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|Title:||Coiled-coil regions play 4 role in the function of the Shigella flexneri O-antigen chain length regulator Wzz(pHS2)|
Van Den Bosch, L.
|Citation:||Microbiology, 2008; 154(Part 4):1104-1116|
|Publisher:||Soc General Microbiology|
|Leanne Purins, Luisa Van Den Bosch, Vanessa Richardson and Renato Morona|
|Abstract:||Regulation of the length of the O-antigen (Oag) chain attached to LPS in Shigella flexneri is important for virulence and is dependent on the inner-membrane protein Wzz. A lack of high-resolution structural data for Wzz has hampered efforts so far to correlate mutations affecting function of Wzz with structure and describe a mechanism for chain length regulation. Here we have used secondary structure prediction to show that the periplasmic domain of the Wzz(pHS2) protein has three regions of significant coiled-coil (CC) potential, two of which lie within an extended helical region. We describe here the first site-directed mutagenesis study to investigate the role of individual predicted CC regions (CCRs) in Wzz function and oligomerization. We found that CCRs 2 and 3 are necessary for wild-type Oag chain length regulation by Wzz(pHS2). The in vivo cross-linking profile of mutants affected in the three CCRs was not altered, indicating that individually each CCR is not required for oligomerization. Interestingly, the CCR3 mutation resulted in a temperature-sensitive phenotype and an inhibitory effect on Oag polymerization. Analysis of Wzz(pHS2) and the mutant constructs in a S. flexneri degP mutant showed that DegP did not affect the function of wild-type Wzz(pHS2) but its presence influenced the phenotype of the Wzz(pHS2) CCR3 mutant. Additionally, the phenotype of the Wzz(pHS2) CCR3 mutant was suppressed by a cis mutation near the putative cytoplasmic C-terminus of Wzz(pHS2).|
|Keywords:||Shigella flexneri; Serine Endopeptidases; O Antigens; Periplasmic Proteins; Bacterial Proteins; Heat-Shock Proteins; Mutagenesis, Site-Directed; Gene Deletion; Suppression, Genetic; Amino Acid Sequence; Base Sequence; Protein Structure, Secondary; Molecular Sequence Data; Hot Temperature|
|Appears in Collections:||Molecular and Biomedical Science publications|
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