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

Abstract

SummaryCapsular 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 2RX2R 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.

The UDP-glucose Dehydrogenase of Escherichia coli K-12 Displays Substrate Inhibition by NAD That Is Relieved by Nucleotide Triphosphates

“…The second BY-kinase, Etk, is encoded at an unlinked locus that is not expressed in all isolates (25) but can contribute to the formation of group 1 CPSs (26). Etk and Wzc are structural homologs (27,28).…”

Tyrosine Phosphorylation and Dephosphorylation in Burkholderia cenocepacia Affect Biofilm Formation, Growth under Nutritional Deprivation, and Pathogenicity

“…1B). These changes could affect the functionality of BCAM1331, as demonstrated in E. coli, where the replacement of similar residues severely reduces Wzc kinase activity and the production of colonic acid (4,56). As an attempt to explain the biofilm complementation results in the ⌬2BYK strain with either pbceF or pM1331 (Fig.…”

Wzy-Dependent Bacterial Capsules as Potential Drug Targets

“…In Gram-negative bacteria, the BY-kinase is expressed as a single membrane-spanning polypeptide, also known as a PCP 2a protein [68], with the C-terminal kinase domain on the cytoplasmic side. Examples of this are Etk and Wzc in E. coli [76,77], but homologous proteins are also evident in other Gram-negative pathogens such as K. pneumoniae [78]. In Gram-positive bacteria, this protein is split into two such as in S. pneumoniae with the membrane spanning CpsC and cytoplasmic CpsD.…”

“…The phosphoregulatory system in particular seems to be well suited for this, given the many studies that have shown its central role in CPS production, and its presence across a wide range of bacterial pathogens [72][73][74]95]. Of the two processes, phosphorylation and dephosphorylation, the effects of BY-kinases are the better studied and have been the focus of recent reviews [66,77]. The success of the last decade in targeting human kinases has made bacterial kinases tractable as drug targets as well.…”