title: Structure-function relationships of the outer membrane translocon Wza investigated by cryo-electron microscopy and mutagenesis.
abstract: The outer membrane protein Wza, from Escherichia coli K30, forms an octameric complex that is essential for capsular polysaccharide export. Homologs of Wza are widespread in gram-negative bacterial pathogens where capsules are critical virulence determinants. Wza is unusual in that it spans the outer membrane using a barrel composed of amphipathic alpha-helices, rather than being a beta-barrel like almost all other outer membrane channels. The transmembrane helical barrel of Wza also forms the external opening to a hydrophilic translocation pathway that spans the periplasm. Here, we have probed the structure and function of the Wza complex using both cryo-electron microscopy and mutagenesis. The helical barrel structure is stable in detergent micelles under mildly acidic conditions but is destabilized at basic pH, although the overall quaternary structure is retained. Truncation of the C-terminal region that forms the helical barrel by 4 residues has no effect on the ability of Wza to oligomerize and support capsule export, but larger truncations of 18, 24 or 35 amino acids abolish its function. The bulk of the C-terminal domain is essential for the stability and assembly of the E. coli Wza complex.
authors.affiliation: Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, University of Manchester, Manchester M1 7DN, UK. bob.ford@manchester.ac.uk
authors.forename: Robert C
authors.initials: RC
authors.lastname: Ford
authors.forename: Anne L
authors.initials: AL
authors.lastname: Brunkan-LaMontagne
authors.forename: Richard F
authors.initials: RF
authors.lastname: Collins
authors.forename: Bradley R
authors.initials: BR
authors.lastname: Clarke
authors.forename: Robert
authors.initials: R
authors.lastname: Harris
authors.forename: James H
authors.initials: JH
authors.lastname: Naismith
authors.forename: Chris
authors.initials: C
authors.lastname: Whitfield
mesh_terms: D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D015153:Blotting, Western; D002942:Circular Dichroism; D020285:Cryoelectron Microscopy / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011135:Polysaccharides, Bacterial / Q000737:chemistry; D055550:Protein Stability; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
chemical_list: D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D011135:Polysaccharides, Bacterial; C404158:Wza protein, E coli