Periplasmic protein-protein contacts in the inner membrane protein Wzc form a tetrameric complex required for the assembly of Escherichia coli group 1 capsules

R F Collins, K Beis, B R Clarke, R C Ford, M Hulley, J H Naismith, C Whitfield

Research output: Contribution to journalArticlepeer-review

59 Citations (Scopus)

Abstract

The K antigenic capsular polysaccharide forms a structural layer, the capsule, on the surfaces of Escherichia coli cells. The capsule provides an important protective covering that helps protect encapsulated bacteria from host immune defenses. The assembly and translocation of the capsule requires proteins in the inner and outer membranes. The inner membrane protein Wzc is a tyrosine autokinase that plays an essential role in what is believed to be a coordinated biosynthesis and secretion process. Mutants lacking Wzc can form K antigen oligosaccharides but are unable to polymerize high molecular weight capsular polymers. Wzc homologs have been identified in exopolymer biosynthesis systems in many different Gram-negative and -positive bacteria. Using single particle averaging on cryo-negatively stained samples, we have produced the first three-dimensional structure of this type of membrane protein in its phosphorylated state at similar to 14 angstrom resolution. Perfluoro-octanoate-PAGE analysis of detergent-solubilized oligomeric Wzc and symmetry analysis of the transmission electron microscopy data clearly demonstrated that Wzc forms a tetrameric complex with C4 rotational symmetry. Viewed from the top of the complex, the oligomer is square with a diameter of similar to 100 angstrom and can be divided into four separate densities. From the side, Wzc is similar to 110 angstrom high and has a distinctive appearance similar to an extracted molar tooth. The upper "crown" region is similar to 55 angstrom high and forms a continuous ring of density. Four unconnected "roots" (similar to 65 angstrom high) emerge from the underside of the crown. We propose that the crown is formed by protein-protein contacts from the four Wzc periplasmic domains, while each root represents an individual cytoplasmic tyrosine autokinase domain.

Original languageEnglish
Pages (from-to)2144-2150
Number of pages7
JournalJournal of Biological Chemistry
Volume281
DOIs
Publication statusPublished - 27 Jan 2006

Keywords

  • OUTER-MEMBRANE
  • TYROSINE AUTOKINASE
  • PSEUDOMONAS-AERUGINOSA
  • ELECTRON-MICROSCOPY
  • FUNCTIONAL-ANALYSIS
  • VIBRIO-CHOLERAE
  • II SECRETION
  • POLYSACCHARIDE
  • TRANSLOCATION
  • EXPRESSION

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