title: Residues located inside the Escherichia coli FepE protein oligomer are essential for lipopolysaccharide O-antigen modal chain length regulation.
abstract: The Escherichia coli O157 : H7 FepE protein regulates lipopolysaccharide (LPS) O-antigen (Oag) chain length to confer a very long modal chain length of >80 Oag repeat units (RUs). The mechanism by which FepE regulates Oag modal chain length and the regions within it that are important for its function remain unclear. Studies on the structure of FepE show that the protein oligomerizes. However, the exact size of the oligomer is in dispute, further hampering our understanding of its mechanism. Guided by information previously obtained for regions known to be important for Oag modal chain length determination in the homologous Shigella flexneri WzzBSF protein, a set of FepE mutant constructs with single amino acid substitutions was created. Analysis of the resulting LPS conferred by these mutant His6-FepE proteins showed that amino acid substitutions of leucine 168 (L168) and aspartic acid 268 (D268) resulted in LPS with consistently shortened Oag chain lengths of <80 Oag RUs. Substitution of FepE's transmembrane cysteine residues did not affect function. Chemical cross-linking experiments on mutant FepE proteins showed no consistent correlation between oligomer size and functional activity, and MS analysis of FepE oligomers indicated that the in vivo size of FepE is consistent with a maximum size of a hexamer. Our findings suggest that different FepE residues, mainly located within the internal cavity of the oligomer, contribute to Oag modal chain length determination but not the oligomeric state of the protein.
authors: Discipline of Microbiology and Immunology, School of Molecular and Biomedical Science, University of Adelaide, Adelaide 5005, Australia.
authors: Elizabeth Ngoc Hoa
authors: ENH
authors: Tran
authors: Discipline of Microbiology and Immunology, School of Molecular and Biomedical Science, University of Adelaide, Adelaide 5005, Australia.
authors: Renato
authors: R
authors: Morona
mesh_terms: D019453:Escherichia coli O157 / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D008070:Lipopolysaccharides / Q000737:chemistry / Q000378:metabolism; D019081:O Antigens / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization; D017433:Protein Structure, Secondary
chemical_list: D029968:Escherichia coli Proteins; D008070:Lipopolysaccharides; D019081:O Antigens