title: Structural and Biochemical Analysis of a Single Amino-Acid Mutant of WzzBSF That Alters Lipopolysaccharide O-Antigen Chain Length in Shigella flexneri.
abstract: Lipopolysaccharide (LPS), a surface polymer of Gram-negative bacteria, helps bacteria survive in different environments and acts as a virulence determinant of host infection. The O-antigen (Oag) component of LPS exhibits a modal chain-length distribution that is controlled by polysaccharide co-polymerases (PCPs). The molecular basis of the regulation of Oag chain-lengths remains unclear, despite extensive mutagenesis and structural studies of PCPs from Escherichia coli and Shigella. Here, we identified a single mutation (A107P) of the Shigella flexneri WzzBSF, by a random mutagenesis approach, that causes a shortened Oag chain-length distribution in bacteria. We determined the crystal structures of the periplasmic domains of wild-type WzzBSF and the A107P mutant. Both structures form a highly similar open trimeric assembly in the crystals, and show a similar tendency to self-associate in solution. Binding studies by bio-layer interferometry reveal cooperative binding of very short (VS)-core-plus-O-antigen polysaccharide (COPS) to the periplasmic domains of both proteins, but with decreased affinity for the A107P mutant. Our studies reveal that subtle and localized structural differences in PCPs can have dramatic effects on LPS chain-length distribution in bacteria, for example by altering the affinity for the substrate, which supports the role of the structure of the growing Oag polymer in this process.
authors.affiliation: School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane Qld 4072, Australia; Institute for Molecular Bioscience, University of Queensland, Brisbane Qld 4072, Australia; Australian Infectious Disease Research Centre, University of Queensland, Brisbane Qld 4072, Australia.
authors.forename: Chiung-Wen
authors.initials: CW
authors.lastname: Chang
authors.affiliation: School of Biological Sciences, Department of Molecular and Cellular Biology, University of Adelaide, Adelaide 5005, Australia.
authors.forename: Elizabeth N H
authors.initials: EN
authors.lastname: Tran
authors.affiliation: School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane Qld 4072, Australia; Institute for Molecular Bioscience, University of Queensland, Brisbane Qld 4072, Australia; Australian Infectious Disease Research Centre, University of Queensland, Brisbane Qld 4072, Australia.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Ericsson
authors.affiliation: School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane Qld 4072, Australia; Institute for Molecular Bioscience, University of Queensland, Brisbane Qld 4072, Australia; Australian Infectious Disease Research Centre, University of Queensland, Brisbane Qld 4072, Australia.
authors.forename: Lachlan W
authors.initials: LW
authors.lastname: Casey
authors.affiliation: School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane Qld 4072, Australia.
authors.forename: Thierry
authors.initials: T
authors.lastname: Lonhienne
authors.affiliation: School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane Qld 4072, Australia; Institute for Molecular Bioscience, University of Queensland, Brisbane Qld 4072, Australia; Australian Infectious Disease Research Centre, University of Queensland, Brisbane Qld 4072, Australia.
authors.forename: Friederike
authors.initials: F
authors.lastname: Benning
authors.affiliation: School of Biological Sciences, Department of Molecular and Cellular Biology, University of Adelaide, Adelaide 5005, Australia.
authors.forename: Renato
authors.initials: R
authors.lastname: Morona
authors.affiliation: School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane Qld 4072, Australia; Institute for Molecular Bioscience, University of Queensland, Brisbane Qld 4072, Australia; Australian Infectious Disease Research Centre, University of Queensland, Brisbane Qld 4072, Australia.
authors.forename: Bostjan
authors.initials: B
authors.lastname: Kobe
mesh_terms: D000596:Amino Acids / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D018360:Crystallography, X-Ray / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D008070:Lipopolysaccharides / Q000235:genetics*; D016296:Mutagenesis / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D019081:O Antigens / Q000235:genetics*; D017434:Protein Structure, Tertiary / Q000235:genetics; D012763:Shigella flexneri / Q000235:genetics*
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008070:Lipopolysaccharides; D019081:O Antigens; C075969:rol protein, Bacteria