Structure and Mechanism of an ATP-Driven Flippase of Lipid-Linked Oligosaccharides

Abstract

The flipping of lipid-linked oligosaccharides (LLOs) that serve as glycan donors in N-linked protein glycosylation is slow and energetically unfavorable and therefore catalyzed by flippases. This is an essential step during protein N-glycosylation both at the endoplasmic reticulum and similarly at the membrane of bacteria like Campylobacter jejuni. Whereas the identity and potential energy source of the relevant flippase in eukaryotes remains under debate, LLO flipping in C. jejuni is catalyzed by the ABC transporter PglK. We propose a mechanism of PglK-catalyzed LLO flipping based on crystal structures in distinct states, a newly devised in vitro flipping assay, and in vivo studies. We conclude that although PglK can adopt inward- and outward-facing conformations in vitro, only outward-facing states are required for flipping. Whereas the pyrophosphate-oligosaccharide head group of LLO enters the translocation cavity and interacts with positively charged side chains, the lipidic polyprenyl tail binds and activates the transporter but remains exposed to the lipid bilayer during the reaction. Our results provide a framework for understanding lipid-linked oligosaccharide flipping. The proposed mechanism is distinct from the classical alternating-access model applied to other transporters.