Seattle Structural Genomics Center Research Articles

Structure of Plasmodium vivaxN-myristoyltransferase with inhibitor IMP-1088: exploring an NMT inhibitor for antimalarial therapy.

Plasmodium vivax, a significant contributor to global malaria cases, poses an escalating health burden on a substantial portion of the world's population. The increasing spread of P. vivax because of climate change underscores the development of new and rational drug-discovery approaches. The Seattle Structural Genomics Center for Infectious Diseases is taking a structure-based approach by investigating essential enzymes such as N-myristoyltransferase (NMT). P.vivax N-myristoyltransferase (PvNMT) is a promising target for the development of novel malaria treatments unlike current drugs, which target only the erythrocytic stages of the parasite. Here, the 1.8 Å resolution ternary structure of PvNMT in complex with myristoyl-CoA and IMP-1088, a validated NMT inhibitor, is reported. IMP-1088 is a validated nonpeptidic inhibitor and aternary complex structure with human NMT has previously been reported. IMP-1088 binds similarly to PvNMT as to human NMT.

Co-crystal structure of Helicobacter pylori biotin protein ligase with biotinyl-5-ATP.

Helicobacter pylori, a type 1 carcinogen that causes human gastric ulcers and cancer, is a priority target of the Seattle Structural Genomics Center for Infectious Disease (SSGCID). These efforts include determining the structures of potential H. pylori therapeutic targets. Here, the purification, crystallization and X-ray structure of one such target, H. pylori biotin protein ligase (HpBPL), are reported. HpBPL catalyzes the activation of various biotin-dependent metabolic pathways, including fatty-acid synthesis, gluconeogenesis and amino-acid catabolism, and may facilitate the survival of H. pylori in the high-pH gastric mucosa. HpBPL is a prototypical bacterial biotin protein ligase, despite having less than 35% sequence identity to any reported structure in the Protein Data Bank. A biotinyl-5-ATP molecule sits in a well conserved cavity. HpBPL shares extensive tertiary-structural similarity with Mycobacterium tuberculosis biotin protein ligase (MtBPL), despite having less than 22% sequence identity. The active site of HpBPL is very similar to that of MtBPL and has the necessary residues to bind inhibitors developed for MtBPL.

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.

Kinetic Characterization of a Crystallized Putative Hydroxybutyrate Dehydrogenase from the Opportunistic Pathogen Burkholderia cenocepacia

Burkholderia cenocepaciais an opportunistic pathogen posing risk among individuals with cystic fibrosis or who are immune compromised. The crystal structure of a putative hydroxybutyrate dehydrogenase (HBDH), based on sequence analysis, from Burkholderia cenocepaciahas been solved by the Seattle Structural Genomics Center for Infectious Disease and deposited in the Protein Database (PDB ID 4TRR). We sought to determine if the protein is an HBDH and to determine the kinetic parameters of the enzyme. HBDHs catalyze the reversible conversion of acetoacetate to hydroxybutyrate utilizing the cofactor NAD(H). The SSGCID kindly provided us the construct encoding a His‐tagged BcHBDH. The protein was overexpressed in E. coli, purified to approximate homogeneity using nickel affinity chromatography, and activity assayed spectrophotometrically. The putative BcHBDH is confirmed to have HBDH activity and the kinetic parameters for the enzyme were determined in both the direction of acetoacetate and hydroxybutyrate formation. BcHBDH has a catalytic efficiency of 2.85x104 mM‐1 sec‐1 and 1.21x105mM‐1 sec‐1 in the direction of hydroxybutyrate formation for acetoacetate and NADH, respectively. In the direction of acetoacetate formation, the enzyme has a catalytic efficiency of 4.1x103 mM‐1 sec‐1 and 2.86x104 mM‐1 sec‐1 for hydroxybutyrate and NAD+, respectively. By comparing both the structure and kinetic parameters to known kinetically characterized and crystallized HBDHs, we conclude the protein from Burkholderia cenocepacia is an HBDH.

Crystal structure of an inorganic pyrophosphatase from Chlamydia trachomatis D/UW-3/Cx.

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections globally and is one of the most commonly reported infections in the United States. There is a need to develop new therapeutics due to drug resistance and the failure of current treatments to clear persistent infections. Structures of potential C. trachomatis rational drug-discovery targets, including C. trachomatis inorganic pyrophosphatase (CtPPase), have been determined bythe Seattle Structural Genomics Center for Infectious Disease. Inorganic pyrophosphatase hydrolyzes inorganic pyrophosphate during metabolism. Furthermore, bacterial inorganic pyrophosphatases have shown promise for therapeutic discovery. Here, a 2.2 Å resolution X-ray structure of CtPPase is reported. The crystal structure of CtPPase reveals shared structural features that may facilitate the repurposing of inhibitors identified for bacterial inorganic pyrophosphatases as starting points for new therapeutics for C. trachomatis.

Crystal structure of a hypothetical protein from Giardia lamblia.

Giardiasis is the most prevalent diarrheal disease globally and affects humans and animals. It is a significant problem in developing countries, the number one cause of travelers' diarrhea and affects children and immunocompromised individuals, especially HIV-infected individuals. Giardiasis is treated with antibiotics (tinidazole and metronidazole) that are also used for other infections such as trichomoniasis. The ongoing search for new therapeutics for giardiasis includes characterizing the structure and function of proteins from the causative protozoan Giardia lamblia. These proteins include hypothetical proteins that share 30% sequence identity or less with proteins of known structure. Here, the atomic resolution structure of a 15.6 kDa protein was determined by molecular replacement. The structure has the two-layer αβ-sandwich topology observed in the prototypical endoribonucleases L-PSPs (liver perchloric acid-soluble proteins) with conserved allosteric active sites containing small molecules from the crystallization solution. This article is an educational collaboration between Hampton University and the Seattle Structural Genomics Center for Infectious Disease.

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.

Naegleria fowleri: Protein structures to facilitate drug discovery for the deadly, pathogenic free-living amoeba

Naegleria fowleri is a pathogenic, thermophilic, free-living amoeba which causes primary amebic meningoencephalitis (PAM). Penetrating the olfactory mucosa, the brain-eating amoeba travels along the olfactory nerves, burrowing through the cribriform plate to its destination: the brain’s frontal lobes. The amoeba thrives in warm, freshwater environments, with peak infection rates in the summer months and has a mortality rate of approximately 97%. A major contributor to the pathogen’s high mortality is the lack of sensitivity of N. fowleri to current drug therapies, even in the face of combination-drug therapy. To enable rational drug discovery and design efforts we have pursued protein production and crystallography-based structure determination efforts for likely drug targets from N. fowleri. The genes were selected if they had homology to drug targets listed in Drug Bank or were nominated by primary investigators engaged in N. fowleri research. In 2017, 178 N. fowleri protein targets were queued to the Seattle Structural Genomics Center of Infectious Disease (SSGCID) pipeline, and to date 89 soluble recombinant proteins and 19 unique target structures have been produced. Many of the new protein structures are potential drug targets and contain structural differences compared to their human homologs, which could allow for the development of pathogen-specific inhibitors. Five of the structures were analyzed in more detail, and four of five show promise that selective inhibitors of the active site could be found. The 19 solved crystal structures build a foundation for future work in combating this devastating disease by encouraging further investigation to stimulate drug discovery for this neglected pathogen.

Toward a structome of Acinetobacter baumannii drug targets.

Acinetobacter baumannii is well known for causing hospital-associated infections due in part to its intrinsic antibiotic resistance as well as its ability to remain viable on surfaces and resist cleaning agents. In a previous publication, A. baumannii strain AB5075 was studied by transposon mutagenesis and 438 essential gene candidates for growth on rich-medium were identified. The Seattle Structural Genomics Center for Infectious Disease entered 342 of these candidate essential genes into our pipeline for structure determination, in which 306 were successfully cloned into expression vectors, 192 were detectably expressed, 165 screened as soluble, 121 were purified, 52 crystalized, 30 provided diffraction data, and 29 structures were deposited in the Protein Data Bank. Here, we report these structures, compare them with human orthologs where applicable, and discuss their potential as drug targets for antibiotic development against A. baumannii.

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

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.

Crystal structure of chorismate mutase from Burkholderia thailandensis.

Burkholderia thailandensis is often used as a model for more virulent members of this genus of proteobacteria that are highly antibiotic-resistant and are potential agents of biological warfare that are infective by inhalation. As part of ongoing efforts to identify potential targets for the development of rational therapeutics, the structures of enzymes that are absent in humans, including that of chorismate mutase from B. thailandensis, have been determined by the Seattle Structural Genomics Center for Infectious Disease. The high-resolution structure of chorismate mutase from B. thailandensis was determined in the monoclinic space group P21 with three homodimers per asymmetric unit. The overall structure of each protomer has the prototypical AroQγ topology and shares conserved binding-cavity residues with other chorismate mutases, including those with which it has no appreciable sequence identity.

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.

Macromolecular crystallography and what it can contribute to antiparasite drug discovery.

This issue of Acta Crystallographica Section F is dedicated towards structural investigations on proteins associated with molecular parasitology, specifically research linked to protozoan pathogens. It is in line with the previous special sections or issues contributed by the RIKEN-UK structural genomics consortium (December 2009), the Joint Center for Structural Genomics in October 2010 (Weiss & Einspahr, 2010 ▶) and the Seattle Structural Genomics Center for Infectious Diseases in September 2011 (Weiss & Einspahr, 2011 ▶). In contrast to the previous issues however, it is thematically much more focused on protozoan pathogens. Interest in these primitive eukaryotic organisms is often to exploit them as model systems and many significant advances have resulted from such research. As examples there are the fundamentally important discoveries relating to RNA editing, antigenic variation and the occurrence of glycosylphosphatidylinositol tags that anchor proteins on the cell surface, all accrued from research with the African trypanosome. However, a major impetus to study protists is the drastic toll they take on human life. The World Health Organization provide documentation and sobering statistics on the diseases and their impact on human health. For example http://www.who.int/mediacentre/factsheets/fs375/en/ is their recently updated information sheet on Leishmaniasis. Researchers have sought to understand the processes that allow pathogens to exist, to invade a host, to evade the immune response, and to cause debilitating and often fatal diseases. Extending from such investigations and alongside drug discovery efforts enter structural biology, in particular crystallography and the high quality chemical information it can provide. Remember that most drugs are smallish organic molecules that exert their beneficial effect by interacting with certain macromolecules. The discovery of such molecules has transformed human existence. Indeed the emergence of the modern pharmaceutical industry, founded as it was on the basis of antimicrobial and analgesic agents, might be considered one of the major developments of the last 100 years. Different disciplines contribute to drug discovery, and increasingly the search is on for new targets, better technologies and model systems to assist the process. Structural studies contribute a key role by providing accurate chemical information to guide assessment of targets, the development of reliable structure–activity relationships or templates for computational design projects in the search for lead compounds. There are numerous excellent studies published in which specific parasite systems have been targeted and dissected. In addition structural genomics efforts have involved cohorts of protist proteins and the Protein Data Bank has been greatly enriched in this area with both NMR and crystal structures. We now present an issue of fundamental studies in molecular parasitology and also research directed toward protozoan drug target characterization. To set the scene there is an overview and perspective from Professor Wim Hol. In this he also details a few of his favourite examples where structural information contributes to the search for improved therapies. We commissioned Wim for an overview because of his outstanding contributions and infectious enthusiasm for the area; he continues to be an inspirational champion for the role of crystallography in drug discovery. There is also a short review from Professor Inari Kursula on cytoskeletal proteins related to the infection process. We thought it relevant to address this aspect and would highlight this as one in which there may be opportunities in future drug discovery work. We are pleased to also have a number of communications from structural genomics efforts which sit comfortably alongside more focused projects. Section F’s mandate is to allow the structural biology community to efficiently publish their data and we hope this special issue will serve to inform our community and perhaps others in the life sciences or medicinal chemistry areas, generate interest and provide encouragement to consider the journal as a natural home for results in molecular parasitology. We look forward to seeing more structural information on parasite proteins, more advances in basic biology and results to support future efforts in early stage drug discovery.

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.