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Structure of the SecY channel during initiation of protein translocation

Nature volume 506, pages 102–106 (2014)Cite this article

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Abstract

Many secretory proteins are targeted by signal sequences to a protein-conducting channel, formed by prokaryotic SecY or eukaryotic Sec61 complexes, and are translocated across the membrane during their synthesis1,2. Crystal structures of the inactive channel show that the SecY subunit of the heterotrimeric complex consists of two halves that form an hourglass-shaped pore with a constriction in the middle of the membrane and a lateral gate that faces the lipid phase3,4,5. The closed channel has an empty cytoplasmic funnel and an extracellular funnel that is filled with a small helical domain, called the plug. During initiation of translocation, a ribosome–nascent chain complex binds to the SecY (or Sec61) complex, resulting in insertion of the nascent chain. However, the mechanism of channel opening during translocation is unclear. Here we have addressed this question by determining structures of inactive and active ribosome–channel complexes with cryo-electron microscopy. Non-translating ribosome–SecY channel complexes derived from Methanocaldococcus jannaschii or Escherichia coli show the channel in its closed state, and indicate that ribosome binding per se causes only minor changes. The structure of an active E. coli ribosome–channel complex demonstrates that the nascent chain opens the channel, causing mostly rigid body movements of the amino- and carboxy-terminal halves of SecY. In this early translocation intermediate, the polypeptide inserts as a loop into the SecY channel with the hydrophobic signal sequence intercalated into the open lateral gate. The nascent chain also forms a loop on the cytoplasmic surface of SecY rather than entering the channel directly.

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Figure 1: Structures of non-translating ribosome–channel complexes.
Figure 2: Purification of a RNC–channel complex.
Figure 3: Structure of the active SecY channel.
Figure 4: Path of the nascent chain.

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Accession codes

Primary accessions

Electron Microscopy Data Bank

Protein Data Bank

Data deposits

Electron density maps have been submitted to the Electron Microscopy Data Bank (http://www.emdatabank.org/) under accession numbers EMD-5691, EMD-5692 and EMD-5693, and modelled structures to the Protein Data Bank (http://www.rcsb.org/pdb/home/home.do) under accession numbers 3J43, 3J44, 3J45, 3J46 and 1VVK.

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Acknowledgements

We thank K. Matlack and T. Guettler for reading the manuscript. This work was supported by National Institutes of Health grants GM067887 to J.C.G., GM080139 to S.J.L., GM052586 to T.A.R. and GM45377 to C.W.A. T.A.R. is a Howard Hughes Institute investigator.

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Authors and Affiliations

  1. Department of Cell Biology and Howard Hughes Medical Institute, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA,

    Eunyong Park, Weikai Li, Andrew Whynot & Tom A. Rapoport

  2. Department of Physiology and Biophysics, Boston University School of Medicine, 700 Albany Street, Boston, Massachusetts 02118-2526, USA,

    Jean-François Ménétret & Christopher W. Akey

  3. School of Physics, Georgia Institute of Technology, Atlanta, 30332, Georgia, USA

    James C. Gumbart

  4. Verna and Marrs McLean Department of Biochemistry and Molecular Biology, National Center for Macromolecular Imaging, Baylor College of Medicine, 1 Baylor Plaza, Houston, Texas 77030, USA,

    Steven J. Ludtke

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Contributions

E.P. designed and purified RNC–channel complexes, J.-F.M. and C.W.A. obtained and analysed the electron microscopy data, J.C.G. helped with MDFF and channel modelling, S.J.L. helped with data analysis, W.L. and A.W. purified M. jannaschii components, and T.A.R., E.P. and C.W.A. wrote the paper.

Corresponding authors

Correspondence to Tom A. Rapoport or Christopher W. Akey.

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Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-22 and Supplementary Tables 1-3. (PDF 15975 kb)

Conformational changes in the E. coli SecY channel during translocation - part I

The video illustrates conformational changes in the SecY channel that occur when a nascent chain with a signal sequence is bound in a looped configuration. The nascent chain has been omitted for clarity. A morph was generated between models of the closed and active SecY complex in Chimera, with the channel viewed from the front. The N-terminal half of SecY is in light blue, the C-terminal half is in red, SecE is dark blue, SecG is brown, and the plug is yellow. Six isoleucines that line the pore are shown as ball and stick representations in dark grey. Ribosomal helices H6, H7, H50 and H50 are shown in white; proteins L23, and L24 are green, and L29 is dark grey. The position and conformation of the 6/7 loop was fixed during the simulation. Note that SecE tilts to accommodate movements of the two halves of SecY, and helix 8b moves downwards towards the lateral gate, while helix 9 remains anchored to the large ribosomal subunit. These movements open the lateral gate between the two halves of SecY. (MOV 5307 kb)

Conformational changes in the E. coli SecY channel during translocation - part II

The video illustrates conformational changes in SecY channel that occur when a nascent chain with a signal sequence is bound in a looped configuration. The channel is viewed from the top. The nascent chain has been omitted for clarity. Note that the N-terminal half of SecY rotates and tilts backwards, approximating a rigid body movement that also involves SecG. However, helix 3 bends and the junction between helix 5 and 6 also moves to accommodate these large movements. These movements create a large translocation pore with access to the opened lateral gate. (MOV 5618 kb)

Visualizing the nascent chain in the active E. coli SecY channel

The video illustrates the orientation of a closed SecY channel beneath the ribosome and then shows a progression from a closed to an open SecY. The nascent chain containing the signal sequence helix (residues 1-73; in green) is bound in a looped configuration and a cytoplasmic loop resides in a V-shaped canyon bounded by the 6/7 connection and helix 10. The N-terminal half of SecY is in light blue, the C-terminal half is in red, SecE is dark blue, SecG is brown, and the plug is yellow. Six isoleucines that line the pore are shown as ball and stick representations in dark grey. Ribosomal helices H6, H7, H50 and H59 are shown in white; ribosomal proteins are colored as follows: L23 (purple), L29 (dark grey) and L29 (yellow). The extended nascent chain exiting the tunnel couldinteract with the 6/7 loop to help stabilize the translocating ribosomechannel complex. (MOV 12322 kb)

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Park, E., Ménétret, JF., Gumbart, J. et al. Structure of the SecY channel during initiation of protein translocation. Nature 506, 102–106 (2014). https://doi.org/10.1038/nature12720

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