Fig. 7: The YxY motif regulates the activity of Wzy.

a Immunoblot (anti-K30 CPS) of whole cell lysates separated by SDS-PAGE. The wildtype E. coli serotype K30 strain (E69) was used as a positive control, and E. coli CWG285 (Δwzc) was used as a negative control. WT, WT_N711Y, 3YE, 3YE_N711Y represent plasmids encoding wild-type Wzc and derivatives with the N711Y, Y715E/Y717E/Y718E and Y715E/Y717E/Y718E/N711Y mutations, respectively. As CPS does not migrate strictly according to size, protein standard is not useful to indicate the molecular weight. These data are representative of three biological replicates. b Corresponding western immunoblot of purified Wzc proteins probed with anti-pTyr antibody (top) and anti-hexahistidine antibody (bottom). The experiment represents one of three technical replicates. c Western immunoblot of purified Wzc proteins using anti-pTyr antibody (top) and anti-hexahistidine antibody (bottom). The mutations are identified above each lane. The C-terminal sequences of the tyrosine-rich portion of YF_713Y, YF_N711Y and YF_713Y/N711Y are shown below. The data shows one of three technical replicates. d The ratchet mechanism. The YxY motif acts as a clamp stabilizing the octamer but also ordering the C-terminal tail (top). The tyrosine-rich tails in the octamer are phosphorylated and move unidirectionally through the active sites of adjacent monomers (top right). Phosphorylation progressively destabilizes the octamer which, after the fourth phosphorylation, disassembles (top right and bottom). Wzc regulates the polymerization activity of Wzy (purple) and is thought to form a complex with the translocon Wza (green). The cycling of the structural arrangement of the octamer is driven by (auto)phosphorylation and dephosphorylation (catalysed by Wzb) and the cycling is required for the production and export of CPS (bottom).