Molecular basis of lipid-linked oligosaccharide recognition and processing by bacterial oligosaccharyltransferase

Abstract

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.

Structure of the yeast oligosaccharyltransferase complex gives insight into eukaryotic N-glycosylation

“…Subcomplex II includes the catalytic subunit STT3, which is homologous to the functionally and structurally related ssOST enzymes PglB from Campylobacter lari and AglB from Archaeoglobus fulgidus (8,9,18). Our structure confirms that yeast STT3 contains a similar TM topology with 13 TM helices ( Fig.…”

The atomic structure of a eukaryotic oligosaccharyltransferase complex

“…2b, 5c), was previously shown to be lethal when mutated to alanine in Stt3 40 . The corresponding W215A mutation in AglB reduced the activity 11 , and the PglB equivalent Y196 directly interact with LLO 12 . The lower half of this groove is lined with numerous hydrophobic residues and is occupied by phospholipid PL8 in our structure (Fig.…”

“…The EL5 loop of PglB is disordered in the absence of the donor, but becomes ordered when both donor and acceptor substrates are present 12,13,41 (Fig. 5d).…”

“…5d). It was proposed that EL5 disordering allows the donor to diffuse under it into the catalytic site 12 . Eukaryotic LLO is much larger, both in the dolichol-binding transmembrane region and the OS-binding lumenal region: the yeast dolichol contains 14–18 repeating isoprene units, twice that of bacterial LLO 42 , and the yeast OS contains 14 sugars compared with the 7-sugar bacterial OS.…”

Cryo-EM Structures and Regulation of Arabinofuranosyltransferase AftD from Mycobacteria

“…,terminal 22 arabinofuranose(s) of arabinogalactan/lipoarabinomannan, or the divalent cation likely required for 23 catalysis(Sharma et al, 1981). The map shows poorly resolved densities for the flexible EL4 loop 24 around this site for apo-AftD, and we expect this region to become ordered to assist in substrate 1 binding, as shown to occur in PglB and ArnT(Napiórkowska et al, 2017; Petrou et al, 2016). N-2 EL4 should become ordered to trap DPA (State 2' inFigure 7), being closest to the putative DPA 3 binding site (Figures S5B, S5D, S5E and S5F).…”