Schistosoma mansoni venom allergen-like protein 4 (SmVAL4) is a novel lipid-binding SCP/TAPS protein that lacks the prototypical CAP motifs.

Schistosomiasis is a parasitic disease that affects over 200 million people. Vaccine candidates have been identified, including Schistosoma mansoni venom allergen-like proteins (SmVALs) from the SCP/TAPS (sperm-coating protein/Tpx/antigen 5/pathogenesis related-1/Sc7) superfamily. The first SmVAL structure, SmVAL4, was refined to a resolution limit of 2.16 Å. SmVAL4 has a unique structure that could not be predicted from homologous structures, with longer loops and an unusual C-terminal extension. SmVAL4 has the characteristic α/ß-sandwich and central SCP/TAPS cavity. Furthermore, SmVAL4 has only one of the signature CAP cavity tetrad amino-acid residues and is missing the histidines that coordinate divalent cations such as Zn(2+) in other SCP/TAPS proteins. SmVAL4 has a cavity between α-helices 1 and 4 that was observed to bind lipids in tablysin-15, suggesting the ability to bind lipids. Subsequently, SmVAL4 was shown to bind cholesterol in vitro. Additionally, SmVAL4 was shown to complement the in vivo sterol-export phenotype of yeast mutants lacking their endogenous CAP proteins. Expression of SmVAL4 in yeast cells lacking endogenous CAP function restores the block in sterol export. These studies suggest an evolutionarily conserved lipid-binding function shared by CAP proteins such as SmVAL4 and yeast CAP proteins such as Pry1.

Structural and biochemical analyses of alanine racemase from the multidrug-resistant Clostridium difficile strain 630.

Clostridium difficile, a Gram-positive, spore-forming anaerobic bacterium, is the leading cause of infectious diarrhea among hospitalized patients. C. difficile is frequently associated with antibiotic treatment, and causes diseases ranging from antibiotic-associated diarrhea to life-threatening pseudomembranous colitis. The severity of C. difficile infections is exacerbated by the emergence of hypervirulent and multidrug-resistant strains, which are difficult to treat and are often associated with increased mortality rates. Alanine racemase (Alr) is a pyridoxal-5'-phosphate (PLP)-dependent enzyme that catalyzes the reversible racemization of L- and D-alanine. Since D-alanine is an essential component of the bacterial cell-wall peptidoglycan, and there are no known Alr homologs in humans, this enzyme is being tested as an antibiotic target. Cycloserine is an antibiotic that inhibits Alr. In this study, the catalytic properties and crystal structures of recombinant Alr from the virulent and multidrug-resistant C. difficile strain 630 are presented. Three crystal structures of C. difficile Alr (CdAlr), corresponding to the complex with PLP, the complex with cycloserine and a K271T mutant form of the enzyme with bound PLP, are presented. The structures are prototypical Alr homodimers with two active sites in which the cofactor PLP and cycloserine are localized. Kinetic analyses reveal that the K271T mutant CdAlr has the highest catalytic constants reported to date for any Alr. Additional studies are needed to identify the basis for the high catalytic activity. The structural and activity data presented are first steps towards using CdAlr for the development of structure-based therapeutics for C. difficile infections.

Structure of monomeric Na-GST-3, a glutathione S-transferase from the major human hookworm parasite Necator americanus.

Necator americanus is the major cause of human hookworm infection, which is a global cause of anemia in the developing world. Ongoing efforts to control hookworm infection include the identification of candidate vaccine antigens as well as potential therapeutic targets from the infective L3 larval stages and adult stages of the parasite. One promising family of proteins are the adult-stage-secreted cytosolic glutathione S-transferases (GSTs). Nematode GSTs facilitate the inactivation and degradation of a variety of electrophilic substrates (drugs) via the nucleophilic addition of reduced glutathione. Parasite GSTs also play significant roles in multi-drug resistance and the modulation of host immune defense mechanisms. Here, the structure of Na-GST-3, one of three GSTs secreted by adult-stage N. americanus, is reported. Unlike most GST structures, the Na-GST-3 crystal contains a monomer in the asymmetric unit. However, the monomer forms a prototypical GST dimer across the crystallographic twofold. A glutathione from the fermentation process is bound to the monomer. The overall binding cavity of Na-GST-3 is reminiscent of that of other N. americanus GSTs and is larger and capable of binding a wider array of ligands than GSTs from organisms that have other major detoxifying mechanisms. Furthermore, despite having low sequence identity to the host GST, Na-GST-3 has a greater tertiary-structure similarity to human sigma-class GST than was observed for the other N. americanus GSTs.

Structure of 2-keto-3-deoxy-D-manno-octulosonate-8-phosphate synthase from Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a major cause of opportunistic infection and is resistant to most antibiotics. As part of efforts to generate much-needed new antibiotics, structural studies of enzymes that are critical for the virulence of P. aeruginosa but are absent in mammals have been initiated. 2-Keto-3-deoxy-D-manno-octulosonate-8-phosphate synthase (KDO8Ps), also known as 2-dehydro-3-deoxyphosphooctonate aldolase, is vital for the survival and virulence of P. aeruginosa. This enzyme catalyzes a key step in the synthesis of the lipopolysaccharide (LPS) of most Gram-negative bacteria: the condensation reaction between phosphoenolpyruvate (PEP) and arabinose 5-phosphate to produce 2-keto-3-deoxy-D-manno-octulosonate-8-phosphate (KDO8P). This step is vital for the proper synthesis and assembly of LPS and the survival of P. aeruginosa. Here, the recombinant expression, purification and crystal structure of KDO8Ps from P. aeruginosa are presented. Orthorhombic crystals were obtained by vapor diffusion in sitting drops in the presence of 1 mM phosphoenlpyruvate. The structure reveals the prototypical α/ß TIM-barrel structure expected from this family of enzymes and contains a tetramer in the asymmetric unit.

KLIPP - a precision CRISPR approach to target structural variant junctions in cancer.

Current cancer therapies typically give rise to dose-limiting normal tissue toxicity. We have developed KLIPP, a precision cancer approach that specifically kills cancer cells using CRISPR/Cas9 technology. The approach consists of guide RNAs that target cancer-specific structural variant junctions to nucleate two parts of a dCas9-conjugated endonuclease, Fok1, leading to its activation. We show that KLIPP causes induction of DNA double strand breaks (DSBs) at the targeted junctions and cell death. When cancer cells were grown orthotopically in mice, activation of Fok1 at only two junctions led to the disappearance of tumor cells in 7/11 mice. This therapeutic approach has high specificity for tumor cells and is independent of tumor-specific drivers. Individualized translation of KLIPP to patients would be transformative and lead to consistent and simplified cancer treatment decisions.

TRIM29 promotes bladder cancer invasion by regulating the intermediate filament network and focal adhesion.

Bladder cancer is a common malignancy whose lethality is determined by invasive potential. We have previously shown that TRIM29, also known as ATDC, is transcriptionally regulated by TP63 in basal bladder cancers where it promotes invasive progression and metastasis, but the molecular events which promote invasion and metastasis downstream of TRIM29 remained poorly understood. Here we identify stimulation of bladder cancer migration as the specific role of TRIM29 during invasion. We show that TRIM29 physically interacts with K14 + intermediate filaments which in turn regulates focal adhesion stability. Further, we find that both K14 and the focal adhesion protein, ZYX are required for bladder cancer migration and invasion. Taken together, these results establish a role for TRIM29 in the regulation of cytoskeleton and focal adhesions during invasion and identify a pathway with therapeutic potential.

ATDC mediates a TP63-regulated basal cancer invasive program.

Basal subtype cancers are deadly malignancies but the molecular events driving tumor lethality are not completely understood. Ataxia-telangiectasia group D complementing gene (ATDC, also known as TRIM29), is highly expressed and drives tumor formation and invasion in human bladder cancers but the factor(s) regulating its expression in bladder cancer are unknown. Molecular subtyping of bladder cancer has identified an aggressive basal subtype, which shares molecular features of basal/squamous tumors arising in other organs and is defined by activation of a TP63-driven gene program. Here, we demonstrate that ATDC is linked with expression of TP63 and highly expressed in basal bladder cancers. We find that TP63 binds to transcriptional regulatory regions of ATDC and KRT14 directly, increasing their expression, and that ATDC and KRT14 execute a TP63-driven invasive program. In vivo, ATDC is required for TP63-induced bladder tumor invasion and metastasis. These results link TP63 and the basal gene expression program to ATDC and to aggressive tumor behavior. Defining ATDC as a molecular determinant of aggressive, basal cancers may lead to improved biomarkers and therapeutic approaches.

Crystal Structure of Borrelia turicatae protein, BTA121, a differentially regulated gene in the tick-mammalian transmission cycle of relapsing fever spirochetes.

Tick-borne relapsing fever (RF) borreliosis is a neglected disease that is often misdiagnosed. RF species circulating in the United States include Borrelia turicatae, which is transmitted by argasid ticks. Environmental adaptation by RF Borrelia is poorly understood, however our previous studies indicated differential regulation of B. turicatae genes localized on the 150 kb linear megaplasmid during the tick-mammalian transmission cycle, including bta121. This gene is up-regulated by B. turicatae in the tick versus the mammal, and the encoded protein (BTA121) is predicted to be surface localized. The structure of BTA121 was solved by single-wavelength anomalous dispersion (SAD) using selenomethionine-derivative protein. The topology of BTA121 is unique with four helical domains organized into two helical bundles. Due to the sequence similarity of several genes on the megaplasmid, BTA121 can serve as a model for their tertiary  structures. BTA121 has large interconnected tunnels and cavities that can accommodate ligands, notably long parallel helices, which have a large hydrophobic central pocket. Preliminary in-vitro studies suggest that BTA121 binds lipids, notably palmitate with a similar order of binding affinity as tablysin-15, a known palmitate-binding protein. The reported data will guide mechanistic studies to determine the role of BTA121 in the tick-mammalian transmission cycle of B. turicatae.

Identification, Characterization, and Structure of Tm16 from Trichuris muris.

Trichuriasis is a disease of poverty for which excretory and secretory (ES) products that induce the protective immunity are being investigated as candidate vaccines antigens. In this study, ES products of T. muris and immune sera were produced. The immune sera recognized more than 20 proteins on a 2D-gel of ES products of T. muris adult worms. Tm16 was one of the proteins identified by mass spectrometry. Tm16 shares 57% sequence identity with Ov16, an immunodominant diagnostic antigen from Onchocerca volvulus. Recombinant Tm16 with a carboxyl terminal hexahistidine was produced using Pichia pastoris. Polyclonal antibodies against rTm16 were generated by one-prime and two-boost immunization of three female Balb/c mice with 25 µg of recombinant Tm16 emulsified with ISA720 adjuvant. These polyclonal antibodies confirmed that Tm16 is localized to the ES products and the soluble fraction of the adult worm. Additionally, the high-resolution crystal structure of Tm16 was solved by molecular replacement. Tm16 belongs to the phosphatidylethanolamine-binding-like protein (PEBP1) family and this is the first structure of a PEBP1 from a parasite.

Structure of SALO, a leishmaniasis vaccine candidate from the sand fly Lutzomyia longipalpis.

BACKGROUND: Immunity to the sand fly salivary protein SALO (Salivary Anticomplement of Lutzomyia longipalpis) protected hamsters against Leishmania infantum and L. braziliensis infection and, more recently, a vaccine combination of a genetically modified Leishmania with SALO conferred strong protection against L. donovani infection. Because of the importance of SALO as a potential component of a leishmaniasis vaccine, a plan to produce this recombinant protein for future scale manufacturing as well as knowledge of its structural characteristics are needed to move SALO forward for the clinical path. METHODOLOGY/PRINCIPAL FINDINGS: Recombinant SALO was expressed as a soluble secreted protein using Pichia pastoris, rSALO(P), with yields of 1g/L and >99% purity as assessed by SEC-MALS and SDS-PAGE. Unlike its native counterpart, rSALO(P) does not inhibit the classical pathway of complement; however, antibodies to rSALO(P) inhibit the anti-complement activity of sand fly salivary gland homogenate. Immunization with rSALO(P) produces a delayed type hypersensitivity response in C57BL/6 mice, suggesting rSALO(P) lacked anti-complement activity but retained its immunogenicity. The structure of rSALO(P) was solved by S-SAD at Cu-Kalpha to 1.94 Å and refined to Rfactor 17%. SALO is ~80% helical, has no appreciable structural similarities to any human protein, and has limited structural similarity in the C-terminus to members of insect odorant binding proteins. SALO has three predicted human CD4+ T cell epitopes on surface exposed helices. CONCLUSIONS/SIGNIFICANCE: The results indicate that SALO as expressed and purified from P. pastoris is suitable for further scale-up, manufacturing, and testing. SALO has a novel structure, is not similar to any human proteins, is immunogenic in rodents, and does not have the anti-complement activity observed in the native salivary protein which are all important attributes to move this vaccine candidate forward to the clinical path.

Structural and functional characterization of the CAP domain of pathogen-related yeast 1 (Pry1) protein.

The production, crystal structure, and functional characterization of the C-terminal cysteine-rich secretory protein/antigen 5/pathogenesis related-1 (CAP) domain of pathogen-related yeast protein-1 (Pry1) from Saccharomyces cerevisiae is presented. The CAP domain of Pry1 (Pry1CAP) is functional in vivo as its expression restores cholesterol export to yeast mutants lacking endogenous Pry1 and Pry2. Recombinant Pry1CAP forms dimers in solution, is sufficient for in vitro cholesterol binding, and has comparable binding properties as full-length Pry1. Two crystal structures of Pry1CAP are reported, one with Mg(2+) coordinated to the conserved CAP tetrad (His208, Glu215, Glu233 and His250) in spacegroup I41 and the other without divalent cations in spacegroup P6122. The latter structure contains four 1,4-dioxane molecules from the crystallization solution, one of which sits in the cholesterol binding site. Both structures reveal that the divalent cation and cholesterol binding sites are connected upon dimerization, providing a structural basis for the observed Mg(2+)-dependent sterol binding by Pry1.

Expression, purification, crystallization and crystallographic study of Lutzomyia longipalpis LJL143.

Leishmaniasis is a neglected vector-borne disease with a global prevalence of over 12 million cases and 59,000 annual deaths. Transmission of the parasite requires salivary proteins, including LJL143 from the New World sandfly Lutzomyia longipalpis. LJL143 is a known marker of sandfly exposure in zoonotic hosts. LJL143 was crystallized from soluble protein expressed using Pichia pastoris. X-ray data were collected to 2.6 Šresolution from orthorhombic crystals belonging to space group P2(1)2(1)2(1), with average unit-cell parameters a = 57.39, b = 70.24, c = 79.58 Å. The crystals are predicted to have a monomer in the asymmetric unit, with an estimated solvent content of 48.5%. LJL143 has negligible homology to any reported structures, so the phases could not be determined by molecular replacement. All attempts at S-SAD failed and future studies include experimental phase determination using heavy-atom derivatives.

Structure of type II dehydroquinase from Pseudomonas aeruginosa.

Pseudomonas aeruginosa causes opportunistic infections and is resistant to most antibiotics. Ongoing efforts to generate much-needed new antibiotics include targeting enzymes that are vital for P. aeruginosa but are absent in mammals. One such enzyme, type II dehydroquinase (DHQase), catalyzes the interconversion of 3-dehydroquinate and 3-dehydroshikimate, a necessary step in the shikimate pathway. This step is vital for the proper synthesis of phenylalanine, tryptophan, tyrosine and other aromatic metabolites. The recombinant expression, purification and crystal structure of catalytically active DHQase from P. aeruginosa (PaDHQase) are presented. Cubic crystals belonging to space group F23, with unit-cell parameters a=b=c=125.39 Å, were obtained by vapor diffusion in sitting drops and the structure was refined to an R factor of 16% at 1.74 Šresolution. PaDHQase is a prototypical type II DHQase with the classical flavodoxin-like α/ß topology.

Bacillus thuringiensis Cry5B protein is highly efficacious as a single-dose therapy against an intestinal roundworm infection in mice.

BACKGROUND: Intestinal parasitic nematode diseases are one of the great diseases of our time. Intestinal roundworm parasites, including hookworms, whipworms, and Ascaris, infect well over 1 billion people and cause significant morbidity, especially in children and pregnant women. To date, there is only one drug, albendazole, with adequate efficacy against these parasites to be used in mass drug administration, although tribendimidine may emerge as a second. Given the hundreds of millions of people to be treated, the threat of parasite resistance, and the inadequacy of current treatments, new anthelmintics are urgently needed. Bacillus thuringiensis (Bt) crystal (Cry) proteins are the most common used biologically produced insecticides in the world and are considered non-toxic to vertebrates. METHODS/PRINCIPAL FINDINGS: Here we study the ability of a nematicidal Cry protein, Cry5B, to effect a cure in mice of a chronic roundworm infection caused by the natural intestinal parasite, Heligmosomoides bakeri (formerly polygyrus). We show that Cry5B produced from either of two Bt strains can act as an anthelmintic in vivo when administered as a single dose, achieving a approximately 98% reduction in parasite egg production and approximately 70% reduction in worm burdens when delivered per os at approximately 700 nmoles/kg (90-100 mg/kg). Furthermore, our data, combined with the findings of others, suggest that the relative efficacy of Cry5B is either comparable or superior to current anthelmintics. We also demonstrate that Cry5B is likely to be degraded quite rapidly in the stomach, suggesting that the actual dose reaching the parasites is very small. CONCLUSIONS/SIGNIFICANCE: This study indicates that Bt Cry proteins such as Cry5B have excellent anthelmintic properties in vivo and that proper formulation of the protein is likely to reveal a superior anthelmintic.