A common mechanism of Sec61 translocon inhibition by small molecules

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.

Extended Data Figure 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. 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 stabilize 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. 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 feature (cyan) is visible at the lateral gate.

Extended Data Figure 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 Figure 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 schematics 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 Figure 4.. FSC curve and local resolution maps of inhibitor-bound Sec complexes.

As in Extended Data Figure 3 e and f, but for all other inhibitor-bound structures.

Extended Data Figure 5.. Comparison between the structures of cotransin-bound human and chimeric Sec complexes.

The high-resolution structure of the cotransin-bound chimeric Sec complex (ribbon representation for Sec61 and stick representation for cotransin) is docked into the low-resolution cotransin-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 are essentially superimposable between the two structures. Dashed lines, lateral gate helices (TM2b, TM3, and TM7).

Extended Data Figure 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- and ipomoeassin F- bound structures, respectively, whereas the representation in green is the structure with the indicated inhibitor.

Extended Data Figure 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 Figure 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 more clear 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 Figure 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 Figure 10.. Comparison with the mycolactone model by Gérard et al.

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. For example, 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. Other notable discrepancies include a one-residue shift in the helical register of the Sec61α TM7, which forms a lateral gate in Gérard et al.

Figure 1.. Cryo-EM structures of the human Sec61 complex inhibited by various small-molecule inhibitors.

a, Architecture of the Sec61 channel and overall model for gating and substrate engagement. b, Design of a human-yeast chimeric Sec complex. Parts derived from human and yeast proteins are outlined with solid and dashed lines, respectively. Note that except for the cytosolic L6/7 and L8/9 loops, Sec61α is from the human SEC61A1 protein sequence. Hs, Homo sapiens; Sc, Saccharomyces cerevisiae; J, J-domain. c, 2.7-Å-resolution cryo-EM map of the chimeric Sec complex in an apo state (Class 1, unsharpened map). The lateral gate helices are indicated by dashed lines and TM numbers. The region outlined by a rectangle indicates the inhibitor-binding site (also see d–k). d–k, Views into the inhibitor-binding site of Sec61α of apo and inhibitor-bound structures. Cryo-EM maps (semi-transparent surface) and atomic models were overlaid. Inhibitor and plug densities are shown in cyan and purple, respectively. Dashed lines indicate lateral gate helices TMs 2b, 3, and 7 as in c.

Figure 2.. Structural plasticity of the inhibitor-binding pocket.

a–c, The inhibitor-binding pocket of Sec61 and bound inhibitors are shown in surface (protein) and sphere (inhibitors) representations. Shown are for cotransin CP2 (a), decatransin (b), and ipomoeassin F (c). For other inhibitors, see Extended Data Fig. 6. Conserved polar amino acids N300 and Q127 at the inhibitor binding site (also see Fig. 3) are indicated in light and dark orange, respectively. Note that part (cinnamate moiety) of ipomoeassin is deeply buried inside the channel and invisible in this representation. d, Superposition of the Sec61 structures bound to cotransin CP2 (red), decatransin (green), and ipomoeassin F (pink). Note differences in the lateral gate opening due to the varying position of the N-terminal half of Sec61α, particularly TMs 2b and 3.

Figure 3.. Maps for interactions between Sec61 and inhibitors.

a, The chemical structure of cotransin CP2 and the positions of amino acids (ovals) of Sec61 in the immediate vicinity are drawn in a two-dimensional representation. Different colors were used for ovals to indicate regions in Sec61α: purple–plug, brown–pore ring, gray–lateral gate, light and dark oranges–polar cluster Q127/N300, and white–others. In the cotransin CP2 chemical structure, main lipid-exposed parts are in green whereas channel-facing parts are in blue. Moieties in orange and red interact with N300 and Q127 respectively. b–g, As in a, but drawn for decatransin (b), apratoxin F (c), mycolactone (d), ipomoeassin F (e), CADA (f), and ESI (g). Dashed lines indicate putative hydrogen bonds. Note that in the mycolactone-bound structure, a water molecule coordinated by Sec61 and mycolactone was observed in the pocket. For 3D structures, see Extended Data Fig. 8.

Figure 4.. Inhibitor-resistant mutations.

a, Positions of mutations tested with yeast Sec61 were mapped onto the cotransin CP2-bound structure (also see Supplementary Table 3). Left, front view; right, cutaway side view. Cotransin CP2 (cyan) and amino acid side chains are shown as spheres. Red and pale green spheres indicate positions in which mutation to Asp or Trp develops high and no cotransin CP2 resistance, respective. Magenta, positions of other resistant mutations previously reported^,. b, As in a, but with ipomoeassin-F-resistant mutations. Yellow spheres additionally show positions that give rise to moderate ipomoeassin F resistance. c–e, Effects of Sec61 lateral gate polar amino acid mutations on yeast growth inhibition by cotransin CP2, decatransin, and ipomoeassin F (residue numbers are according to yeast Sec61). Shown are means, s.e.m., and fitted curves (n=3 independent experiments for cotransin CP2 and decatransin; n=4 independent experiments for ipomoeassin F). f–j, Dose-response curves for indicated inhibitors from viability assays of cultured human (HEK293) cells expressing the indicated Sec61α variant (residue numbers are according to human SEC61A1; means and s.e.m., n=4 independent experiments). k, Inhibition of expression of CD4 in HEK293 by CADA (means and s.e.m., n=4 independent experiments).

Figure 5.. Proposed model for Sec61 inhibition.

a, General model for the mechanism of Sec61 inhibitors. Inhibitors bind to Sec61 in a partially open conformation and preclude the plug from opening. This prevents substrate polypeptide insertion. b, A proposed model for client-specific inhibition. Certain client-specific inhibitors may allow an interaction between strong signals (e.g., TM signal anchors) and the channel such that the signal sequence/anchor is wedged into the partially open lateral gate. This would further open the lateral gate to cause release of the inhibitor. Inhibitors forming less interactions with the pore and plug, rendering the lateral gate into a more open conformation, and/or displaying a weaker overall affinity are likely to be overcome by this manner.