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Mol Microbiol. 2010 Sep;77(5):1315-25. doi: 10.1111/j.1365-2958.2010.07291.x.

Identification of structural and molecular determinants of the tyrosine-kinase Wzc and implications in capsular polysaccharide export.

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1
Institut de Biologie et Chimie des Protéines, CNRS, Université Lyon 1, Université de Lyon, 69367 Lyon, France.

Abstract

Capsular polysaccharides are well-established virulence factors of pathogenic bacteria. Their biosynthesis and export are regulated within the transmembrane polysaccharide assembly machinery by the autophosphorylation of atypical tyrosine-kinases, named BY-kinases. However, the accurate functioning of these tyrosine-kinases remains unknown. Here, we report the crystal structure of the non-phosphorylated cytoplasmic domain of the tyrosine-kinase Wzc from Escherichia coli in complex with ADP showing that it forms a ring-shaped octamer. Mutational analysis demonstrates that a conserved EX(2) RX(2) R motif involved in subunit interactions is essential for polysaccharide export. We also elucidate the role of a putative internal regulatory tyrosine and we show that BY-kinases from proteobacteria autophosphorylate on their C-terminal tyrosine cluster via a single-step intermolecular mechanism. This structure-function analysis also allows us to demonstrate that two different parts of a conserved basic region called the RK-cluster are essential for polysaccharide export and for kinase activity respectively. Based on these data, we revisit the dichotomy made between BY-kinases from proteobacteria and firmicutes and we propose a unique process of oligomerization and phosphorylation. We also reassess the function of BY-kinases in the capsular polysaccharide assembly machinery.

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