Mapping the architecture of the initiating phosphoglycosyl transferase from S. enterica O-antigen biosynthesis in a liponanoparticle.

Bacterial cell surface glycoconjugates are critical for cell survival and for interactions between bacteria and their hosts. Consequently, the pathways responsible for their biosynthesis have untapped potential as therapeutic targets. The localization of many glycoconjugate biosynthesis enzymes to the membrane represents a significant challenge for expressing, purifying, and characterizing these enzymes. Here, we leverage cutting-edge methods to stabilize, purify, and structurally characterize WbaP, a phosphoglycosyl transferase (PGT) from Salmonella enterica (LT2) O-antigen biosynthesis without extraction from the lipid bilayer. Results from these experiments establish WbaP as a homodimer, reveal the structural elements responsible for oligomerization, shed light on the role of a domain of unknown function embedded within WbaP, and identify conserved structural motifs between PGTs and functionally unrelated UDP-sugar dehydratases. The approach developed here is generalizable and provides a toolkit for studying small membrane proteins embedded in liponanoparticles beyond PGTs.