Two-State Exchange Dynamics in Membrane-Embedded Oligosaccharyltransferase Observed in Real-Time by High-Speed AFM

Yuki Kawasaki, Hirotaka Ariyama, Hajime Motomura, Daisuke Fujinami, Daisuke Noshiro, Toshio Ando, Daisuke Kohda

研究成果: Contribution to journalArticle査読

抄録

Oligosaccharyltransferase (OST) is a membrane-bound enzyme that catalyzes the transfer of oligosaccharide chains from lipid-linked oligosaccharides (LLO) to asparagine residues in polypeptide chains. Using high-speed atomic force microscopy (AFM), we investigated the dynamic properties of OST molecules embedded in biomembranes. An archaeal single-subunit OST protein was immobilized on a mica support via biotin–avidin interactions and reconstituted in a lipid bilayer. The distance between the top of the protein molecule and the upper surface of the lipid bilayer was monitored in real-time. The height of the extramembranous part exhibited a two-step variation with a difference of 1.8 nm. The high and low states are designated as state 1 and state 2, respectively. The transition processes between the two states fit well to single exponential functions, suggesting that the observed dynamic exchange is an intrinsic property of the archaeal OST protein. The two sets of cross peaks in the NMR spectra of the protein supported the conformational changes between the two states in detergent-solubilized conditions. Considering the height values measured in the AFM measurements, state 1 is closer to the crystal structure, and state 2 has a more compact form. Subsequent AFM experiments indicated that the binding of the sugar donor LLO decreased the structural fluctuation and shifted the equilibrium almost completely to state 1. This dynamic behavior is likely necessary for efficient catalytic turnover. Presumably, state 2 facilitates the immediate release of the bulky glycosylated polypeptide product, thus allowing OST to quickly prepare for the next catalytic cycle.

本文言語英語
ページ(範囲)5951-5965
ページ数15
ジャーナルJournal of Molecular Biology
432
22
DOI
出版ステータス出版済み - 11 6 2020

All Science Journal Classification (ASJC) codes

  • 構造生物学
  • 分子生物学

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