| Abstract | The O-antigen component of the lipopolysaccharide (LPS) represents a population of polysaccharide molecules with a non-random (modal) chain length distribution. The number of the repeat O-units in each individual O-antigen polymer depends on the Wzz chain-length regulator, an inner membrane protein belonging to the polysaccharide co-polymerase (PCPs) family. Different Wzz proteins confer vastly different ranges of modal lengths (4 to >100 repeat units) despite having remarkably conserved structural folds. The molecular mechanism responsible for the selective preference for a certain number of O-units is unknown. Guided by the three-dimensional structures of PCPs we constructed a panel of chimeric molecules containing parts of two closely related Wzz proteins from Salmonella enterica and Shigella flexneri, which confer different O-antigen chain length distribution. Analysis of the O-antigen length distribution imparted by each chimera revealed regions spanning amino acids 67-95, 200-255 and 269-274 as primarily affecting the length distribution. We also showed that there is no synergy between these regions. In particular, the region 269-274 also influenced chain length distribution mediated by two distantly related PCPs, WzzB and FepE. Furthermore, from the 3 regions uncovered in this study, region 269-274 appeared to be critical for the stability of the oligomeric form of Wzz, as determined by crosslinking experiments. Together, our data suggest that chain length determination depends on regions that likely contribute to stabilize a supramolecular complex. |
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