Crystal Structures of SecYEG in Lipidic Cubic Phase Elucidate a Precise Resting and a Peptide-Bound State

Takaharu Mori,Yasunori Sugano,Yoshiki Tanaka,Tomoya Tsukazaki,Arata Furukawa,Yuji Sugita,Mizuki Takemoto,Kaoru Kumazaki,Osamu Nureki,Tsukasa Kusakizako,Ayako Kashima,Ryuichiro Ishitani

doi:10.1016/j.celrep.2015.10.025

Abstract

The bacterial SecYEG translocon functions as a conserved protein-conducting channel. Conformational transitions of SecYEG allow protein translocation across the membrane without perturbation of membrane permeability. Here, we report the crystal structures of intact SecYEG at 2.7-Å resolution and of peptide-bound SecYEG at 3.6-Å resolution. The higher-resolution structure revealed that the cytoplasmic loop of SecG covers the hourglass-shaped channel, which was confirmed to also occur in the membrane by disulfide bond formation analysis and molecular dynamics simulation. The cytoplasmic loop may be involved in protein translocation. In addition, the previously unknown peptide-bound crystal structure of SecYEG implies that interactions between the cytoplasmic side of SecY and signal peptides are related to lateral gate opening at the first step of protein translocation. These SecYEG structures therefore provide a number of structural insights into the Sec machinery for further study.

Highlights

More than 30% of newly synthesized proteins in the cell are transported via the Sec translocon, a process that is mediated by Sec factors (Deshaies et al, 1991; Gardel et al, 1990; Ito, 1984; Nishiyama et al, 1994; Oliver and Beckwith, 1981; Rapoport, 2007; Riggs et al, 1988)
The previously unknown peptide-bound crystal structure of SecYEG implies that interactions between the cytoplasmic side of SecY and signal peptides are related to lateral gate opening at the first step of protein translocation
A signal peptide is proposed to bind to the cytoplasmic side of the lateral gate at the first step, which triggers the opening of the lateral gate, a detailed view of this interaction remains unclear

Summary

Graphical Abstract

The Sec translocon is an essential protein-conducting channel composed of SecY/E/G in bacteria and Sec61a/g/b in eukaryotes. Tanaka et al solve highresolution and peptide-bound SecYEG structures in a lipid environment, providing notable insights into the cytoplasmic side of the Sec translocon. Highlights d Crystal structures of full-length and peptide-bound SecYEG were determined.

SUMMARY

INTRODUCTION

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EXPERIMENTAL PROCEDURES