Tethering an N-glycosylation sequon-containing peptide creates a catalytically competent oligosaccharyltransferase complex

Shunsuke Matsumoto, Yuya Taguchi, Atsushi Shimada, Mayumi Igura, Daisuke Kohda

研究成果: Contribution to journalArticle査読

25 被引用数 (Scopus)

抄録

Oligosaccharyltransferase (OST) transfers an oligosaccharide chain to the Asn residue in the Asn-X-Ser/Thr sequon in proteins, where X is not proline. A sequon was tethered to an archaeal OST enzyme via a disulfide bond. The positions of the cysteine residues in the OST protein and the sequon-containing acceptor peptide were selected by reference to the eubacterial OST structure in a noncovalent complex with an acceptor peptide. We determined the crystal structure of the cross-linked OST - sequon complex. The Ser/Thr-binding pocket recognizes the Thr residue in the sequon, and the catalytic structure termed the "carboxylate dyad" interacted with the Asn residue. Thus, the recognition and the catalytic mechanism of the sequon are conserved between the archaeal and eubacterial OSTs. We found that the tethered peptides in the complex were efficiently glycosylated in the presence of the oligosaccharide donor. The stringent requirements are greatly relaxed in the cross-linked state. The two conserved acidic residues in the catalytic structure were each dispensable, although the double mutation abolished the activity. A Gln residue at the Asn position in the sequon functioned as an acceptor, and the hydroxy group at position +2 was not required. In the standard assay using short free peptides, strong amino acid preferences were observed at the X position, but the preferences, except for Pro, completely disappeared in the cross-linked state. By skipping the initial binding process and stabilizing the complex state, the catalytically competent cross-linked complex offers a unique system for studying the oligosaccharyl transfer reaction. (Figure Presented).

本文言語英語
ページ(範囲)602-611
ページ数10
ジャーナルBiochemistry
56
4
DOI
出版ステータス出版済み - 1 31 2017

All Science Journal Classification (ASJC) codes

  • 生化学

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