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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Electron Microscopy Data Bank
Protein Data Bank
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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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.
The authors declare no competing financial interests.
This file contains Supplementary Figures 1-22 and Supplementary Tables 1-3. (PDF 15975 kb)
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)
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)
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, J., 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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