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Context-dependent protein folding of a virulence peptide in the bacterial and host environments: structure of an SycH-YopH chaperone-effector complex.

ABSTRACT: Yersinia pestis injects numerous bacterial proteins into host cells through an organic nanomachine called the type 3 secretion system. One such substrate is the tyrosine phosphatase YopH, which requires an interaction with a cognate chaperone in order to be effectively injected. Here, the first crystal structure of a SycH-YopH complex is reported, determined to 1.9 Å resolution. The structure reveals the presence of (i) a nonglobular polypeptide in YopH, (ii) a so-called ?-motif in YopH and (iii) a conserved hydrophobic patch in SycH that recognizes the ?-motif. Biochemical studies establish that the ?-motif is critical to the stability of this complex. Finally, since previous work has shown that the N-terminal portion of YopH adopts a globular fold that is functional in the host cell, aspects of how this polypeptide adopts radically different folds in the host and in the bacterial environments are analysed.

SUBMITTER: Vujanac M

PROVIDER: S-EPMC3606036 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Context-dependent protein folding of a virulence peptide in the bacterial and host environments: structure of an SycH-YopH chaperone-effector complex.

Vujanac Milos M Stebbins C Erec CE

Acta crystallographica. Section D, Biological crystallography 20130309 Pt 4

Yersinia pestis injects numerous bacterial proteins into host cells through an organic nanomachine called the type 3 secretion system. One such substrate is the tyrosine phosphatase YopH, which requires an interaction with a cognate chaperone in order to be effectively injected. Here, the first crystal structure of a SycH-YopH complex is reported, determined to 1.9 Å resolution. The structure reveals the presence of (i) a nonglobular polypeptide in YopH, (ii) a so-called β-motif in YopH and (iii ...[more]

PMID: 23519663

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Project description:Riemerella anatipestifer is a major pathogenic agent of duck septicemic and exudative diseases. Recent studies have shown that the R. anatipestifer type IX secretion system (T9SS) is a crucial factor in bacterial virulence. The AS87_RS04190 protein was obviously missing from the secreted proteins of the T9SS mutant strain Yb2ΔgldM. A bioinformatic analysis indicated that the AS87_RS04190 protein contains a T9SS C-terminal domain sequence and encodes a putative subtilisin-like serine protease (SspA). To determine the role of the putative SspA protein in R. anatipestifer pathogenesis and proteolysis, we constructed two strains with an sspA mutation and complementation, respectively, and determined their median lethal doses, their bacterial loads in infected duck blood, and their adherence to and invasion of cells. Our results demonstrate that the SspA protein functions in bacterial virulence. It is also associated with the bacterial protease activity and has a conserved catalytic triad structure (Asp126, His158, and Ser410), which is necessary for protein function. The optimal reactive pH and temperature were determined to be 7.0 and 50°C, respectively, and Km and Vmax were determined to be 10.15 mM and 246.96 U/mg, respectively. The enzymatic activity of SspA is activated by Ca2+, Mg2+, and Mn2+ and inhibited by Cu2+ and EDTA. SspA degrades gelatin, fibrinogen, and bacitracin LL-37. These results demonstrate that SspA is an effector protein of T9SS and functions in R. anatipestifer virulence and its proteolysis of gelatin, fibrinogen, and bacitracin LL-37. IMPORTANCE In recent years, Riemerella anatipestifer T9SS has been reported to act as a virulence factor. However, the functions of the proteins secreted by R. anatipestifer T9SS are not entirely clear. In this study, a secreted subtilisin-like serine protease SspA was shown to be associated with R. anatipestifer virulence, host complement evasion, and degradation of gelatin, fibrinogen, and LL-37. The enzymatic activity of recombinant SspA was determined, and its Km and Vmax were 10.15 mM and 246.96 U/mg, respectively. Three conserved sites (Asp126, His158, and Ser410) are necessary for the protein's function. The median lethal dose of the sspA-deleted mutant strain was reduced >10,000-fold, indicating that SspA is an important virulence factor. In summary, we demonstrate that the R. anatipestifer AS87_RS04190 gene encodes an important T9SS effector, SspA, which plays an important role in bacterial virulence.

Dataset Information

Context-dependent protein folding of a virulence peptide in the bacterial and host environments: structure of an SycH-YopH chaperone-effector complex.

Publications

Context-dependent protein folding of a virulence peptide in the bacterial and host environments: structure of an SycH-YopH chaperone-effector complex.

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