Structural and functional analysis of essential pre-mRNA splicing factor Prp19p

U-box-containing Prp19p is an integral component of the Prp19p-associated complex (the nineteen complex, or NTC) that is essential for activation of the spliceosome. Prp19p makes numerous protein-protein contacts with other NTC components and is required for NTC stability. Here we show that Prp19p forms a tetramer in vitro and in vivo and we map the domain required for its oligomerization to a central tetrameric coiled-coil. Biochemical and in vivo analyses are consistent with Prp19p tetramerization providing an interaction surface for a single copy of its binding partner, Cef1p. Electron microscopy showed that the isolated Prp19p tetramer is an elongated particle consisting of four globular WD40 domains held together by a central stalk consisting of four N-terminal U-boxes and four coiled-coils. These structural and functional data provide a basis for understanding the role of Prp19p as a key architectural component of the NTC.     

Structural and functional characterization of Staphylococcus aureus dihydrodipicolinate synthase

Lysine biosynthesis is crucial for cell-wall formation in bacteria. Enzymes involved in lysine biosynthesis are thus potential targets for anti-microbial therapeutics. Dihydrodipicolinate synthase (DHDPS) catalyzes the first step of this pathway. Unlike its homologues, Staphylococcus aureus DHDPS is a dimer both in solution and in the crystal and is not feedback inhibited by lysine. The crystal structure of S. aureus DHDPS in the free and substrate bound forms provides a structural rationale for its catalytic mechanism. The structure also reveals unique conformational features of the S. aureus enzyme that could be crucial for the design of specific non-competitive inhibitors.     

Structural switching of Staphylococcus aureus Clp protease: a key to understanding protease dynamics

ATP-dependent Clp protease (ClpP) is an attractive new target for the development of anti-infective agents. The ClpP protease consists of two heptameric rings that enclose a large chamber containing 14 proteolytic active sites. Recent studies indicate that ClpP likely undergoes conformational switching between an extended and degraded active state required for substrate proteolysis and a compacted and catalytically inactive state allowing product release. Here, we present the wild-type ClpP structures in two distinct states from Staphylococcus aureus. One structure is very similar to those solved ClpP structures in the extended states. The other is strikingly different from both the extended and the compacted state as observed in ClpP from other species; the handle domain of this structure kinks to take on a compressed conformation. Structural analysis and molecular dynamic simulations show that the handle domain predominantly controls the way in which degradation products exit the chamber through dynamic conformational switching from the extended state to the compressed state. Given the highly conserved sequences among ClpP from different species, this compressed conformation is unexpected and novel, which is potentially valuable for understanding the enzymatic dynamics and the acting mechanisms of ClpP.     

Structural characterization of the alpha-hemolysin monomer from Staphylococcus aureus

Alpha-hemolysin from Staphylococcus aureus is secreted as a water-soluble monomer and assembles on membranes to oligomerize into a homo-heptameric, water-filled pore. These pores lead to lysis and cell death. Although the structure of the heptameric pore is solved by means of X-ray crystallography, structures of intermediate states-from the soluble monomer to all potential "pre-pore" structures-are yet unknown. Here, we propose a model of the monomeric alpha-hemolysin in solution based on molecular modeling, verified by small angle X-ray scattering data. This structure reveals details of the monomeric conformation of the alpha-hemolysin, for example inherent flexibility, along with definite differences in comparison to the structures used as templates.     

Structural analysis of the surface polysaccharide of Staphylococcus aureus M.

The chemical structure of the surface polysaccharide from Staphylococcus aureus M was investigated by a combination of methanolytic, hydrolytic, and chromatographic techniques. The repeating unit that was most consistent with the data was a hexasaccharide composed of N-acetyl-D-aminogalacturonic acid, N-acetyl-D-fucosamine, and taurine in molar ratios of 4:2:1. A disaccharide was isolated and characterized, by combined gas-liquid chromatography-mass spectrometry, as N-acetyl-D-aminogalacturonyl-(1 leads to 3)-N-acetyl-D-fucosamine. Taurine is linked to a carboxyl group of N-acetyl-D-aminogalacturonic acid via an amide bond.     

Structural and evolutionary relationships of beta-lactamase transposons from Staphylococcus aureus

A comparison of the beta-lactamase elements detected on three classes of large plasmids together with the chromosomes of penicillin-resistant Staphylococcus aureus revealed substantial physical and genetic relatedness. In most cases, beta-lactamase production could be associated with the presence of a DNA segment of approximately 6.7 kb. Analysis showed that the plasmid-borne determinants constitute nearly identical transposons or transposon-like elements. An element indistinguishable from one of these, Tn4002, which is carried by the pSK1 family of plasmids in clinical isolates from Australian hospitals, was also identified on the staphylococcal chromosome and is implicated in an evolutionary cycle of transposition between chromosomal and extrachromosomal sites in Australian strains of multiresistant S. aureus.     

Computational structural and functional analysis of hypothetical proteins of Staphylococcus aureus

Genome sequencing projects has led to an explosion of large amount of gene products in which many are of hypothetical proteins with unknown function. Analyzing and annotating the functions of hypothetical proteins is important in Staphylococcus aureus which is a pathogenic bacterium that cause multiple types of diseases by infecting various sites in humans and animals. In this study, ten hypothetical proteins of Staphylococcus aureus were retrieved from NCBI and analyzed for their structural and functional characteristics by using various bioinformatics tools and databases. The analysis revealed that some of them possessed functionally important domains and families and protein-protein interacting partners which were ABC transporter ATP-binding protein, Multiple Antibiotic Resistance (MAR) family, export proteins, Helix-Turn-helix domains, arsenate reductase, elongation factor, ribosomal proteins, Cysteine protease precursor, Type-I restriction endonuclease enzyme and plasmid recombination enzyme which might have the same functions in hypothetical proteins. The structural prediction of those proteins and binding sites prediction have been done which would be useful in docking studies for aiding in the drug discovery.     

Structural analysis of Staphylococcus aureus serine/threonine kinase PknB

Effective treatment of infections caused by the bacterium Staphylococcus aureus remains a worldwide challenge, in part due to the constant emergence of new strains that are resistant to antibiotics. The serine/threonine kinase PknB is of particular relevance to the life cycle of S. aureus as it is involved in the regulation of purine biosynthesis, autolysis, and other central metabolic processes of the bacterium. We have determined the crystal structure of the kinase domain of PknB in complex with a non-hydrolyzable analog of the substrate ATP at 3.0 ? resolution. Although the purified PknB kinase is active in solution, it crystallized in an inactive, autoinhibited state. Comparison with other bacterial kinases provides insights into the determinants of catalysis, interactions of PknB with ligands, and the pathway of activation.     

Dimerization of long hibernation promoting factor from Staphylococcus aureus: Structural analysis and biochemical characterization

Staphylococcus aureus hibernation promoting factor (SaHPF) is responsible for the formation of 100S ribosome dimers, which in turn help this pathogen to reduce energy spent under unfavorable conditions. Ribosome dimer formation strongly depends on the dimerization of the C-terminal domain of SaHPF (CTD^SaHPF). In this study, we solved the crystal structure of CTD^SaHPF at 1.6 Å resolution and obtained a precise arrangement of the dimer interface. Residues Phe¹⁶⁰, Val¹⁶², Thr¹⁷¹, Ile¹⁷³, Tyr¹⁷⁵, Ile¹⁸⁵ andThr¹⁸⁷ in the dimer interface of SaHPF protein were mutated and the effects were analyzed for the formation of 100S disomes of ribosomes isolated from S. aureus. It was shown that substitution of any of single residues Phe¹⁶⁰, Val¹⁶², Ile¹⁷³, Tyr¹⁷⁵ and Ile¹⁸⁵ in the SaHPF homodimer interface abolished the ribosome dimerization in vitro.     

Development and binding characteristics of phosphonate inhibitors of SplA protease from Staphylococcus aureus

Staphylococcus aureus is responsible for a variety of human infections, including life-threatening, systemic conditions. Secreted proteome, including a range of proteases, constitutes the major virulence factor of the bacterium. However, the functions of individual enzymes, in particular SplA protease, remain poorly characterized. Here, we report development of specific inhibitors of SplA protease. The design, synthesis, and activity of a series of α-aminoalkylphosphonate diaryl esters and their peptidyl derivatives are described. Potent inhibitors of SplA are reported, which may facilitate future investigation of physiological function of the protease. The binding modes of the high-affinity compounds Cbz-Phe^P-(OC₆H₄−4-SO₂CH₃)₂ and Suc-Val-Pro-Phe^P-(OC₆H₅)₂ are revealed by high-resolution crystal structures of complexes with the protease. Surprisingly, the binding mode of both compounds deviates from previously characterized canonical interaction of α-aminoalkylphosphonate peptidyl derivatives and family S1 serine proteases.     

Interactions between lipoteichoic acid and peptidoglycan from Staphylococcus aureus: a structural and functional analysis

The cell wall of Gram-positive bacteria contains lipoteichoic acid (LTA) and peptidoglycan (PepG), which synergise to cause shock and organ failure in animals, and to activate human blood to release proinflammatory cytokines. The structural elements within LTA and PepG that are essential for the observed synergism are discussed.     

基于功能宏基因组学技术的金黄色葡萄球菌生物被膜抑制物的发现与活性分析

宏基因组学方法直接提取环境中的全部微生物基因组DNA,并使其得到功能性表达,为微生物天然产物的开发利用提供了新的方法。利用功能宏基因组学技术,使用大肠杆菌-链霉菌穿梭载体构建四川峨眉山土壤宏基因组文库,并将文库菌中所携带的环境DNA接合转移到链霉菌宿主中。通过活性筛选获得两个具有抗菌活性的阳性链霉菌克隆,其发酵粗提物对金黄色葡萄球菌生物被膜的形成均有很好的抑制作用,当浓度达到2 MIC(Minimum inhibitory concentration)时,抑制作用超过90%;同时,两种粗提物样品对金黄色葡萄球菌生物被膜也存在显著的清除作用,其中EM110样品对金黄色葡萄球菌生物被膜的清除率高于EM123样品。本文通过功能宏基因组学技术,直接从土壤中筛选获得了对金黄色葡萄球菌生物被膜有较强抑制及清除作用的活性物质。     

The essential catalytic redox couple in arsenate reductase from Staphylococcus aureus

Arsenate reductase (ArsC) encoded by Staphylococcus aureus arsenic-resistance plasmid pI258 reduces intracellular As(V) (arsenate) to the more toxic As(III) (arsenite), which is subsequently extruded from the cell. ArsC couples to thioredoxin, thioredoxin reductase, and NADPH to be enzymatically active. A novel purification method leads to high production levels of highly pure enzyme. A reverse phase method was introduced to systematically analyze and control the oxidation status of the enzyme. The essential cysteinyl residues and redox couple in arsenate reductase were identified by a combination of site-specific mutagenesis and endoprotease-digest mass spectroscopy analysis. The secondary structures, as determined with CD, of wild-type ArsC and its Cys mutants showed a relatively high helical content, independent of the redox status. Mutation of Cys 10, 82, and 89 led to redox-inactive enzymes. ArsC was oxidized in a single catalytic cycle and subsequently digested with endoproteinases ArgC, AspN, and GluC. From the peptide-mass profiles, cysteines 82 and 89 were identified as the redox couple of ArsC necessary to reduce arsenate to arsenite.     

Structural and functional role of threonine 112 in a superantigen Staphylococcus aureus enterotoxin B.

Bacterial superantigens are potent T-cell stimulatory protein molecules produced by Staphylococcus aureus and Streptococcus pyogenes. Their superantigenic activity can be attributed to their ability to cross-link major histocompatibility complex class II molecules with T-cell receptors (TCRs) to form a tri-molecular complex. Each superantigen is known to interact with a specific V(beta) element of TCR. Staphylococcal enterotoxin B (SEB, a superantigen), a primary cause of food poisoning, is also responsible for a significant percentage of non-menstrual associated toxic shock syndrome in patients with a variety of staphylococcal infections. Structural studies have elucidated a binding cavity on the toxin molecule essential for TCR binding. To understand the crucial residues involved in binding, mutagenesis analysis was performed. Our analysis suggest that mutation of a conserved residue Thr(112) to Ser (T112S) in the binding cavity induces a selective reduction in the affinity for binding one TCR V(beta) family and can be attributed to the structural differences in the native and mutant toxins. We present a detailed comparison of the mutant structure determined at 2.0 A with the previously reported native SEB and SEB-TCR V(beta) complex structures.     

Identification of an essential glycoprotease in Staphylococcus aureus

The emergence of multi-drug resistant bacterial pathogens is generating enormous public health concern, and highlights an urgent need for new, alternative agents for treating multi-drug-resistant pathogens. The gene products essential for bacterial growth in vitro and survival during infection constitute an initial set of protein targets for the development of antibacterial agents. In this study, we employed regulated gene expression approaches and demonstrated that a putative glycoprotease (Gcp) is required for staphylococcal growth in the culture. We found that Staphylococcus aureus becomes more sensitive to the Zn(2+) ion under the downregulation of Gcp expression in vitro. Bioinformatic analyses demonstrated that Gcp is conserved in many Gram-positive pathogens and exists in a variety of Gram-negative pathogens. Our results indicate that Gcp is a potential novel target for the development of antimicrobials against S. aureus infection.     

Cleavage by protease from Staphylococcus aureus V8: an improvement in the sequence analysis of human hemoglobin variants

Protease from Staphylococcus aureus V8 cleaves either at glutamic residues or at both aspartic and glutamic residues, depending on the experimental conditions. In structural analyses of human hemoglobin variants, the specificity of this enzyme is of considerable interest to localize substitutions occurring in medium or large size peptides as it cleaves in smaller fragments which may be unambiguously characterized. It may also recognize the replacement of an acidic residue by the corresponding amide, or vice versa, avoiding protein sequence analysis. The various aspects of the use of protease V8 are illustrated by the study of four alpha chain hemoglobin variants concerning peptides alpha T-9 and alpha T-12b.