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

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 EX₂RX₂R 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.