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.

Oligosaccharyltransferase (OST) is a membrane-integral enzyme that catalyzes the transfer of glycans from lipid-linked oligosaccharides (LLOs) onto asparagine side chains, the first step in protein N-glycosylation. Here, we report the X-ray structure of a single-subunit OST, PglB from Campylobacter lari, trapped in an intermediate state bound to an acceptor peptide and a synthetic LLO analog. The structure reveals the role of the external loop EL5, present in all OST enzymes, in substrate recognition. Whereas the N-terminal half of EL5 binds LLO, the C-terminal half interacts with the acceptor peptide. The glycan moiety of LLO must thread under EL5 to access the active site. Reducing EL5 mobility decreases the catalytic rate of OST when full-size heptasaccharide LLO is provided, but not for a monosaccharide-containing LLO analog. Our results define the chemistry of a ternary complex state, assign functional roles to conserved OST motifs, and provide opportunities for glycoengineering by rational design of PglB.

The initial transfer of a complex glycan in protein N-glycosylation is catalyzed by oligosaccharyltransferase (OST), which is generally a multisubunit membrane protein complex in the endoplasmic reticulum but a single-subunit enzyme (ssOST) in some protists. To investigate the reaction mechanism of ssOST, we recombinantly expressed, purified and characterized the STT3A protein from Trypanosoma brucei (TbSTT3A). We analyzed the in vitro activity of TbSTT3A by synthesizing fluorescently labeled acceptor peptides as well as lipid-linked oligosaccharide (LLO) analogs containing a chitobiose moiety coupled to oligoprenyl carriers of distinct lengths (C10, C15, C20 and C25) and with different double bond stereochemistry. We found that in addition to proline, charged residues at the +1 position of the sequon inhibited glycan transfer. An acidic residue at the −2 position significantly increased catalytic turnover but was not essential, in contrast to the bacterial OST. While all synthetic LLO analogs were processed by TbSTT3A, the length of the polyprenyl tail, but not the stereochemistry of the double bonds, determined their apparent affinity. We also synthesized phosphonate analogs of the LLOs, which were found to be competitive inhibitors of the reaction, although with lower apparent affinity to TbSTT3A than the active pyrophosphate analogs.

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The membrane-associated, processive and retaining glycosyltransferase PglH from Campylobacter jejuni is part of the biosynthetic pathway of the lipid-linked oligosaccharide (LLO) that serves as the glycan donor in bacterial protein N-glycosylation. Using an unknown counting mechanism, PglH catalyzes the transfer of exactly three α1,4 N-acetylgalactosamine (GalNAc) units to the growing LLO precursor, GalNAc-α1,4-GalNAc-α1,3-Bac-α1-PP-undecaprenyl. Here, we present crystal structures of PglH in three distinct states, including a binary complex with UDP-GalNAc and two ternary complexes containing a chemo-enzymatically generated LLO analog and either UDP or synthetic, nonhydrolyzable UDP-CH2-GalNAc. PglH contains an amphipathic helix (“ruler helix”) that has a dual role of facilitating membrane attachment and glycan counting. The ruler helix contains three positively charged side chains that can bind the pyrophosphate group of the LLO substrate and thus limit the addition of GalNAc units to three. These results, combined with molecular dynamics simulations, provide the mechanism of glycan counting by PglH.