Structural basis for the function of a minimembrane protein subunit of yeast oligosaccharyltransferase

Abstract

N-glycosylation of proteins is an essential, highly conserved modification reaction that occurs in all eukaryotes and some prokaryotes. This process is catalyzed by oligosaccharyltransferase (OT), a multisubunit enzyme localized in the endoplasmic reticulum. Complete loss of N-glycosylation is lethal in all organisms. In Saccharomyces cerevisiae, OT is composed of nine nonidentical membrane proteins. Here, we report the atomic structure of an OT subunit from S. cerevisiae, Ost4p. This unusually small membrane protein containing only 36 residues folds into a well formed, kinked helix in the model-membrane solvent system used in this study. The residues critical for the OT activity and the stability of Stt3p-Ost4p-Ost3p subcomplex are located in helix ␣2, the larger cytosolic half of this kinked helix. The residues known to disrupt Ost4p-Stt3p complex form a well defined ridge in the 3D structure. Taking together prior mutational studies and the NMR structure of Ost4p, we propose that in the OT complex Stt3p is packed against the ␣2-helix of Ost4p by using a ''ridges-into-grooves'' model, with Met-18, Leu-21, and Ile-24 as the packing interface on one face, whereas Ost3p is involved in interactions with Met-19, Thr-20, Ile-22, and Val-23 on the other face.

Oxidoreductase activity of oligosaccharyltransferase subunits Ost3p and Ost6p defines site-specific glycosylation efficiency

“…The high-resolution structures we present here are unique in representing soluble, biochemically active domains of the eukaryotic OTase. In combination with the previously reported structures of the small transmembrane protein Ost4p from yeast (40), the soluble C-terminal domain of archaeal Stt3p from Pyrococcus furiosus (41), and the cryo-EM structure of the yeast OTase complex (42), our results provide a significant step toward a unified structural understanding of the mechanisms controlling N-glycosylation activity and substrate selection by the eukaryotic OTase.…”

Unraveling the Mechanism of Protein N-Glycosylation

“…It was later shown by NMR structural analysis that this particular segment actually localizes in the second half of the transmembrane domain of Ost4p, the ␣2 helix (residues 15-28) ( Fig. 2) (22). The three-dimensional structure of Ost4p also revealed that this ␣2 helix is connected to another helix segment, the ␣1 helix formed by residues 10 -14, via a kink of 37°b etween residues Phe-14 and Gly-15.…”

The atomic structure of a eukaryotic oligosaccharyltransferase complex

“…Crystal structures of the Ost6 lumenal domain revealed a thioredoxin fold (TRX) 17,18 . The structures of Ost4 were solved by NMR 19,20 . Biochemical studies suggested that Ost1 and Wbp1 recognize acceptor and donor substrates, respectively 8,21,22 .…”