Structural genomics of infectious disease drug targets: the SSGCID

An introduction and overview of the focus, goals and overall mission of the Seattle Structural Genomics Center for Infectious Disease (SSGCID) is given.

Gene Composer in a structural genomics environment

For structural biology applications, protein-construct engineering is guided by comparative sequence analysis and structural information, which allow the researcher to better define domain boundaries for terminal deletions and nonconserved regions for surface mutants. A database software application called Gene Composer has been developed to facilitate construct design.

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

An overview of one salvage strategy for high-value SSGCID targets is given.

Immobilized metal-affinity chromatography protein-recovery screening is predictive of crystallographic structure success

An overview of the methods used for high-throughput cloning and protein-expression screening of SSGCID hexahistidine recombinant proteins is provided. It is demonstrated that screening for recombinant proteins that are highly recoverable from immobilized metal-affinity chromatography improves the likelihood that a protein will produce a structure.

Expression of proteins in Escherichia coli as fusions with maltose-binding protein to rescue non-expressed targets in a high-throughput protein-expression and purification pipeline

The rescue of protein-expression levels by cloning genes into MBP-fusion vector is described.

High-throughput protein production and purification at the Seattle Structural Genomics Center for Infectious Disease

An overview of the standard SSGCID protein-purification protocol is given and success rates and cleavage alternatives are discussed.

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

The Protein Maker instrument addresses a critical bottleneck in structural genomics by allowing automated purification and buffer testing of multiple protein targets in parallel with a single instrument. Here, the use of this instrument to (i) purify multiple influenza-virus proteins in parallel for crystallization trials and (ii) identify optimal lysis-buffer conditions prior to large-scale protein purification is described.

Salvage and storage of infectious disease protein targets in the SSGCID high-throughput crystallization pathway using microfluidics

SSGCID protein crystals were salvaged and stored using the MPCS Plug Maker and CrystalCards when high-throughput traditional sitting-drop vapor diffusion initially failed.

Wheat germ cell-free expression system as a pathway to improve protein yield and solubility for the SSGCID pipeline

A set of 44 protein targets was used to test expression in the wheat germ cell-free system, the vast majority of which were expressed and soluble in this system; further increases in solubility were achieved by addition of the NVoy polymer.

Structures of phosphopantetheine adenylyltransferase from Burkholderia pseudomallei

Phosphopantetheine adenylyltransferase (PPAT) reversibly converts ATP and 4′-phosphopantetheine into dephospho-coenzyme A and pyrophosphate. Crystal structures are presented of PPAT from B. pseudomallei, the pathogenic bacterium that causes melioidosis.

Structures of a putative ζ-class gluta­thione S-transferase from the pathogenic fungus Coccidioides immitis

The pathogenic fungus C. immitis causes coccidioidomycosis, a potentially fatal disease. Here, apo and glutathione-bound crystal structures of a previously uncharacterized protein from C. immitis that appears to be a ζ-class glutathione S-transferase are presented.

An ensemble of structures of Burkholderia pseudomallei 2,3-bis­phosphoglycerate-dependent phosphoglycerate mutase

An ensemble of crystal structures are reported for 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase from B. pseudomallei. The structures include two vanadate complexes, revealing the structure of a close analogue of the transition state for phosphate transfer.

Structure of fructose bis­phosphate aldolase from Bartonella henselae bound to fructose 1,6-bis­phosphate

While other aldolases crystallize readily in the apo form, diffraction-quality crystals of B. henselae aldolase could only be obtained in the presence of the native substrate. The quaternary structure is tetrameric, as is typical of aldolases.

Structure of fructose bis­phosphate aldolase from Encephalitozoon cuniculi

The eukaryotic parasite E. cuniculi expresses a fructose bisphosphate aldolase that crystallizes readily in the presence of the partial substrate analog phosphate. This aldolase–phosphate structure and that of the sugar-bound Schiff base are reported. E. cuniculi aldolase displays a dimeric structure rather than the expected tetrameric quaternary structure.

Probing conformational states of glutaryl-CoA de­hydrogenase by fragment screening

The first crystal structure is reported of a glutaryl-CoA dehydrogenase in the apo state without flavin adenine dinucleotide cofactor bound. Additional structures with small molecules complexed in the catalytic active site were obtained by fragment-based screening.

Inhibitor-bound complexes of di­hydrofolate reductase-thymidylate synthase from Babesia bovis

Structural characterization of the bifunctional enzyme dihydrofolate reductase-thymidylate synthase from B. bovis in the apo state and complexed with antifolate inhibitors in both enzymatic active sites is reported.

Structure of a Nudix hydrolase (MutT) in the Mg²⁺-bound state from Bartonella henselae, the bacterium responsible for cat scratch fever

B. henselae is the etiological agent responsible for cat scratch fever (bartonellosis). The crystal structure of the smaller of the two Nudix hydrolases encoded in the genome of B. henselae, Bh-MutT, was determined to 2.1 Å resolution.

Structure of a cyclin-dependent kinase from Giardia lamblia

Crystal structures of a cyclin-dependent kinase from G. lamblia are presented in both apo and AMP-bound forms.

Structure of thymidylate kinase from Ehrlichia chaffeensis

A 2.15 Å resolution apo structure of thymidylate kinase from E. chaffeensis is reported.

Structure of triosephosphate isomerase from Cryptosporidium parvum

The crystal structure of the ubiquitous glycolytic enzyme triosephosphate isomerase from C. parvum in the open-loop conformation was determined at a resolution of 1.55 Å.

Structure of nitrilotri­acetate monooxygenase component B from Mycobacterium thermoresistibile

The 1.6 Å resolution crystal structure of nitrilotriacetate monooxygenase component B (NTA-MoB) from M. thermoresistibile is presented, revealing a highly conserved C-terminal tail that may modulate the activity of NTA-MoB in mycobacteria.

BrabA.11339.a: anomalous diffraction and ligand binding guide towards the elucidation of the function of a `putative β-lactamase-like protein' from Brucella melitensis

The structure of a β-lactamase-like protein from B. melitensis was solved independently using two data sets with anomalous signal. Anomalous Fourier maps could confirm the identity of two metal ions in the active site. AMP-bound and GMP-bound structures provide hints to the possible function of the protein.

Structure of aldose reductase from Giardia lamblia

The 1.75 Å resolution crystal structure of aldose reductase from G. lamblia, the etiological agent of giardiasis, is reported.

Structure of 3-keto­acyl-(acyl-carrier-protein) reductase from Rickettsia prowazekii at 2.25 Å resolution

The R. prowazekii 3-ketoacyl-(acyl-carrier-protein) reductase is similar to those from other prokaryotic pathogens but differs significantly from the mammalian orthologue, strengthening its case as a potential drug target.

Structure of fumarate hydratase from Rickettsia prowazekii, the agent of typhus and suspected relative of the mitochondria

Fumarate hydratase is an enzyme of the tricarboxylic acid cycle, one of the metabolic pathways characteristic of the mitochondria. The structure of R. prowazekii class II fumarate hydratase is reported at 2.4 Å resolution and is compared with the available structure of the human homolog.

Solution structure of an arsenate reductase-related protein, YffB, from Brucella melitensis, the etiological agent responsible for brucellosis

B. melitensis is a NIAID Category B microorganism that is responsible for brucellosis and is a potential agent for biological warfare. Here, the solution structure of the 116-residue arsenate reductase-related protein Bm-YffB (BR0369) from this organism is reported.

NMR structure of an acyl-carrier protein from Borrelia burgdorferi

The high-resolution NMR structure of the acyl-carrier protein from the pathogen B. burgdorferi determined to a r.m.s. deviation of 0.4 Å over the protein backbone is reported. The NMR structure was determined using multidimensional NMR spectroscopy and consists of four α-helices and two 3₁₀-helices. Structural comparison reveals that this protein is highly similar to the acyl-carrier protein from A. aeolicus.

Comparative analysis of glutaredoxin domains from bacterial opportunistic pathogens

NMR structures of the glutaredoxin (GLXR) domains from Br. melitensis and Ba. henselae have been determined as part of the SSGCID initiative. Comparison of the domains with known structures reveals overall structural similarity between these proteins and previously determined E. coli GLXR structures, with minor changes associated with the position of helix 1 and with regions that diverge from similar structures found in the closest related human homolog.

Solution-state NMR structure and biophysical characterization of zinc-substituted rubredoxin B (Rv3250c) from Mycobacterium tuberculosis

One third of the world's human population is infected with M. tuberculosis, the etiological agent responsible for tuberculosis (TB). Here, the solution structure of the small iron-binding protein from this organism, rubredoxin B (Rv3250c), is reported in the zinc-substituted form.

Structure of the cysta­thionine γ-synthase MetB from Mycobacterium ulcerans

Cystathionine γ-synthase (CGS) is a transferase that catalyzes the reaction between O⁴-succinyl-L-homoserine and L-cysteine to produce L-­cystathionine and succinate. The crystal structure of CGS from M. ulcerans is presented covalently linked to the cofactor pyridoxal phosphate (PLP). A second structure contains PLP as well as a highly ordered HEPES molecule in the active site acting as a pseudo-ligand. This is the first structure ever reported from the pathogen M. ulcerans.