The outer membrane of Gram-negative bacteria has an external leaflet that is predominantly composed of the essential endotoxin lipopolysaccharide, which provides a selective and protective permeation barrier, against antimicrobials in particular. Lipopolysaccharide is a glycolipid with three constituents, lipid A, which provides the lipidic anchor to the membrane, the core oligosaccharide, and an extended, species-dependent variable polysaccharide known as the Oantigen. The final and crucial step in the biosynthesis of lipopolysaccharide is the addition of the O-antigen to the lipid A core oligosaccharide, via a glycosyltransferase reaction catalyzed by the O-antigen ligase WaaL, a 44kDa integral membrane enzyme.
We have used single-particle cryo-electron microscopy combined with fragment antigen-binding technology to determine the structure of WaaL from Cupriavidus metallidurans, both in the apo state and in complex with its lipid carrier undecaprenyl pyrophosphate. The structure reveals that
WaaL comprises 12 transmembrane helices and a predominantly α-helical periplasmic region, which we show contains many of the conserved residues that are required for catalysis. We observe a conserved fold within the GT-C family of glycosyltransferases and hypothesize that they have a
common mechanism for shuttling the undecaprenyl-based carrier to and from the active site. Our structures, combined with genetic, biochemical, bioinformatics and molecular dynamics simulation experiments, provide molecular details on how the ligands come in apposition, and allows us to propose a mechanism for catalysis. Together, our work offers a structural basis for lipopolysaccharide biosynthesis by a member of the GT-C superfamily of glycosyltransferases.