Structure of the human signal peptidase complex reveals the determinants for signal peptide cleavage
Liaci, A Manuel; Steigenberger, Barbara; Telles de Souza, Paulo Cesar; Tamara, Sem; Gröllers-Mulderij, Mariska; Ogrissek, Patrick; Marrink, Siewert J; Scheltema, Richard A; Förster, Friedrich
(2021) Molecular Cell, volume 81, issue 19, pp. 3934 - 3948.e11
(Article)
Abstract
The signal peptidase complex (SPC) is an essential membrane complex in the endoplasmic reticulum (ER), where it removes signal peptides (SPs) from a large variety of secretory pre-proteins with exquisite specificity. Although the determinants of this process have been established empirically, the molecular details of SP recognition and removal remain
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elusive. Here, we show that the human SPC exists in two functional paralogs with distinct proteolytic subunits. We determined the atomic structures of both paralogs using electron cryo-microscopy and structural proteomics. The active site is formed by a catalytic triad and abuts the ER membrane, where a transmembrane window collectively formed by all subunits locally thins the bilayer. Molecular dynamics simulations indicate that this unique architecture generates specificity for SPs based on the length of their hydrophobic segments.
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Keywords: ER translocon, crosslinking mass spectrometry, cryo-EM, membrane thinning, molecular dynamics simulations, protein maturation, protein secretion, secretory pathway, signal peptidase complex, signal peptide, Taverne, Molecular Biology, Cell Biology
ISSN: 1097-2765
Publisher: Cell Press
Note: Funding Information: Research was supported by the ERC Consolidator grant 724425 (Biogenesis and Degradation of Endoplasmic Reticulum Proteins, to F.F.), the research program TA with project number 741.018.201 (to R.A.S. and F.F.) partly financed by the Dutch Research Council (NWO), and the ERC advanced grant ?COMP-MICR-CROW-MEM? (to S.J.M.), the European Union Horizon 2020 program INFRAIA project Epic-XS (project 823839), and the Netherlands Electron Microscopy Infrastructure. This work benefited from access to the NKI Protein Facility, an Instruct-NL and Instruct-ERIC center, the cellular protein chemistry laboratory at Utrecht University, and computing time at the National Computing Facilities Foundation (NCF) of the NWO. We thank Dr. Robert-Jan Lebbink, Dr. Patrique Praest (UMC Utrecht), and Prof. Dr. Thijn Brummelkamp (NKI Amsterdam) for providing the cell lines used to characterize SEC11 expression; Dr. Stuart Howes (Utrecht University) for EM support; Susanne Br?kner (NKI Amsterdam) for support with nanoDSF; Panagiotis Drougkas (Utrecht University) for help with protein purification; Prof. Dr Ineke Braakman, Guus van Zadelhoff, Dr. Juliette Fedry, and Lena Th?richen for help with activity assays; and Prof. Dr. Richard Zimmerman for critically reading the manuscript. A.M.L. and F.F. conceived the project. A.M.L. designed the expression constructs. A.M.L. and M.G.-M. cloned constructs and point mutants. A.M.L. expressed cells and established a protein purification protocol. A.M.L. and P.O. performed protein purification and sample purification for cryo-EM, and MS. B.S. S.T. R.A.S. A.M.L. and F.F. conceived the MS experiments. B.S. R.A.S. and S.T. performed the MS experiments and data analysis. A.M.L. and P.O. solved the structures of SPC-A and SPC-C, including sample preparation, data collection, data processing, and refinement. A.M.L. and F.F. generated, refined, and analyzed the atomic models. A.M.L. conceived and performed the stability and activity assays. P.C.T.d.S. A.M.L. S.J.M. and F.F. conceived the MD simulation experiments, which P.C.T.d.S. performed and analyzed. M.G.-M. grew cells for the shotgun analysis. A.M.L. B.S. S.T. P.C.T.d.S. S.J.M. R.A.S. and F.F. wrote the manuscript and prepared the figures with help from all co-authors. The authors declare no competing interests. Funding Information: Research was supported by the ERC Consolidator grant 724425 (Biogenesis and Degradation of Endoplasmic Reticulum Proteins, to F.F.), the research program TA with project number 741.018.201 (to R.A.S. and F.F.) partly financed by the Dutch Research Council (NWO), and the ERC advanced grant “ COMP-MICR-CROW-MEM ” (to S.J.M.), the European Union Horizon 2020 program INFRAIA project Epic-XS (project 823839 ), and the Netherlands Electron Microscopy Infrastructure . This work benefited from access to the NKI Protein Facility, an Instruct-NL and Instruct-ERIC center, the cellular protein chemistry laboratory at Utrecht University, and computing time at the National Computing Facilities Foundation (NCF) of the NWO. We thank Dr. Robert-Jan Lebbink, Dr. Patrique Praest (UMC Utrecht), and Prof. Dr. Thijn Brummelkamp (NKI Amsterdam) for providing the cell lines used to characterize SEC11 expression; Dr. Stuart Howes (Utrecht University) for EM support; Susanne Brükner (NKI Amsterdam) for support with nanoDSF; Panagiotis Drougkas (Utrecht University) for help with protein purification; Prof. Dr Ineke Braakman, Guus van Zadelhoff, Dr. Juliette Fedry, and Lena Thärichen for help with activity assays; and Prof. Dr. Richard Zimmerman for critically reading the manuscript. Publisher Copyright: © 2021 Elsevier Inc.
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