Global signal peptide profiling reveals principles of selective Sec61 inhibition.

Cotransins target the Sec61 translocon and inhibit the biogenesis of an undefined subset of secretory and membrane proteins. Remarkably, cotransin inhibition depends on the unique signal peptide (SP) of each Sec61 client, which is required for cotranslational translocation into the endoplasmic reticulum. It remains unknown how an SP's amino acid sequence and biophysical properties confer sensitivity to structurally distinct cotransins. Here we describe a fluorescence-based, pooled-cell screening platform to interrogate nearly all human SPs in parallel. We profiled two cotransins with distinct effects on cancer cells and discovered a small subset of SPs, including the oncoprotein human epidermal growth factor receptor 3 (HER3), with increased sensitivity to the more selective cotransin, KZR-9873. By comparing divergent mouse and human orthologs, we unveiled a position-dependent effect of arginine on SP sensitivity. Our multiplexed profiling platform reveals how cotransins can exploit subtle sequence differences to achieve SP discrimination.

Signal peptide mimicry primes Sec61 for client-selective inhibition.

Preventing the biogenesis of disease-relevant proteins is an attractive therapeutic strategy, but attempts to target essential protein biogenesis factors have been hampered by excessive toxicity. Here we describe KZR-8445, a cyclic depsipeptide that targets the Sec61 translocon and selectively disrupts secretory and membrane protein biogenesis in a signal peptide-dependent manner. KZR-8445 potently inhibits the secretion of pro-inflammatory cytokines in primary immune cells and is highly efficacious in a mouse model of rheumatoid arthritis. A cryogenic electron microscopy structure reveals that KZR-8445 occupies the fully opened Se61 lateral gate and blocks access to the lumenal plug domain. KZR-8445 binding stabilizes the lateral gate helices in a manner that traps select signal peptides in the Sec61 channel and prevents their movement into the lipid bilayer. Our results establish a framework for the structure-guided discovery of novel therapeutics that selectively modulate Sec61-mediated protein biogenesis.

Electronic and Steric Control of n→π* Interactions: Stabilization of the α-Helix Conformation without a Hydrogen Bond.

The preferred conformations of peptides and proteins are dependent on local interactions that bias the conformational ensemble. The n→π* interaction between consecutive carbonyls promotes compact conformations, including the α-helix and polyproline II helix. In order to further understand the n→π* interaction and to develop methods to promote defined conformational preferences through acyl N-capping motifs, a series of peptides was synthesized in which the electronic and steric properties of the acyl group were modified. Using NMR spectroscopy, van't Hoff analysis of enthalpies, X-ray crystallography, and computational investigations, we observed that more electron-rich donor carbonyls (pivaloyl, iso-butyryl, propionyl) promote stronger n→π* interactions and more compact conformations than acetyl or less electron-rich donor carbonyls (methoxyacetyl, fluoroacetyl, formyl). X-ray crystallography indicates a strong, electronically tunable preference for the α-helix conformation, as observed directly on the φ and ψ torsion angles. Electron-donating acyl groups promote the α-helical conformation, even in the absence of the hydrogen bonding that stabilizes the α-helix. In contrast, electron-withdrawing acyl groups led to more extended conformations. More sterically demanding groups can promote trans amide bonds independent of the electronic effect on n→π* interactions. Chloroacetyl groups additionally promote n→π* interactions through the interaction of the chlorine lone pair with the proximal carbonyl π*. These data provide additional support for an important role of n→π* interactions in the conformational ensemble of disordered or unfolded proteins. Moreover, this work suggests that readily incorporated acyl N-capping motifs that modulate n→π* interactions may be employed rationally to promote conformational biases in peptides, with potential applications in molecular design and medicinal chemistry.

The Distinct Conformational Landscapes of 4S-Substituted Prolines That Promote an endo Ring Pucker.

4-Substitution on proline directly impacts protein main chain conformational preferences. The structural effects of N-acyl substitution and of 4-substitution were examined by NMR spectroscopy and X-ray crystallography on minimal molecules with a proline 4S-nitrobenzoate. The effects of N-acyl substitution on conformation were attenuated in the 4S-nitrobenzoate context, due to the minimal role of the n→π* interaction in stabilizing extended conformations. By X-ray crystallography, an extended conformation was observed for most molecules. The formyl derivative adopted a δ conformation that is observed at the i+2 position of ß-turns. Computational analysis indicated that the structures observed crystallographically represent the inherent conformational preferences of 4S-substituted prolines with electron-withdrawing 4-position substituents. The divergent conformational preferences of 4R- and 4S-substituted prolines suggest their wider structure-specific application in molecular design. In particular, the proline endo ring pucker favored by 4S-substituted prolines uniquely promotes the δ conformation [(ϕ, ψ) ≈(-80°, 0°)] found in ß-turns. In contrast to other acyl capping groups, the pivaloyl group strongly promoted trans amide bond and polyproline II helix conformation, with a close n→π* interaction in the crystalline state, despite the endo ring pucker, suggesting its special capabilities in promoting compact conformations in ϕ due to its strongly electron-donating character.