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J. Biol. Chem., Vol. 281, Issue 4, 2144-2150, January 27, 2006

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Periplasmic Protein-Protein Contacts in the Inner Membrane Protein Wzc Form a Tetrameric Complex Required for the Assembly of Escherichia coli Group 1 Capsules^*

Richard F. Collins1, Konstantinos Beis, Bradley R. Clarke¶, Robert C. Ford, Martyn Hulley, James H. Naismith2, and Chris Whitfield, Recipient of a Canada Research Chair and financial support from the Canadian Institutes of Health Research¶3

From the Faculty of Life Science, University of Manchester, Manchester, M60 1QD, United Kingdom, Centre for Biomolecular Sciences, University of St. Andrews, North Haugh, St. Andrews, Fife KY16 9ST, United Kingdom, and ^¶Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada

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 14 Å 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 100 Å and can be divided into four separate densities. From the side, Wzc is 110 Å high and has a distinctive appearance similar to an extracted molar tooth. The upper "crown" region is 55 Å high and forms a continuous ring of density. Four unconnected "roots" (65 Å 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.

Received for publication, July 25, 2005 , and in revised form, September 15, 2005.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

² Recipient of a Biotechnology and Biological Sciences Research Council career development fellowship.

¹ To whom correspondence may be addressed. E-mail: Richard.Collins{at}manchester.ac.uk. ³ To whom correspondence may be addressed. E-mail: cwhitfie{at}uoguelph.ca.

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