Abstract
The Sec61 complex forms a protein-conducting channel in the endoplasmic reticulum membrane that is required for secretion of soluble proteins and production of many membrane proteins. Several natural and synthetic small molecules specifically inhibit Sec61, generating cellular effects that are useful for therapeutic purposes, but their inhibitory mechanisms remain unclear. Here we present near-atomic-resolution structures of human Sec61 inhibited by a comprehensive panel of structurally distinct small molecules—cotransin, decatransin, apratoxin, ipomoeassin, mycolactone, cyclotriazadisulfonamide and eeyarestatin. All inhibitors bind to a common lipid-exposed pocket formed by the partially open lateral gate and plug domain of Sec61. Mutations conferring resistance to the inhibitors are clustered at this binding pocket. The structures indicate that Sec61 inhibitors stabilize the plug domain in a closed state, thereby preventing the protein-translocation pore from opening. Our study provides the atomic details of Sec61–inhibitor interactions and the structural framework for further pharmacological studies and drug design.
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Data availability
EM maps and models are available through EM Data Bank (EMDB) and Protein Data Bank (PDB) under the following accession codes: EMD-27581 and PDB-8DNV for the apo class 1 structure, EMD-27582 and PDB-8DNW for the apo class 2 structure, EMD-27583 and PDB-8DNX for the cotransin CP2-bound complex, EMD-27584 and PDB-8DNY for the decatransin-bound complex, EMD-27585 and PDB-8DNZ for the apratoxin F-bound complex, EMD-27586 and PDB-8DO0 for the mycolactone bound complex, EMD-27587 and PDB-8DO1 for ipomoeassin F-bound complex, EMD-27588 and PDB-8DO2 for the CADA-bound complex, and EMD-27589 and PDB-8DO3 for the eeyarestatin I-bound complex. Additional full Sec complex maps were also deposited to EMDB (for accession codes, see Supplementary Table 1). Source data are provided with this paper.
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Acknowledgements
We thank D. Toso for support for electron microscope operation, G. Zong for ipomoeassin F synthesis, P. Mathys and R. Riedl for help acquiring the IC50 data. E.P. was supported by the Vallee Scholars Program, Pew Biomedical Scholars Program and Hellman Fellowship. S.I. and L.W. were supported by a National Institutes of Health training grant (5T32GM008295). M.S and T.J. were supported by the Swiss National Science Foundation (31003A-182519). N.B. was supported by Fondation Raoul Follereau and Fondation Pour Le Développement De La Chimie Des Substances Naturelles Et Ses Applications. W.Q.S. (synthesis of ipomoeassin F) was supported by an AREA grant from National Institutes of Health (GM116032). C.F. and L.X. were supported by the Ohio State University.
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Contributions
E.P. conceived the project and supervised the cryo-EM study. L.W. and S.I. cloned the chimeric Sec construct and prepared protein samples. S.I., L.W. and E.P. collected and analyzed cryo-EM data and built atomic models. L.W. performed the human cell-based assays. R.S. helped purification of the human Sec complex and cloning of the chimeric Sec complex. T.J., M.S. and D.H. performed the yeast mutational study. D.H. provided cotransin CP2 and decatransin. C.F. and L.X. provided apratoxin F. W.Q.S. provided ipomoeassin F. N.B. provided mycolactone. All authored contributed to interpretation of results. E.P. wrote the manuscript with input from all authors.
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During the revision of the manuscript, the Park lab (E.P. and L.W.) signed a sponsored research collaboration agreement with Kezar Life Sciences. The remaining authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Cryo-EM analysis of the yeast and human Sec complexes.
a, A schematic of the single-particle cryo-EM analysis of the yeast Sec (ScSec) complex incubated with cotransin CP2. Note that the particles were sorted into two 3D classes, with and without Sec62, due to partial occupancy of Sec62. b, 3D reconstructions of the ScSec complex with and without ScSec62 (shown in yellow). No cotransin-like density was observed in either class. For this experiment, we used a pore ring mutant (PM; M90L/T185I/M294I/M450L) that stabilizes the plug towards a closed conformation. c, Purification of the human Sec (HsSec) complex. Shown is a Superose 6 size-exclusion chromatography elution profile with fractions analyzed on a Coomassie-stained SDS gel. Note that under the used purification condition, HsSec62 does not co-purify at a stoichiometric ratio or stably comigrate with the Sec61–Sec63 complex. The fractions indicated by gray shade were used for cryo-EM. MW standards: Tg, thyroglobulin; F, ferritin; Ald, aldolase. The experiment was repeated twice independently with similar results. d, A schematic of the single-particle analysis of HsSec complex incubated with cotransin CP2. Due to a poor refinement result from nonuniform refinement in cryoSPARC, the final reconstruction was obtained by the ab-initio refinement function of cryoSPARC (see f). e, Representative 2D classes of the HsSec complex. Diffuse cytosolic features of Sec63 (green arrowheads) suggest its flexibility or disorderedness. f, The 3D reconstruction of the HsSec complex. A putative cotransin CP2 feature (cyan) is visible at the lateral gate.
Extended Data Fig. 2 Cryo-EM analysis of the chimeric Sec complex in an apo form.
a, Purification of the chimeric Sec complex reconstituted in a peptidisc. Left, Superose 6 elution profile; right, Coomassie-stained SDS gel of the peak fraction. The fraction marked by gray shade was used for cryo-EM. Asterisks, putative species of glycosylated ScSec71. The experiment was repeated at least four times independently with similar results. b, A schematic of the cryo-EM analysis of the chimeric Sec complex in an apo state. c and d, Distributions of particle view orientations in the final reconstructions of Classes 1 (c) and 2 (d). e and f, Fourier shell correlation (FSC) curves and local resolution maps of the final reconstructions. g, Superimposition of the Class 1 and 2 atomic models (based on the cytosolic domains) shows a slight difference in relative positions between Sec63–Sec71–Sec72 and the Sec61 complex. h, Side views showing the contact between the engineered cytosolic loops of Sec61α and the FN3 domain of ScSec63. Note that in Apo Class 2, the contact is more poorly packed than Class 1.
Extended Data Fig. 3 Cryo-EM analysis of the chimeric Sec complex in an inhibitor (apratoxin F)-bound form.
a, Images of a representative micrograph and particles of the apratoxin F-bound chimeric Sec complex. Scale bar, 10 nm. b, A schematic of the cryo-EM analysis of the apratoxin F-bound chimeric Sec complex. c, Representative 2D classes of the apratoxin F-bound Sec complex. d, Distribution of particle view orientations in the final reconstruction. e, The FSC curve and local resolution map of the final reconstruction (full Sec complex map). f, As in e, but for the map from focused (local) refinement. g, Segmented density maps of the apratoxin F-bound Sec61α subunit. h, Segmented density features of bound natural inhibitors.
Extended Data Fig. 5 Comparison between the structures of cotransin CP2-bound human and chimeric Sec complexes.
The high-resolution structure of the cotransin CP2-bound chimeric Sec complex (ribbon representation for Sec61 and stick representation for cotransin CP2) is docked into the low-resolution cotransin CP2-bound human Sec complex structure (the semi-transparent gray density map; also see Extended Data Fig. 1f). The features of Sec61α and the bound cotransin CP2 are essentially superimposable between the two structures. Dashed lines indicate lateral gate helices (TM2b, TM3, and TM7).
Extended Data Fig. 6 Variation in the extent of lateral gate opening in inhibitor-bound structures.
As in Fig. 2 a and b, but showing other inhibitor-bound structures. In all panels showing a lateral gate comparison, cylindrical representations in red and pink are the cotransin CP2- and ipomoeassin F- bound structures, respectively, whereas the representation in green is the structure with the indicated inhibitor.
Extended Data Fig. 7 Conformational flexibility of the chimeric Sec complex allows ipomoeassin F binding.
Binding of ipomoeassin F causes a narrower opening of the Sec61 lateral gate compared to the apo complex structures (also see Fig. 2), and this is enabled by disengagement of the Sec61 channel from TM3 (Class 1; panel a) or FN3 domain (Class 2; panel b) of Sec63. For comparison, the structures of the apo complex are also shown.
Extended Data Fig. 8 3D maps for interactions between Sec61 and inhibitors.
Shown are stereo-views into the inhibitor-binding site. Inhibitors and adjacent protein side chains are shown in a stick representation together with Cα traces for TM2b, TM3, TM7, and the plug. The views are roughly similar between the different structures but adjusted for each structure for clearer representations. The following colors are used to differentiate parts: brown, pore ring residues; magenta, plug; lighter orange; N300, darker orange, Q127. All inhibitors are shown in cyan with certain atom-dependent coloring (nitrogen-blue, oxygen-red, sulfur-yellow, and chlorine-green).
Extended Data Fig. 9 Generation of HEK293 cell lines with expression of additional SEC61A1 and effects of CADA in CD4 expression.
a, Expression of indicated human Sec61A1 in stable HEK293 (T-Rex-293) cells was confirmed by western-blotting with anti-HA-tag and anti-Sec61A1 antibodies. b, Human CD4 with a C-terminal Strep-tag was expressed in the indicated HEK293 cell lines by transient transfection, and the CD4 expression level after treating cells with the indicated concentrations of CADA was measured by SDS-PAGE and western-blotting. Four replicates were performed, and the dose-response curves are shown in Fig. 4k.
Extended Data Fig. 10 Comparison with the mycolactone and CK147 structures by others.
a, Chemical structure of mycolactone A/B. b, Structure of mycolactone-bound Sec61 in the current study. c, Structure of mycolactone-bound Sec61 in Gérard et al. (ref. 35). Note that the position and orientation of mycolactone are markedly different between the two structures. The southern chain of mycolactone is buried into the cytosolic funnel of Sec61 in our study, whereas it is in the membrane in the study by Gérard et al. Another notable discrepancy is a one-residue-shifted helical register of the Sec61α TM7 in Gérard et al, which includes the N300 residue. d, Chemical structure of CADA. e, Chemical structure of CK147. f, Structure of CADA-bound Sec61 in the current study. In the lower panel, the surface was clipped along the dashed line shown in the upper panel. g, As in f, but shown is the structure of CK147-bound Sec61 in Pauwels et al. (ref. 36). Note that in this structure, CK147 is almost completely buried within Sec61α. Our CADA-Sec61 structure (f) does not show such a pocket as the space is occupied by the loops flanking the plug helix.
Supplementary information
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Supplementary Tables 1–3 and Figs. 1 and 2.
Supplementary Table
Statistical source data for Supplementary Table 3.
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Source Data Fig. 4
Statistical source data for Fig. 4.
Source Data Extended Data Fig. 1
Unprocessed gel image of Extended Data Fig. 1c.
Source Data Extended Data Fig. 2
Unprocessed gel image of Extended Data Fig. 2a.
Source Data Extended Data Fig. 9
Unprocessed blot images of Extended Data Fig. 9.
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Itskanov, S., Wang, L., Junne, T. et al. A common mechanism of Sec61 translocon inhibition by small molecules. Nat Chem Biol 19, 1063–1071 (2023). https://doi.org/10.1038/s41589-023-01337-y
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DOI: https://doi.org/10.1038/s41589-023-01337-y
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