RESUMO
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 the 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 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 ß-sheet fold containing 2 domains. A structure of the lufaxin-C3bB complex obtained via cryo-electron microscopy (EM) 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 in which 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 to 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 a rationale for the development of a bispecific inhibitor to treat complement-related diseases in which thrombosis is a prominent manifestation.
Assuntos
Coagulação Sanguínea , Fator B do Complemento , Microscopia Crioeletrônica , Fator B do Complemento/química , Fator B do Complemento/metabolismo , Ativação do Complemento , Serina Endopeptidases , Complemento C3b/químicaRESUMO
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 αßα 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.
Assuntos
Moléculas de Adesão Celular , Proteínas Secretadas pela Próstata , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Esteróis , Animais , Cálcio/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Humanos , Masculino , Mamíferos/metabolismo , Próstata/metabolismo , Proteínas Secretadas pela Próstata/genética , Proteínas Secretadas pela Próstata/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Esteróis/antagonistas & inibidores , Esteróis/metabolismoRESUMO
This reflective overview describes the benefits of participation in authentic undergraduate research for students at a Historically Black College and University (HBCU). The department of chemistry and biochemistry at Hampton University has an undergraduate research environment that empowers and fosters a success-oriented research experience for our diverse students. By engaging undergraduate students in research early in their careers, we successfully motivate students to make informed decisions about pursuing STEM careers and entering graduate schools with high confidence. Our structured undergraduate research experiences are created within an inclusive environment that instills a sense of belonging and recognizes the talent all our students bring to STEM. We reflect on our experiences using faculty-student research collaborations within nurturing support systems that leverage African American culture while setting high expectations to improve scientific skills and retain our HBCU students in STEM.
RESUMO
Despite causing considerable damage to host tissue at the onset of parasitism, invasive helminths establish remarkably persistent infections in both animals and plants. Secretions released by these obligate parasites during host invasion are thought to be crucial for their persistence in infection. Helminth secretions are complex mixtures of molecules, most of which have unknown molecular targets and functions in host cells or tissues. Although the habitats of animal- and plant-parasitic helminths are very distinct, their secretions share the presence of a structurally conserved group of proteins called venom allergen-like proteins (VALs). Helminths abundantly secrete VALs during several stages of parasitism while inflicting extensive damage to host tissue. The tight association between the secretion of VALs and the onset of parasitism has triggered a particular interest in this group of proteins, as improved knowledge on their biological functions may assist in designing novel protection strategies against parasites in humans, livestock, and important food crops.
Assuntos
Alérgenos/imunologia , Produtos Agrícolas/imunologia , Proteínas de Helminto/imunologia , Helmintos/imunologia , Interações Hospedeiro-Parasita/imunologia , Infecções por Nematoides/parasitologia , Peçonhas/imunologia , Animais , Infecções por Nematoides/imunologiaRESUMO
Members of the CAP superfamily (cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins), also known as SCP superfamily (sperm-coating proteins), have been implicated in many physiological processes, including immune defenses, venom toxicity, and sperm maturation. Their mode of action, however, remains poorly understood. Three proteins of the CAP superfamily, Pry1, -2, and -3 (pathogen related in yeast), are encoded in the Saccharomyces cerevisiae genome. We have shown previously that Pry1 binds cholesterol in vitro and that Pry function is required for sterol secretion in yeast cells, indicating that members of this superfamily may generally bind sterols or related small hydrophobic compounds. On the other hand, tablysin-15, a CAP protein from the horsefly Tabanus yao, has been shown to bind leukotrienes and free fatty acids in vitro Therefore, here we assessed whether the yeast Pry1 protein binds fatty acids. Computational modeling and site-directed mutagenesis indicated that the mode of fatty acid binding is conserved between tablysin-15 and Pry1. Pry1 bound fatty acids with micromolar affinity in vitro, and its function was essential for fatty acid export in cells lacking the acyl-CoA synthetases Faa1 and Faa4. Fatty acid binding of Pry1 is independent of its capacity to bind sterols, and the two sterol- and fatty acid-binding sites are nonoverlapping. These results indicate that some CAP family members, such as Pry1, can bind different lipids, particularly sterols and fatty acids, at distinct binding sites, suggesting that the CAP domain may serve as a stable, secreted protein domain that can accommodate multiple ligand-binding sites.
Assuntos
Proteínas de Ligação a Ácido Graxo/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Acil Coenzima A/química , Acil Coenzima A/genética , Acil Coenzima A/metabolismo , Coenzima A Ligases/química , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Simulação por Computador , Proteínas de Ligação a Ácido Graxo/química , Proteínas de Ligação a Ácido Graxo/genética , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Mutagênese Sítio-Dirigida , Domínios Proteicos , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
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 2.16â 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.
Assuntos
Alérgenos/química , Lipídeos/química , Schistosoma mansoni/química , Peçonhas/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Cromatografia Líquida , Cristalografia por Raios X , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
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.
Assuntos
Alanina Racemase/química , Clostridioides difficile/enzimologia , Farmacorresistência Bacteriana Múltipla , Sequência de Aminoácidos , Cromatografia em Gel , Clostridioides difficile/efeitos dos fármacos , Cristalografia por Raios X , Dimerização , Dados de Sequência Molecular , Conformação Proteica , Proteínas Recombinantes/química , Homologia de Sequência de AminoácidosRESUMO
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.
RESUMO
Macrophage migration inhibitory factor (MIF) is a catalytic cytokine and an upstream mediator of the inflammatory pathway. MIF has broad regulatory properties, dysregulation of which has been implicated in the pathology of multiple immunological diseases. Inhibition of MIF activity with small molecules has proven beneficial in a number of disease models. Known small molecule MIF inhibitors typically bind in the tautomerase site of the MIF trimer, often covalently modifying the catalytic proline. Allosteric MIF inhibitors, particularly those that associate with the protein by noncovalent interactions, could reveal novel ways to block MIF activity for therapeutic benefit and serve as chemical probes to elucidate the structural basis for the diverse regulatory properties of MIF. In this study, we report the identification and functional characterization of a novel allosteric MIF inhibitor. Identified from a high throughput screening effort, this sulfonated azo compound termed p425 strongly inhibited the ability of MIF to tautomerize 4-hydroxyphenyl pyruvate. Furthermore, p425 blocked the interaction of MIF with its receptor, CD74, and interfered with the pro-inflammatory activities of the cytokine. Structural studies revealed a unique mode of binding for p425, with a single molecule of the inhibitor occupying the interface of two MIF trimers. The inhibitor binds MIF mainly on the protein surface through hydrophobic interactions that are stabilized by hydrogen bonding with four highly specific residues from three different monomers. The mode of p425 binding reveals a unique way to block the activity of the cytokine for potential therapeutic benefit in MIF-associated diseases.
Assuntos
Antígenos de Diferenciação de Linfócitos B/metabolismo , Compostos Azo , Fibroblastos/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Oxirredutases Intramoleculares , Fatores Inibidores da Migração de Macrófagos , Azul Tripano/química , Azul Tripano/farmacologia , Regulação Alostérica/efeitos dos fármacos , Antígenos de Diferenciação de Linfócitos B/química , Compostos Azo/química , Compostos Azo/farmacologia , Células Cultivadas , Fibroblastos/citologia , Antígenos de Histocompatibilidade Classe II/química , Humanos , Oxirredutases Intramoleculares/antagonistas & inibidores , Oxirredutases Intramoleculares/química , Oxirredutases Intramoleculares/metabolismo , Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Fatores Inibidores da Migração de Macrófagos/química , Fatores Inibidores da Migração de Macrófagos/metabolismo , Ligação Proteica/efeitos dos fármacos , Estrutura Quaternária de ProteínaRESUMO
Methicillin-resistant Staphylococcus aureus is estimated to cause more U.S. deaths annually than HIV/AIDS. The emergence of hypervirulent and multidrug-resistant strains has further amplified public health concern and accentuated the need for new classes of antibiotics. RNA degradation is a required cellular process that could be exploited for novel antimicrobial drug development. However, such discovery efforts have been hindered because components of the Gram-positive RNA turnover machinery are incompletely defined. In the current study we found that the essential S. aureus protein, RnpA, catalyzes rRNA and mRNA digestion in vitro. Exploiting this activity, high through-put and secondary screening assays identified a small molecule inhibitor of RnpA-mediated in vitro RNA degradation. This agent was shown to limit cellular mRNA degradation and exhibited antimicrobial activity against predominant methicillin-resistant S. aureus (MRSA) lineages circulating throughout the U.S., vancomycin intermediate susceptible S. aureus (VISA), vancomycin resistant S. aureus (VRSA) and other Gram-positive bacterial pathogens with high RnpA amino acid conservation. We also found that this RnpA-inhibitor ameliorates disease in a systemic mouse infection model and has antimicrobial activity against biofilm-associated S. aureus. Taken together, these findings indicate that RnpA, either alone, as a component of the RNase P holoenzyme, and/or as a member of a more elaborate complex, may play a role in S. aureus RNA degradation and provide proof of principle for RNA catabolism-based antimicrobial therapy.
Assuntos
Anti-Infecciosos/farmacologia , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ribonuclease P/antagonistas & inibidores , Infecções Estafilocócicas/prevenção & controle , Staphylococcus aureus , Animais , Anti-Infecciosos/uso terapêutico , Feminino , Células Hep G2 , Humanos , Camundongos , Modelos Biológicos , Ribonuclease P/fisiologia , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/uso terapêutico , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/patologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidade , Vancomicina/farmacologia , Vancomicina/uso terapêutico , Virulência/efeitos dos fármacos , Virulência/genéticaRESUMO
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.
Assuntos
Glutationa Transferase/química , Proteínas de Helminto/química , Necator americanus/enzimologia , Sequência de Aminoácidos , Animais , Sítios de Ligação/fisiologia , Cristalografia por Raios X , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Dados de Sequência Molecular , Necator americanus/genéticaRESUMO
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 1â 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.
Assuntos
Aldeído Liases/química , Pseudomonas aeruginosa/enzimologia , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Homologia Estrutural de ProteínaRESUMO
We had previously reported that Acanthamoeba castellanii (ACA) contains a mimicry epitope for proteolipid protein 139-151 capable of inducing central nervous system (CNS) autoimmunity in SJL/J mice. We now present evidence that ACA also contains a mimicry epitope for myelin basic protein (MBP) 89-101, a derivative from amoebic nicotinamide adenine dinucleotide dehydrogenase subunit 2 (NAD). The epitope, NAD 108-120, contains a discontinuous stretch of six amino acids in the core region (VVFFKNIILIGFL) sharing 46% identity with MBP 89-101 (VHFFKNIVTPRTP; identical residues are underlined). SJL mice immunized with NAD 108-120 develop encephalomyelitis similar to the disease induced by the cognate peptide. We demonstrate that NAD 108-120 induces T cells that cross-react with MBP 89-101; the antigen-sensitized T cells, which produce predominantly T helper (T(h)) 1 and T(h)17 cytokines, transfer disease in naive SJL recipients reminiscent of the disease induced with MBP 89-101. This is the first report to demonstrate that a solitary microbe can induce CNS autoimmunity by generating cross-reactive T cells for multiple myelin antigens.
Assuntos
Acanthamoeba castellanii/imunologia , Antígenos de Protozoários/metabolismo , Encefalomielite Autoimune Experimental/imunologia , Mimetismo Molecular/imunologia , Esclerose Múltipla/imunologia , Proteína Básica da Mielina/metabolismo , NADH Desidrogenase/metabolismo , Fragmentos de Peptídeos/metabolismo , Animais , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Autoimunidade , Células Cultivadas , Sistema Nervoso Central/imunologia , Reações Cruzadas , Epitopos de Linfócito T/imunologia , Humanos , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos , Proteína Básica da Mielina/genética , Proteína Básica da Mielina/imunologia , NADH Desidrogenase/genética , NADH Desidrogenase/imunologia , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Células Th1/imunologia , Células Th1/metabolismo , Células Th1/patologia , Células Th17/imunologia , Células Th17/metabolismo , Células Th17/patologiaRESUMO
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 â¼97% 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.
Assuntos
Anopheles , Infecções por Flavobacteriaceae , Sequência de Aminoácidos , Animais , Anopheles/metabolismo , Cristalografia por Raios X , Glutamato-tRNA Ligase/química , Glutamato-tRNA Ligase/genética , Glutamato-tRNA Ligase/metabolismoRESUMO
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 1.80â 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.
Assuntos
Burkholderiaceae/enzimologia , NAD/química , Redutases-Desidrogenases de Cadeia Curta/química , Redutases-Desidrogenases de Cadeia Curta/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Coenzimas/química , Coenzimas/metabolismo , Cristalografia por Raios X , Modelos Moleculares , NAD/metabolismo , Conformação ProteicaRESUMO
Paraburkholderia xenovorans degrades organic wastes, including polychlorinated biphenyls. The atomic structure of a putative dehydrogenase/reductase (SDR) from P. xenovorans (PxSDR) was determined in space group P21 at a resolution of 1.45â Å. PxSDR shares less than 37% sequence identity with any known structure and assembles as a prototypical SDR tetramer. As expected, there is some conformational flexibility and difference in the substrate-binding cavity, which explains the substrate specificity. Uniquely, the cofactor-binding cavity of PxSDR is not well conserved and differs from those of other SDRs. PxSDR has an additional seven amino acids that form an additional unique loop within the cofactor-binding cavity. Further studies are required to determine how these differences affect the enzymatic functions of the SDR.
Assuntos
Burkholderiaceae , Redutases-Desidrogenases de Cadeia Curta , Cristalografia por Raios X , Oxirredutases/química , Redutases-Desidrogenases de Cadeia Curta/metabolismo , Especificidade por SubstratoRESUMO
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 by the 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 2.2â 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.
Assuntos
Chlamydia trachomatis , Pirofosfatase Inorgânica , Chlamydia trachomatis/metabolismo , Cristalografia por Raios X , Pirofosfatase Inorgânica/metabolismo , Estados UnidosRESUMO
Burkholderia pseudomallei infection causes melioidosis, which is often fatal if untreated. There is a need to develop new and more effective treatments for melioidosis. This study reports apo and cofactor-bound crystal structures of the potential drug target betaine aldehyde dehydrogenase (BADH) from B. pseudomallei. A structural comparison identified similarities to BADH from Pseudomonas aeruginosa which is inhibited by the drug disulfiram. This preliminary analysis could facilitate drug-repurposing studies for B. pseudomallei.
Assuntos
Proteínas de Bactérias/química , Betaína-Aldeído Desidrogenase/química , Burkholderia pseudomallei/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Betaína-Aldeído Desidrogenase/genética , Betaína-Aldeído Desidrogenase/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Conformação Proteica , Pseudomonas aeruginosa/enzimologiaRESUMO
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 â¼95% 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.
Assuntos
Brucella canis , Brucella suis , Brucelose , Tetra-Hidrofolato Desidrogenase , Brucelose/microbiologia , Cristalografia por Raios X , Humanos , Tetra-Hidrofolato Desidrogenase/genéticaRESUMO
The name of one of the authors in Beard et al. [(2022), Acta Cryst. F78, 59-65] is corrected.