X-ray structure of a bacterial oligosaccharyltransferase

Abstract

Asparagine-linked glycosylation is a post-translational modification of proteins containing the conserved sequence motif Asn-X-Ser/Thr. The attachment of oligosaccharides is implicated in diverse processes such as protein folding and quality control, organism development or host-pathogen interactions. The reaction is catalysed by oligosaccharyltransferase (OST), a membrane protein complex located in the endoplasmic reticulum. The central, catalytic enzyme of OST is the STT3 subunit, which has homologues in bacteria and archaea. Here we report the X-ray structure of a bacterial OST, the PglB protein of Campylobacter lari, in complex with an acceptor peptide. The structure defines the fold of STT3 proteins and provides insight into glycosylation sequon recognition and amide nitrogen activation, both of which are prerequisites for the formation of the N-glycosidic linkage. We also identified and validated catalytically important, acidic amino acid residues. Our results provide the molecular basis for understanding the mechanism of N-linked glycosylation.

The atomic structure of a eukaryotic oligosaccharyltransferase complex

“…The 13-TMH topology is consistent with a recent characterization of yeast and mouse Stt3 and is similar to the prokaryotic homologues 13,27 . The Stt3 lumenal domain is a mixed α/β fold, similar to the prokaryotic counterparts 10–13,28 , but it is different from the NMR structure of the yeast Stt3 that was derived from proteins refolded in the presence of the denaturing sodium dodecyl sulfate 29 . The structural conservation between Stt3 and prokaryotic enzymes is remarkable, considering their ~20% sequence identity.…”

“…The structures of the bacterial PglB and the archaeal AglB provided first insights into the glycosyl transfer reaction 10–13 . However, prokaryotic protein N-glycosylation is simpler and occurs post-translationally.…”

“…The bacterial enzymes have an extended acceptor sequon of DXNXS/T 30 . The −2 position D is stabilized by R331 in the PglB crystal structure 13 . PglB R331 is replaced by D362 in yeast Stt3, explaining the shorter eukaryotic sequon of NXS/T.…”

“…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).…”

Deglycosylation systematically improves N-glycoprotein identification in liquid chromatography–tandem mass spectrometry proteomics for analysis of cell wall stress responses in Saccharomyces cerevisiae lacking Alg3p

“…The mature Glu 3 Man 9 GlcNAc 2 glycan is efficiently transferred to asparagines in nascent polypeptides by the multiprotein enzyme complex oligosaccharyltransferase (OTase) [2,3]. OTase preferentially binds asparagines located in 'glycosylation sequons' (N-x-S/T; x≠P) at its peptide acceptor binding site, resulting in efficient modification of such sequences [4,5]. The presence of N-glycans on nascent polypeptides assists productive protein folding in the ER directly by increasing the solubility of polypeptides, and indirectly through lectin-chaperone folding and quality control systems [6].…”