Structure and mechanism of an active lipid-linked oligosaccharide flippase

Abstract

The flipping of membrane-embedded lipids containing large, polar head groups is slow and energetically unfavourable, and is therefore catalysed by flippases, the mechanisms of which are unknown. A prominent example of a flipping reaction is the translocation of lipid-linked oligosaccharides that serve as donors in N-linked protein glycosylation. In Campylobacter jejuni, this process is catalysed by the ABC transporter PglK. Here we present a mechanism of PglK-catalysed lipid-linked oligosaccharide flipping based on crystal structures in distinct states, a newly devised in vitro flipping assay, and in vivo studies. PglK can adopt inward- and outward-facing conformations in vitro, but only outward-facing states are required for flipping. While the pyrophosphate-oligosaccharide head group of lipid-linked oligosaccharides 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. The proposed mechanism is distinct from the classical alternating-access model applied to other transporters.

Conformational dynamics of the ABC transporter McjD seen by single‐molecule FRET

“…The crystal structures of exporters display varying degrees of NBD disengagement in the absence of nucleotides (Ward et al , 2007; Lin et al , 2015; Perez et al , 2015; Bountra et al , 2017). These data were questioned as a detergent artifact mistaken for dynamic NBDs.…”

“…DEER data of ABC transporters in liposomes and bicelles show broad lines for apo NBDs (Dong et al , 2005; Borbat et al , 2007; Zou et al , 2009; Bountra et al , 2017), further suggesting that in the absence of nucleotides the NBDs can have different degrees of disengagement. Upon nucleotide binding, the NBDs dimerize and display nearly identical distances across different transporters (Ward et al , 2007; Lin et al , 2015; Perez et al , 2015; Bountra et al , 2017). Our smFRET data in detergent and liposomes revealed that the NBDs of McjD adopt very similar disengagement with E* values of 0.3 and larger E* values of 0.5 upon nucleotide binding.…”

“…All ABC transporters share a common architecture consisting of a transmembrane domain (TMD) for substrate recognition and transport, and a nucleotide‐binding domain (NBD) that converts the chemical energy of ATP into conformational changes for transport (Beis, 2015). The structures of several homodimeric (Dawson & Locher, 2006; Ward et al , 2007; Perez et al , 2015) and heterodimeric ABC transporters (Hohl et al , 2012; Noll et al , 2017) revealed distinct conformations and suggest, in combination with biophysical studies (e.g., EPR and NMR; Dong et al , 2005; Zou et al , 2009; Bountra et al , 2017; Timachi et al , 2017; Barth et al , 2018), that they undergo large conformational changes during transport. Their complex architecture is, however, a fundamental hurdle to fully understand the coupling between conformational changes, substrate binding, ATP binding and hydrolysis, and transport.…”

“…Based on the available structural, biophysical, and biochemical data for exporters, two transport mechanisms have been proposed, the alternating access mechanism (Dawson & Locher, 2006; Ward et al , 2007; Choudhury et al , 2014; Bountra et al , 2017) and the outward‐only mechanism (Perez et al , 2015). ABC exporters that use the alternating access mechanism switch between inward‐ and outward‐facing states with transmembrane helices intertwining, which exposes the ligand binding site alternatively to the inside or outside of the membrane, a process that is believed to be coupled to ATP binding to hydrolysis.…”

“…ABC exporters that use the alternating access mechanism switch between inward‐ and outward‐facing states with transmembrane helices intertwining, which exposes the ligand binding site alternatively to the inside or outside of the membrane, a process that is believed to be coupled to ATP binding to hydrolysis. The outward‐only mechanism was proposed in light of the structural and functional data of the lipid‐linked oligosaccharide flippase PglK from Campylobacter jejuni (Perez et al , 2015); here the inward‐facing conformation is not required for substrate translocation since it can be directly recruited from the membrane and ATP hydrolysis at the NBDs is transmitted to the TMD to drive substrate release.…”