Carbon-nitrogen hydrolases (CNHs) are members of the diverse nitrilase superfamily of enzymes that facilitate cellular adaptation to environmental stress by metabolizing nitrogen, detoxifying xenobiotics and catabolizing environmentally derived metabolites. Helicobacter pylori CNH (HpCNH) may contribute to metabolic flexibility under acid stress, detoxification of reactive nitrogen species or nutrient scavenging in the nutrient-limited gastric environment. Here, we report the 2.1 Å resolution crystal structure of a CNH from H. pylori strain G27 (PDB entry 6mg6). HpCNH adopts the characteristic nitrilase-superfamily αββα-sandwich core and contains the conserved catalytic cysteine typical of enzymatically active CNHs. The overall structure and active site of HpCNH are most similar to those of carbamoylputrescine amidohydrolase from the plant Medicago truncatula. Despite structural variations in loop regions, including near the active site, HpCNH retains the key residues required to bind putrescine and the prototypical N-carbamoylputrescine amidase active site.

Trypanosoma brucei, the causative agent of Human African Trypanosomiasis (HAT), relies exclusively on purine salvage for nucleotide biosynthesis, making its nucleotide-processing enzymes attractive drug targets. Here, we present a comprehensive structural and functional characterization of T. brucei's nucleoside diphosphate kinase B (TbNDPK), a key enzyme in nucleotide homeostasis. Circular dichroism and fluorescence spectroscopy revealed that TbNDPK is highly stable under thermal and chemical stress and undergoes nucleotide-induced conformational changes. This study also presents high-resolution crystal structures of the apo enzyme and complexes with UDP, CDP, and GDP, showing a conserved hexameric fold, with induced-fit binding via a flexible loop involving Phe59 and key active-site residues. Enzymatic assays revealed substrate preferences for UDP and GDP, while deoxyribonucleotide diphosphates were processed with significantly reduced efficiency. Molecular dynamics simulations revealed ligand-dependent flexibility and subunit-specific nucleotide dynamics, indicating potential asymmetry and cooperative communication within the hexamer. Collectively, these findings position TbNDPK as a thermostable, catalytically efficient, and structurally distinct enzyme optimized for ribonucleotide metabolism and support its potential as a selective target for future antitrypanosomal drug discovery.

Plasmodium vivax is a major cause of malaria globally and has recently been transmitted locally in the USA. P. vivax produces homologs of host proteins, including cytokines such as macrophage migration inhibitory factor (MIF). MIF regulates both adaptive and innate immune responses and contributes to the pathogenesis of parasitic infections, including malaria. Plasma concentrations of P. vivax MIF (PvMIF) correlate with the severity of P. vivax malaria. Plasmodium spp. MIFs have been recognized as candidate malaria vaccines. PvMIF, like other protozoan MIFs, binds to host CD74 and can suppress host MIF-CD74 signaling. The production, crystallization and 1.8 Å resolution structure of PvMIF (PDB entry 9b0m, pdb_00009b0m) are reported. PvMIF crystallized in space group P63 with a single molecule in the asymmetric unit. The biological unit of PvMIF is the prototypical MIF trimer.

Trichomonas vaginalis causes trichomoniasis, the most common non-viral sexually transmitted disease in humans. T. vaginalis pyrophosphate-dependent phosphofructokinase ( Tv PPi-PFK) is a putative target for rational, structure-based drug discovery, given its absence in mammals and its importance for parasite survival. Tv PPi-PFK is a cytosolic enzyme that catalyzes the phosphorylation of fructose-6-phosphate using pyrophosphate (PPi) as the phosphoryl donor. This reversible reaction, catalyzed by Tv PPi-PFK, is the first committed step in glycolysis. Its reverse reaction is vital for gluconeogenesis in T. vaginalis . The purification, crystallization, structure determination, and crystal structures of Tv PPi-PFK are reported. Tv PPi-PFK is the first reported eukaryotic PPi-PFK structure. Tv PPi-PFK retains the overall PPi-PFK topology observed in bacterial PPi-PFK including conserved motifs essential for pyrophosphate binding and PPi-PFK catalytic activity. In addition to the catalytic PPi-PFK binding sites, Tv PPi-PFK has two additional ligand binding sites. The first binds AMP usurped during protein production and helps stabilize the Tv PPi-PFK tetramer. A second ligand binding site was observed in proximity to the AMP-binding site and accommodates sugar phosphates soaked into preformed crystals. This sugar phosphates binding site is distinct from the Tv PPi-PFK active site that binds fructose-6-phosphate. Future mutagenesis and activity studies are planned to determine the relevance of both sites. Synopsis:The production, crystallization, and crystal structures of a pyrophosphate-dependent phosphofructokinase from Trichomonas vaginalis ( Tv PPi-PFK) are reported. Tv PPi-PFK has a prototypical PPi-PFK active site as well as unexpected AMP and sugar-phosphate binding sites at the dimer interface.

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.

Entamoeba histolytica causes amebiasis, a neglected disease that kills ∼100 000 people globally each year. Due to emerging drug resistance, E. histolytica is one of the target organisms for structure-based drug discovery by the Seattle Structural Genomics Center for Infectious Disease (SSGCID). Purification, crystallization and three structures of the putative drug target endoribonuclease L-PSP from E. histolytica (EhL-PSP) are presented. EhL-PSP has a two-layer α/β-sandwich with structural homology to endoribonuclease L-PSP. All three structures reveal the prototypical YjgF/YER057c/UK114 family trimer topology with accessible allosteric active sites. Citrate molecules from the crystallization solution are bound to the allosteric site in two of the three reported structures. The large allosteric site of EhL-PSP is well conserved with bacterial YjgF/YER057c/UK114 family members and could be targeted for inhibition, drug discovery or repurposing.

The focused issue on Empowering education through structural genomics is introduced. The virtual issue is available at https://journals.iucr.org/special_issues/2024/educationsg.

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 a ternary complex structure with human NMT has previously been reported. IMP-1088 binds similarly to PvNMT as to human NMT.

Proteins belonging to the CAP superfamily are present in all kingdoms of life and have been implicated in various processes, including sperm maturation and cancer progression. They are mostly secreted glycoproteins and share a unique conserved CAP domain. The precise mode of action of these proteins, however, has remained elusive. Saccharomyces cerevisiae expresses three members of this protein family, which bind sterols in vitro and promote sterol secretion from cells. This sterol-binding and export function of yeast Pry proteins is conserved in the mammalian cysteine-rich secretory protein (CRISP) proteins and other CAP superfamily members. CRISP3 is an abundant protein of the human seminal plasma and interacts with alpha-1-B glycoprotein (A1BG), a human plasma glycoprotein that is upregulated in different types of cancers. Here, we examined whether the interaction between CRISP proteins and A1BG affects the sterol-binding function of CAP family members. Coexpression of A1BG with CAP proteins abolished their sterol export function in yeast and their interaction inhibits sterol-binding in vitro. We map the interaction between A1BG and CRISP2 to the third of five repeated immunoglobulin-like domains within A1BG. Interestingly, the interaction between A1BG and CRISP2 requires magnesium, suggesting that coordination of Mg2+ by the highly conserved tetrad residues within the CAP domain is essential for a stable interaction between the two proteins. The observation that A1BG modulates the sterol-binding function of CRISP2 has potential implications for the role of A1BG and related immunoglobulin-like domain containing proteins in cancer progression and the toxicity of reptile venoms containing CRISP proteins.

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.

The unicellular parasitic protozoan Trichomonas vaginalis causes trichomoniasis, the most prevalent nonviral sexually transmitted disease globally. T. vaginalis evades host immune responses by producing homologs of host proteins, including cytokines such as macrophage migration inhibitory factor. T. vaginalis macrophage migration inhibitory factor (TvMIF) helps to facilitate the survival of T. vaginalis during nutritional stress conditions, increases prostate cell proliferation and invasiveness, and induces inflammation-related cellular pathways, thus mimicking the ability of human MIF to increase inflammation and cell proliferation. The production, crystallization and three structures of N-terminally hexahistidine-tagged TvMIF reveal a prototypical MIF trimer with a topology similar to that of human homologs (hMIF-1 and hMIF-2). The N-terminal tag obscures the expected pyruvate-binding site. The similarity of TvMIF to its human homologs can be exploited for structure-based drug discovery.

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 a PvNMT-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.

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.

Helicobacter pylori is one of the most common bacterial infections; over two-thirds of the world's population is infected by early childhood. Persistent H. pylori infection results in gastric ulcers and cancers. Due to drug resistance, there is a need to develop alternative treatments to clear H. pylori. The Seattle Structural Genomics Center for Infectious Disease (SSGCID) conducts structure-function analysis of potential therapeutic targets from H. pylori. Glutamyl-tRNA synthetase (GluRS) is essential for tRNA aminoacylation and is under investigation as a bacterial drug target. The SSGCID produced, crystallized and determined the apo structure of H. pylori GluRS (HpGluRS). HpGluRS has the prototypical bacterial GluRS topology and has similar binding sites and tertiary structures to other bacterial GluRS that are promising drug targets. Residues involved in glutamate binding are well conserved in comparison with Pseudomonas aeruginosa GluRS (PaGluRS), which has been studied to develop promising new inhibitors for P. aeruginosa. These structural similarities can be exploited for drug discovery and repurposing to generate new antibacterials to clear persistent H. pylori infection and reduce gastric ulcers and cancer.

The Asojo laboratory investigates the chemistry of life processes, including deciphering the underlying mechanisms and structural basis of immune and suppression evasion by parasites, infectious agents, or host cells in cancer or reproduction as a preamble for the rational design of vaccines and therapeutics. Over the past two decades, we have used protein crystallography research to engage diverse students in chemistry and biochemistry. We will share approaches for incorporating structural biology into the formal curriculum at Hampton University, a predominantly undergraduate institution and historically black college and university (1). We will discuss the successes and failures in fully integrating pre-ba ccalaureate students in crystallography research as authors of peer-reviewed manuscripts (2 - 8). We will also discuss the transformative effect of crystallography on our students and department.

Inhibitors of complement and coagulation are present in the saliva of a variety of blood feeding arthropods that transmit parasitic and viral pathogens. Here we describe the structure and mechanism of action of the sand fly salivary protein lufaxin, which inhibits formation of the central alternative C3 convertase (C3bBb) and inhibits coagulation factor Xa (fXa). Surface plasmon resonance experiments show that lufaxin stabilizes the binding of the serine protease factor B (FB) to C3b but does not detectably bind either C3b or FB alone. The crystal structure of the inhibitor reveals a novel all β-sheet fold containing two domains. A structure of the lufaxin-C3bB complex obtained by cryo-electron microscopy shows that lufaxin binds via its N-terminal domain at an interface containing elements of both C3b and FB. By occupying this spot, the inhibitor locks FB into a closed conformation where proteolytic activation of FB by FD cannot occur. C3bB-bound lufaxin binds fXa at a separate site in its C-terminal domain. In the cryo-EM structure of a C3bB-lufaxin-fXa complex the inhibitor binds both targets simultaneously and lufaxin inhibits fXa through substrate-like binding of a C-terminal peptide at the active site as well as other interactions in this region. Lufaxin inhibits complement activation in ex vivo models of atypical hemolytic uremic syndrome (aHUS) and paroxysmal nocturnal hemoglobinuria (PNH) as well as thrombin generation in plasma, providing rationale for the development of a bispecific inhibitor to treat complement-related diseases in which thrombosis is a prominent manifestation.

Members of the CAP protein superfamily are present in all kingdoms of life and have been implicated in many different processes, including pathogen defense, immune evasion, sperm maturation, and cancer progression. Most CAP proteins are secreted glycoproteins and share a unique conserved alpha beta alpha sandwich fold. The precise mode of action of this class of proteins, however, has remained elusive. Saccharomyces cerevisiae has three CAP family members, termed pathogen related in yeast (Pry). We have previously shown that Pry1 and Pry2 export sterols in vivo and that they bind sterols in vitro. This sterol binding and export function of yeast Pry proteins is conserved in the mammalian CRISP proteins and other CAP superfamily members. CRISP3 is an abundant protein of the human seminal plasma and interacts with prostate secretory protein of 94 amino acids (PSP94), another major protein component in the seminal plasma. Here we examine whether the interaction between CRISP proteins and PSP94 affects the sterol binding function of CAP family members. We show that coexpression of PSP94 with CAP proteins in yeast abolished their sterol export function and the interaction between PSP94 and CAP proteins inhibits sterol binding in vitro. In addition, mutations that affect the formation of the PSP94-CRISP2 heteromeric complex restore sterol binding. Of interest, we found the interaction of PSP94 with CRISP2 is sensitive to high calcium concentrations. The observation that PSP94 modulates the sterol binding function of CRISP2 in a calcium-dependent manner has potential implications for the role of PSP94 and CRISP2 in prostate physiology and progression of prostate cancer.

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.

Burkholderia phymatum is an important symbiotic nitrogen-fixing betaproteobacterium. B. phymatum is beneficial, unlike other Burkholderia species, which cause disease or are potential bioagents. Structural genomics studies at the SSGCID include characterization of the structures of short-chain dehydrogenases/reductases (SDRs) from multiple Burkholderia species. The crystal structure of a short-chain dehydrogenase from B. phymatum (BpSDR) was determined in space group C2221 at a resolution of 1.80 Å. BpSDR shares less than 38% sequence identity with any known structure. The monomer is a prototypical SDR with a well conserved cofactor-binding domain despite its low sequence identity. The substrate-binding cavity is unique and offers insights into possible functions and likely inhibitors of the enzymatic functions of BpSDR.