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1.  Structural characterization of a hypothetical protein: a potential agent involved in trimethylamine metabolism in Catenulispora acidiphila 
Catenulispora acidiphila is a newly identified lineage of actinomycetes that produces antimicrobial activities and represents a promising source of novel antibiotics and secondary metabolites. Among the discovered protein coding genes, 68 % were assigned a putative function, while the remaining 32 % are genes encoding “hypothetical” proteins. Caci_0382 is one of the “hypothetical” proteins that has very few homologs. Sequence analysis shows that the protein belongs to the NTF2-like protein family. The structure of Caci_0382 demonstrates that it shares the same fold and has a similar active site as limonene-1,2-epoxide hydrolase, which suggests that it may have a related function. Using a fluorescence thermal shift assay, we identified stabilizing compounds that suggest potential natural ligands of Caci_0382. Using this information, we determined the crystal structure in complex with trimethylamine to provide a better understanding of the function of this uncharacterized protein.
Electronic supplementary material
The online version of this article (doi:10.1007/s10969-014-9176-z) contains supplementary material, which is available to authorized users.
doi:10.1007/s10969-014-9176-z
PMCID: PMC3955178  PMID: 24562475
Trimethylamine; NTF2-like superfamily; Caci_0382; X-ray crystal structure; Structural genomics
2.  Characterization of Fibrinogen Binding by Glycoproteins Srr1 and Srr2 of Streptococcus agalactiae* 
The Journal of Biological Chemistry  2013;288(50):35982-35996.
Background: The serine-rich repeat glycoproteins Srr1 and Srr2 are surface adhesins of Streptococcus agalactiae important for pathogenicity.
Results: Both Srrs bind tandem repeats of the fibrinogen Aα chain, but Srr2 has greater affinity explained by structure-function analysis of the Srrs.
Conclusion: A dock, lock, and latch mechanism describes the Srr-fibrinogen interaction.
Significance: The higher affinity of Srr2 may contribute to the hypervirulence of Srr2-expressing strains.
The serine-rich repeat glycoproteins of Gram-positive bacteria comprise a large family of cell wall proteins. Streptococcus agalactiae (group B streptococcus, GBS) expresses either Srr1 or Srr2 on its surface, depending on the strain. Srr1 has recently been shown to bind fibrinogen, and this interaction contributes to the pathogenesis of GBS meningitis. Although strains expressing Srr2 appear to be hypervirulent, no ligand for this adhesin has been described. We now demonstrate that Srr2 also binds human fibrinogen and that this interaction promotes GBS attachment to endothelial cells. Recombinant Srr1 and Srr2 bound fibrinogen in vitro, with affinities of KD = 2.1 × 10−5 and 3.7 × 10−6 m, respectively, as measured by surface plasmon resonance spectroscopy. The binding site for Srr1 and Srr2 was localized to tandem repeats 6–8 of the fibrinogen Aα chain. The structures of both the Srr1 and Srr2 binding regions were determined and, in combination with mutagenesis studies, suggest that both Srr1 and Srr2 interact with a segment of these repeats via a “dock, lock, and latch” mechanism. Moreover, properties of the latch region may account for the increased affinity between Srr2 and fibrinogen. Together, these studies identify how greater affinity of Srr2 for fibrinogen may contribute to the increased virulence associated with Srr2-expressing strains.
doi:10.1074/jbc.M113.513358
PMCID: PMC3861647  PMID: 24165132
Bacterial Adhesion; Bacterial Pathogenesis; Fibrinogen; Protein Crystallization; Streptococcus; Streptococcus agalactiae; Prot
3.  Structure of the Type III Secretion Effector Protein ExoU in Complex with Its Chaperone SpcU 
PLoS ONE  2012;7(11):e49388.
Disease causing bacteria often manipulate host cells in a way that facilitates the infectious process. Many pathogenic gram-negative bacteria accomplish this by using type III secretion systems. In these complex secretion pathways, bacterial chaperones direct effector proteins to a needle-like secretion apparatus, which then delivers the effector protein into the host cell cytosol. The effector protein ExoU and its chaperone SpcU are components of the Pseudomonas aeruginosa type III secretion system. Secretion of ExoU has been associated with more severe infections in both humans and animal models. Here we describe the 1.92 Å X-ray structure of the ExoU–SpcU complex, a full-length type III effector in complex with its full-length cognate chaperone. Our crystallographic data allow a better understanding of the mechanism by which ExoU kills host cells and provides a foundation for future studies aimed at designing inhibitors of this potent toxin.
doi:10.1371/journal.pone.0049388
PMCID: PMC3498133  PMID: 23166655
4.  Structural characterization and comparison of three acyl-carrier-protein synthases from pathogenic bacteria 
The structural characterization of acyl-carrier-protein synthase (AcpS) from three different pathogenic microorganisms is reported. One interesting finding of the present work is a crystal artifact related to the activity of the enzyme, which fortuitously represents an opportunity for a strategy to design a potential inhibitor of a pathogenic AcpS.
Some bacterial type II fatty-acid synthesis (FAS II) enzymes have been shown to be important candidates for drug discovery. The scientific and medical quest for new FAS II protein targets continues to stimulate research in this field. One of the possible additional candidates is the acyl-carrier-protein synthase (AcpS) enzyme. Its holo form post-translationally modifies the apo form of an acyl carrier protein (ACP), which assures the constant delivery of thioester intermediates to the discrete enzymes of FAS II. At the Center for Structural Genomics of Infectious Diseases (CSGID), AcpSs from Staphylococcus aureus (AcpSSA), Vibrio cholerae (AcpSVC) and Bacillus anthracis (AcpSBA) have been structurally characterized in their apo, holo and product-bound forms, respectively. The structure of AcpSBA is emphasized because of the two 3′,5′-adenosine diphosphate (3′,5′-ADP) product molecules that are found in each of the three coenzyme A (CoA) binding sites of the trimeric protein. One 3′,5′-ADP is bound as the 3′,5′-ADP part of CoA in the known structures of the CoA–AcpS and 3′,5′-ADP–AcpS binary complexes. The position of the second 3′,5′-ADP has never been described before. It is in close proximity to the first 3′,5′-­ADP and the ACP-binding site. The coordination of two ADPs in AcpSBA may possibly be exploited for the design of AcpS inhibitors that can block binding of both CoA and ACP.
doi:10.1107/S0907444912029101
PMCID: PMC3447402  PMID: 22993090
acyl-carrier-protein synthase; acyl carrier protein; type II fatty-acid synthesis; inhibition; 3′,5′-adenosine diphosphate; coenzyme A
5.  A Conserved Surface Loop in Type I Dehydroquinate Dehydratases Positions an Active Site Arginine and Functions in Substrate Binding† 
Biochemistry  2011;50(12):2357-2363.
Dehydroquinate dehydratase (DHQD) catalyzes the third step in the biosynthetic shikimate pathway. We present three crystal structures of the Salmonella enterica type I DHQD which address the functionality of a surface loop that is observed to close over the active site following substrate binding. Two wild type structures with differing loop conformations and kinetic and structural studies of a mutant provide evidence of both direct and indirect mechanisms of loop involvement in substrate binding. In addition to allowing amino acid side chains to establish a direct interaction with the substrate, loop closure necessitates a conformational change of a key active site arginine, which in turn positions the substrate productively. The absence of DHQD in humans and its essentiality in many pathogenic bacteria makes the enzyme a target for the development of nontoxic antimicrobials. The structures and ligand binding insights presented here may inform the design of novel type I DHQD inhibiting molecules.
doi:10.1021/bi102020s
PMCID: PMC3062685  PMID: 21291284
6.  Characterization of the Deoxynucleotide Triphosphate Triphosphohydrolase (dNTPase) Activity of the EF1143 Protein from Enterococcus faecalis and Crystal Structure of the Activator-Substrate Complex* 
The Journal of Biological Chemistry  2011;286(38):33158-33166.
The EF1143 protein from Enterococcus faecalis is a distant homolog of deoxynucleotide triphosphate triphosphohydrolases (dNTPases) from Escherichia coli and Thermus thermophilus. These dNTPases are important components in the regulation of the dNTP pool in bacteria. Biochemical assays of the EF1143 dNTPase activity demonstrated nonspecific hydrolysis of all canonical dNTPs in the presence of Mn2+. In contrast, with Mg2+ hydrolysis required the presence of dGTP as an effector, activating the degradation of dATP and dCTP with dGTP also being consumed in the reaction with dATP. The crystal structure of EF1143 and dynamic light scattering measurements in solution revealed a tetrameric oligomer as the most probable biologically active unit. The tetramer contains four dGTP specific allosteric regulatory sites and four active sites. Examination of the active site with the dATP substrate suggests an in-line nucleophilic attack on the α-phosphate center as a possible mechanism of the hydrolysis and two highly conserved residues, His-129 and Glu-122, as an acid-base catalytic dyad. Structural differences between EF1143 apo and holo forms revealed mobility of the α3 helix that can regulate the size of the active site binding pocket and could be stabilized in the open conformation upon formation of the tetramer and dGTP effector binding.
doi:10.1074/jbc.M111.250456
PMCID: PMC3190883  PMID: 21757692
Allosteric Regulation; Crystal Structure; Enzyme Catalysis; Enzyme Structure; Metalloenzymes; Nucleoside Nucleotide Metabolism; Phosphodiesterases; Deoxynucleotide Triphosphate Triphosphohydrolase

Results 1-6 (6)