Quaternary structure of WzzB and WzzE polysaccharide copolymerases

Bacteria have evolved cellular control mechanisms to ensure proper length specification for surface‐bound polysaccharides. Members of the Polysaccharide Copolymerase (PCP) family are central to this process. PCP‐1 family members are anchored to the inner membrane through two transmembrane helices and contain a large periplasm‐exposed domain. PCPs are known to form homooligomers but their exact stoichiometry is controversial in view of conflicting structural and biochemical data. Several prior investigations addressing this question indicated a nonameric, hexameric, or tetrameric organization of several PCP‐1 family members. In this work, we gathered additional evidence that E.coli WzzB and WzzE PCPs form octameric homo‐oligomeric complexes. Detergent‐solubilized PCPs were purified to homogeneity and subjected to blue native gel analysis, which indicated the presence of a predominant high‐molecular product of over 500 kDa in mass. Molecular mass of WzzE and WzzB‐detergent oligomers was estimated to be 550 kDA by size‐exclusion coupled to multiangle laser light scattering (SEC‐MALLS). Oligomeric organization of purified WzzB and WzzE was further investigated by negative stain electron microscopy and by X‐ray crystallography, respectively. Analysis of EM‐derived molecular envelope of WzzB indicated that the full‐length protein is composed of eight protomers. Crystal structure of LDAO‐solubilized WzzE was solved to 6 Å resolutions and revealed its octameric subunit stoichiometry. In summary, we identified a possible biological unit utilized for the glycan chain length determination by two PCP‐1 family members. This provides an important step toward further unraveling of the mechanistic basis of chain length control of the O‐antigen and the enterobacterial common antigen.