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 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.
Bechet
Emmanuelle
E
Institut de Biologie et Chimie des Protéines, CNRS, Université Lyon 1, Université de Lyon, 69367 Lyon, France.
Gruszczyk
Jakub
J
Terreux
Raphaël
R
Gueguen-Chaignon
Virginie
V
Vigouroux
Armelle
A
Obadia
Brice
B
Cozzone
Alain J
AJ
Nessler
Sylvie
S
Grangeasse
Christophe
C
D000244:Adenosine Diphosphate / Q000737:chemistry*; D020816:Amino Acid Motifs / Q000235:genetics; D018360:Crystallography, X-Ray; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D011135:Polysaccharides, Bacterial / Q000378:metabolism*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D011135:Polysaccharides, Bacterial; D014443:Tyrosine; D000244:Adenosine Diphosphate; D011505:Protein-Tyrosine Kinases; C499615:wzc protein, E coli