies in the fields of genomics, transcriptomics, proteomics, and metabolomics. In turn, the combined study of all these global analyses gave birth to the field of systems biology. Another “totality” field brought to life with new emerging technologies is structural genomics, an effort to determine the three-dimensional structure of every protein encoded in a genome. The Seattle Structural Genomics Center for Infectious Disease (SSGCID) is a specialized structural genomics effort composed of academic (University of Washington), government (Pacific Northwest National Laboratory), not-for-profit (Seattle BioMed), and commercial (Emerald BioStructures) institutions that is funded by the National Institute of Allergy and Infectious Diseases (Federal Contract: HHSN272200700057C and HHSN27220120025C) to apply genome-scale approaches in solving protein structures from biodefense organisms, as well as those causing emerging and re-emerging disease. In five years over 540 structures have been deposited into the Protein Data Bank (PDB) by SSGICD. About one third of all SSGCID structures contain bound ligands, many of which are metabolites or metabolite analogues present in the cell. These proteins structures are the blueprints for the structure-based design of the more » next generation of drugs against bacterial pathogens and other infectious diseases. Many of the selected SSGCID targets are annotated enzymes from known metabolomic pathways essential to cellular vitality since selectively “knocking-out” one of the enzymes in an important pathway with a drug may be fatal to the organism. One reason metabolomic pathways are important is because of the small molecules, or metabolites, produced at various steps in these pathways and identified by metabolomic studies. Unlike genomics, transcriptomics, and proteomics that may be influenced by epigenetic, post-transcriptional, and post-translational modifications, respectively, the metabolites present in the cell at any one time represent downstream biochemical endproducts, and therefore, metabolite profiles may be most closely associated with a phenotype and provide valuable information for infectious disease research. Metabolomic data would be even more useful if it could be linked to the vast amount of structural genomics data. Towards this goal SSGCID has created an automated website (http://apps.sbri.org/SSGCIDTargetStatus/Pathway) that assigns selected SSGCID target proteins to MetaCyc pathways (http://metacyc.org/). Details of this website will be provided here. The SSGCID-Pathway website represents a first big step towards linking metabolites and metabolic pathways to structural genomic data with the goal of accelerating the discovery of new agents to battle infectious diseases. « less

Solution structure of putative oxidoreductase from Ehrlichia chaffeensis. (Seattle Structural Genomics Center for Infectious Disease (SSGCID))

Solution structure of putative oxidoreductase from Ehrlichia chaffeensis. (Seattle Structural Genomics Center for Infectious Disease (SSGCID))

Structural genomics of infectious disease drug targets: the SSGCID.

The Seattle Structural Genomics Center for Infectious Disease (SSGCID) is a consortium of researchers at Seattle BioMed, Emerald BioStructures, the University of Washington and Pacific Northwest National Laboratory that was established to apply structural genomics approaches to drug targets from infectious disease organisms. The SSGCID is currently funded over a five-year period by the National Institute of Allergy and Infectious Diseases (NIAID) to determine the three-dimensional structures of 400 proteins from a variety of Category A, B and C pathogens. Target selection engages the infectious disease research and drug-therapy communities to identify drug targets, essential enzymes, virulence factors and vaccine candidates of biomedical relevance to combat infectious diseases. The protein-expression systems, purified proteins, ligand screens and three-dimensional structures produced by SSGCID constitute a valuable resource for drug-discovery research, all of which is made freely available to the greater scientific community. This issue of Acta Crystallographica Section F, entirely devoted to the work of the SSGCID, covers the details of the high-throughput pipeline and presents a series of structures from a broad array of pathogenic organisms. Here, a background is provided on the structural genomics of infectious disease, the essential components of the SSGCID pipeline are discussed and a survey of progress to date is presented.

Gene design, cloning and protein-expression methods for high-value targets at the Seattle Structural Genomics Center for Infectious Disease

Any structural genomics endeavor, particularly ambitious ones such as the NIAID-funded Seattle Structural Genomics Center for Infectious Disease (SSGCID) and Center for Structural Genomics of Infectious Disease (CSGID), face technical challenges at all points of the production pipeline. One salvage strategy employed by SSGCID is combined gene engineering and structure-guided construct design to overcome challenges at the levels of protein expression and protein crystallization. Multiple constructs of each target are cloned in parallel using Polymerase Incomplete Primer Extension cloning and small-scale expressions of these are rapidly analyzed by capillary electrophoresis. Using the methods reported here, which have proven particularly useful for high-value targets, otherwise intractable targets can be resolved.

NMR structure of an acyl-carrier protein from Borrelia burgdorferi.

Nearly complete resonance assignment and the high-resolution NMR structure of the acyl-carrier protein from Borrelia burgdorferi, a target of the Seattle Structural Genomics Center for Infectious Disease (SSGCID) structure-determination pipeline, are reported. This protein was chosen as a potential target for drug-discovery efforts because of its involvement in fatty-acid biosynthesis, an essential metabolic pathway, in bacteria. It was possible to assign >98% of backbone resonances and >92% of side-chain resonances using multidimensional NMR spectroscopy. The NMR structure was determined to a backbone r.m.s.d. of 0.4 Å and contained four α-helices and two 3(10)-helices. A structure-homology search revealed that this protein is highly similar to the acyl-carrier protein from Aquifex aeolicus.

Gene Composerin a structural genomics environment

The structural genomics effort at the Seattle Structural Genomics Center for Infectious Disease (SSGCID) requires the manipulation of large numbers of amino-acid sequences and the underlying DNA sequences which are to be cloned into expression vectors. To improve efficiency in high-throughput protein structure determination, a database software package, Gene Composer, has been developed which facilitates the information-rich design of protein constructs and their underlying gene sequences. With its modular workflow design and numerous graphical user interfaces, Gene Composer enables researchers to perform all common bioinformatics steps used in modern structure-guided protein engineering and synthetic gene engineering. An example of the structure determination of H1N1 RNA-dependent RNA polymerase PB2 subunit is given.

The Protein Maker: an automated system for high-throughput parallel purification.

The Protein Maker is an automated purification system developed by Emerald BioSystems for high-throughput parallel purification of proteins and antibodies. This instrument allows multiple load, wash and elution buffers to be used in parallel along independent lines for up to 24 individual samples. To demonstrate its utility, its use in the purification of five recombinant PB2 C-terminal domains from various subtypes of the influenza A virus is described. Three of these constructs crystallized and one diffracted X-rays to sufficient resolution for structure determination and deposition in the Protein Data Bank. Methods for screening lysis buffers for a cytochrome P450 from a pathogenic fungus prior to upscaling expression and purification are also described. The Protein Maker has become a valuable asset within the Seattle Structural Genomics Center for Infectious Disease (SSGCID) and hence is a potentially valuable tool for a variety of high-throughput protein-purification applications.