Separase is a caspase-family protease that initiates chromatid segregation by cleaving the kleisin subunits (Scc1 and Rec8) of cohesin, and regulates centrosome duplication and mitotic spindle function through cleavage of kendrin and Slk19. To understand the mechanisms of securin regulation of separase, we used single-particle cryo-electron microscopy (cryo-EM) to determine a near-atomic-resolution structure of the Caenorhabditis elegans separase–securin complex. Separase adopts a triangular-shaped bilobal architecture comprising an N-terminal tetratricopeptide repeat (TPR)-like α-solenoid domain docked onto the conserved C-terminal protease domain. Securin engages separase in an extended antiparallel conformation, interacting with both lobes. It inhibits separase by interacting with the catalytic site through a pseudosubstrate mechanism, thus revealing that in the inhibited separase–securin complex, the catalytic site adopts a conformation compatible with substrate binding. Securin is protected from cleavage because an aliphatic side chain at the P1 position represses protease activity by disrupting the organization of catalytic site residues.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
A prometaphase mechanism of securin destruction is essential for meiotic progression in mouse oocytes
Nature Communications Open Access 14 July 2021
Nature Communications Open Access 15 November 2019
Biology Direct Open Access 27 April 2018
Subscribe to Nature+
Get immediate online access to Nature and 55 other Nature journal
Subscribe to Journal
Get full journal access for 1 year
only $8.25 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Uhlmann, F., Wernic, D., Poupart, M.A., Koonin, E.V. & Nasmyth, K. Cleavage of cohesin by the CD clan protease separin triggers anaphase in yeast. Cell 103, 375–386 (2000).
Waizenegger, I.C., Hauf, S., Meinke, A. & Peters, J.M. Two distinct pathways remove mammalian cohesin from chromosome arms in prophase and from centromeres in anaphase. Cell 103, 399–410 (2000).
Hauf, S., Waizenegger, I.C. & Peters, J.M. Cohesin cleavage by separase required for anaphase and cytokinesis in human cells. Science 293, 1320–1323 (2001).
Santaguida, S. & Amon, A. Short- and long-term effects of chromosome mis-segregation and aneuploidy. Nat. Rev. Mol. Cell Biol. 16, 473–485 (2015).
Kamenz, J. & Hauf, S. Time to split up: dynamics of chromosome separation. Trends Cell Biol. 27, 42–54 (2017).
Ciosk, R. et al. An ESP1/PDS1 complex regulates loss of sister chromatid cohesion at the metaphase to anaphase transition in yeast. Cell 93, 1067–1076 (1998).
Uhlmann, F., Lottspeich, F. & Nasmyth, K. Sister-chromatid separation at anaphase onset is promoted by cleavage of the cohesin subunit Scc1. Nature 400, 37–42 (1999).
Zou, H., McGarry, T.J., Bernal, T. & Kirschner, M.W. Identification of a vertebrate sister-chromatid separation inhibitor involved in transformation and tumorigenesis. Science 285, 418–422 (1999).
Sánchez-Puig, N., Veprintsev, D.B. & Fersht, A.R. Human full-length securin is a natively unfolded protein. Protein Sci. 14, 1410–1418 (2005).
Csizmok, V., Felli, I.C., Tompa, P., Banci, L. & Bertini, I. Structural and dynamic characterization of intrinsically disordered human securin by NMR spectroscopy. J. Am. Chem. Soc. 130, 16873–16879 (2008).
Hellmuth, S. et al. Positive and negative regulation of vertebrate separase by Cdk1-cyclin B1 may explain why securin is dispensable. J. Biol. Chem. 290, 8002–8010 (2015).
Jallepalli, P.V. et al. Securin is required for chromosomal stability in human cells. Cell 105, 445–457 (2001).
Nagao, K., Adachi, Y. & Yanagida, M. Separase-mediated cleavage of cohesin at interphase is required for DNA repair. Nature 430, 1044–1048 (2004).
Hornig, N.C., Knowles, P.P., McDonald, N.Q. & Uhlmann, F. The dual mechanism of separase regulation by securin. Curr. Biol. 12, 973–982 (2002).
Holland, A.J. & Taylor, S.S. Cyclin-B1-mediated inhibition of excess separase is required for timely chromosome disjunction. J. Cell Sci. 119, 3325–3336 (2006).
Bachmann, G. et al. A closed conformation of the Caenorhabditis elegans separase-securin complex. Open Biol. 6, 160032 (2016).
Stemmann, O., Zou, H., Gerber, S.A., Gygi, S.P. & Kirschner, M.W. Dual inhibition of sister chromatid separation at metaphase. Cell 107, 715–726 (2001).
Gorr, I.H., Boos, D. & Stemmann, O. Mutual inhibition of separase and Cdk1 by two-step complex formation. Mol. Cell 19, 135–141 (2005).
Cohen-Fix, O., Peters, J.M., Kirschner, M.W. & Koshland, D. Anaphase initiation in Saccharomyces cerevisiae is controlled by the APC-dependent degradation of the anaphase inhibitor Pds1p. Genes Dev. 10, 3081–3093 (1996).
Funabiki, H. et al. Cut2 proteolysis required for sister-chromatid seperation in fission yeast. Nature 381, 438–441 (1996).
Yamamoto, A., Guacci, V. & Koshland, D. Pds1p, an inhibitor of anaphase in budding yeast, plays a critical role in the APC and checkpoint pathway(s). J. Cell Biol. 133, 99–110 (1996).
Sullivan, M., Hornig, N.C., Porstmann, T. & Uhlmann, F. Studies on substrate recognition by the budding yeast separase. J. Biol. Chem. 279, 1191–1196 (2004).
Alexandru, G., Uhlmann, F., Mechtler, K., Poupart, M.A. & Nasmyth, K. Phosphorylation of the cohesin subunit Scc1 by Polo/Cdc5 kinase regulates sister chromatid separation in yeast. Cell 105, 459–472 (2001).
Viadiu, H., Stemmann, O., Kirschner, M.W. & Walz, T. Domain structure of separase and its binding to securin as determined by EM. Nat. Struct. Mol. Biol. 12, 552–553 (2005).
Lin, Z., Luo, X. & Yu, H. Structural basis of cohesin cleavage by separase. Nature 532, 131–134 (2016).
Pantelic, R.S., Meyer, J.C., Kaiser, U., Baumeister, W. & Plitzko, J.M. Graphene oxide: a substrate for optimizing preparations of frozen-hydrated samples. J. Struct. Biol. 170, 152–156 (2010).
Katis, V.L. et al. Rec8 phosphorylation by casein kinase 1 and Cdc7-Dbf4 kinase regulates cohesin cleavage by separase during meiosis. Dev. Cell 18, 397–409 (2010).
Hellmuth, S. et al. Human chromosome segregation involves multi-layered regulation of separase by the peptidyl-prolyl-isomerase Pin1. Mol. Cell 58, 495–506 (2015).
Waizenegger, I., Giménez-Abián, J.F., Wernic, D. & Peters, J.M. Regulation of human separase by securin binding and autocleavage. Curr. Biol. 12, 1368–1378 (2002).
Zou, H., Stemman, O., Anderson, J.S., Mann, M. & Kirschner, M.W. Anaphase specific auto-cleavage of separase. FEBS Lett. 528, 246–250 (2002).
Holland, A.J., Böttger, F., Stemmann, O. & Taylor, S.S. Protein phosphatase 2A and separase form a complex regulated by separase autocleavage. J. Biol. Chem. 282, 24623–24632 (2007).
Sun, Y. et al. Separase is recruited to mitotic chromosomes to dissolve sister chromatid cohesion in a DNA-dependent manner. Cell 137, 123–132 (2009).
McLuskey, K. & Mottram, J.C. Comparative structural analysis of the caspase family with other clan CD cysteine peptidases. Biochem. J. 466, 219–232 (2015).
Nagao, K. & Yanagida, M. Securin can have a separase cleavage site by substitution mutations in the domain required for stabilization and inhibition of separase. Genes Cells 11, 247–260 (2006).
Buonomo, S.B. et al. Disjunction of homologous chromosomes in meiosis I depends on proteolytic cleavage of the meiotic cohesin Rec8 by separin. Cell 103, 387–398 (2000).
Sullivan, M., Lehane, C. & Uhlmann, F. Orchestrating anaphase and mitotic exit: separase cleavage and localization of Slk19. Nat. Cell Biol. 3, 771–777 (2001).
Matsuo, K. et al. Kendrin is a novel substrate for separase involved in the licensing of centriole duplication. Curr. Biol. 22, 915–921 (2012).
Keusekotten, K. et al. OTULIN antagonizes LUBAC signaling by specifically hydrolyzing Met1-linked polyubiquitin. Cell 153, 1312–1326 (2013).
Wickliffe, K.E., Lorenz, S., Wemmer, D.E., Kuriyan, J. & Rape, M. The mechanism of linkage-specific ubiquitin chain elongation by a single-subunit E2. Cell 144, 769–781 (2011).
Huang, Y. et al. Structural basis of caspase inhibition by XIAP: differential roles of the linker versus the BIR domain. Cell 104, 781–790 (2001).
Riedl, S.J. et al. Structural basis for the inhibition of caspase-3 by XIAP. Cell 104, 791–800 (2001).
Zhang, N. et al. Overexpression of separase induces aneuploidy and mammary tumorigenesis. Proc. Natl. Acad. Sci. USA 105, 13033–13038 (2008).
Mukherjee, M. et al. Overexpression and constitutive nuclear localization of cohesin protease Separase protein correlates with high incidence of relapse and reduced overall survival in glioblastoma multiforme. J. Neurooncol. 119, 27–35 (2014).
Zhang, Z., Yang, J. & Barford, D. Recombinant expression and reconstitution of multiprotein complexes by the USER cloning method in the insect cell-baculovirus expression system. Methods 95, 13–25 (2016).
Li, X. et al. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat. Methods 10, 584–590 (2013).
Zhang, K. Gctf: real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016).
Scheres, S.H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).
Tang, G. et al. EMAN2: an extensible image processing suite for electron microscopy. J. Struct. Biol. 157, 38–46 (2007).
Scheres, S.H. Semi-automated selection of cryo-EM particles in RELION-1.3. J. Struct. Biol. 189, 114–122 (2015).
Elmlund, H., Elmlund, D. & Bengio, S. PRIME: probabilistic initial 3D model generation for single-particle cryo-electron microscopy. Structure 21, 1299–1306 (2013).
Scheres, S.H. Beam-induced motion correction for sub-megadalton cryo-EM particles. eLife 3, e03665 (2014).
Scheres, S.H. & Chen, S. Prevention of overfitting in cryo-EM structure determination. Nat. Methods 9, 853–854 (2012).
Rosenthal, P.B. & Henderson, R. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721–745 (2003).
Kucukelbir, A., Sigworth, F.J. & Tagare, H.D. Quantifying the local resolution of cryo-EM density maps. Nat. Methods 11, 63–65 (2014).
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).
Yang, Z. et al. UCSF Chimera, MODELLER, and IMP: an integrated modeling system. J. Struct. Biol. 179, 269–278 (2012).
Murshudov, G.N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol. Crystallogr. 67, 355–367 (2011).
Adams, P.D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D Biol. Crystallogr. 58, 1948–1954 (2002).
Nicholls, R.A., Fischer, M., McNicholas, S. & Murshudov, G.N. Conformation-independent structural comparison of macromolecules with ProSMART. Acta Crystallogr. D Biol. Crystallogr. 70, 2487–2499 (2014).
Kelley, L.A., Mezulis, S., Yates, C.M., Wass, M.N. & Sternberg, M.J. The Phyre2 web portal for protein modeling, prediction and analysis. Nat. Protoc. 10, 845–858 (2015).
Barton, G.J. ALSCRIPT: a tool to format multiple sequence alignments. Protein Eng. 6, 37–40 (1993).
This work was funded by the Medical Research Council (MC_UP_1201/6 to D.B., and MC_UP_A025_1013 to S.H.W.S.), a Cancer Research UK grant (C576/A14109 to D.B.), Long Term EMBO Fellowships (ALTF 79-2014 to A.B., and ALTF 1229-2013 to T.G.M.) and H2020 Marie-Curie Fellowships (657725 to A.B., and 657990 to T.G.M.). We thank members of the Barford group for helpful discussions; T. Tischer for helpful suggestions and support; C. Savva, S. Chen and G. McMullan for maintaining EM facilities; J. Grimmett and T. Darling for computing; and P. Emsley for constant support with COOT. We acknowledge Diamond for access and support of the cryo-EM facilities at the UK national electron bio-imaging centre (eBIC), proposal EM13708, funded by the Wellcome Trust, MRC and BBSRC.
The authors declare no competing financial interests.
Integrated supplementary information
a, SDS PAGE gel of Ce separase-securin. b, Size exclusion chromatograph of purified complex. C, Typical cryo-electon micrographs of Ce separase-securin in vitreous ice (representative of 234 micrographs) (left) and on graphene oxide grids (representative of 2,559 micrographs) (right). d, Gallery of two-dimensional class averages of Ce separase-securin showing typical views representative of 100 two-dimensional classes collected in vitreous ice on holey carbon grids (Quantifoil Au R1.2/1.3, 300 mesh). The white scale bar corresponds to a distance of 100 Å. e, Gallery of two-dimensional class averages of Ce separase-securin showing different views representative of 100 two-dimensional classes collected on graphene oxide coated grids. The white scale bar corresponds to a distance of 100 Å. f, Gallery of two-dimensional class averages of 2D-class averages of human separase-securin showing different views representative of 100 two-dimensional classes collected on graphene oxide coated grids (Quantifoil Au R1.2/1.3, 300 mesh). The white scale bar corresponds to a distance of 100 Å. g, Angular distribution plot of C. elegans data sets collected in vitreous ice (left) compared to the angular distribution plot of the data used for the final reconstruction (right) is shown.
a, Representative EM density for regions of separase with fitted coordinates. b, Gold standard Fourier Shell Correlation (FSC) curve and FSC curve between cryo-EM map and final atomic model of the Ce separase-securin complex. c, Cross-validation of model refinement by half maps. Shown are FSC curves between the atomic model and the half map (maphalf1) it was refined against and FSC curve between the atomic model and the other half map (maphalf2) that was not used during refinement. d, EM density map colour-coded according to local resolution. e, B-factor plot: Cartoon representation of C. elegans separase-securin complex with main-chain atoms colour-coded according to B-factors from low (blue) to high (red).
Supplementary Figure 3 The α-solenoid domain of Ce separase forms a distorted TPR-like superhelix with 11 TPR motifs.
a, α-solenoid domain of Ce separase shown with its 25 α-helices represented as cylinders and colour-ramped from the N- to C-terminus blue to red. TPR motifs TPR1 and TPR11, and α-helices are labeled, as are the positions of insert-1 and insert-2. H2, H16 and H25 are not part of the TPR superhelix. b, An idealized TPR superhelix of eleven TPR motifs superimposed onto the Ce separase α-solenoid TPR-like domain. The Ce separase α-solenoid domain is considerably compressed compared to the idealized TPR superhelix of eleven TPR motifs. H2, H16 and H25 of the Ce separase α-solenoid do not share counterparts in the eleven-motif TPR superhelix.
a, Schematic of C. elegans separase and b, human separase structures. Human separase comprises an α-helical domain N-terminal to the common TPR domain not present in C. elegans separase. Auto-cleavage and phosphorylation sites in human separase are indicated. c, Structure-based alignment of the TPR domains of C. elegans and human separase. α-helices and corresponding TPR motifs, and Insert-1 and Insert-2 are shown. Human auto-cleavage sites from29,30. Phosphorylation sites from17,28. Sequence alignment figure generated using ALSCRIPT61. The species abbreviations are: Ce (Caenorhabditis elegans), Hs (Homo sapiens).
a, Side by side comparison of CeSPD and CtSPD with the L4 loop highlighted in pink (Ce) and purple (Ct). b, Superposition of Ce and CtSPD domains with the L4 loop highlighted in pink (Ce) and purple (Ct).
Secondary structure elements of CeSPD and CtSPD are shown above and below the alignment, respectively. Residues conserved in all aligned sequences are highlighted in red. Residues binding to the securin pseudo-substrate motif P sites P1 to P6 are boxed in wheat, yellow-green, light green, green, cyan and light blue. Catalytic site residues are marked with a red asterisk. The species abbreviations are: Ce (Caenorhabditis elegans), Hs (Homo sapiens), Mm (Mus musculus), Dr (Danio rerio), Sc (Saccharomyces cerevisiae), Ct (Chaetomium thermophilum).
Sequence alignment of the separase-binding motif (SBM) of securin. Secondary structure elements are shown above the alignment. Securin pseudo-substrate motif sites P1 to P6 are indicated. Invariant residues are boxed in dark red, conserved residues in light red. b, Sequence alignment of cleavage sites of separase substrates (Scc1, Rec8, kendrin and Slk19) and the securin pseudo-substrate motif. Sites P1 to P6 are indicated. Separase cleaves substrates immediately C-terminal to an Arg residue at P1 (labeled).
The P1-binding pockets of C. elegans separase and CtSPD were superimposed and shown with the corresponding C. elegans EM density. EM density for selected separase residues is shown in grey and Met126Sec density is coloured red. Whereas there is a good fit for the C. elegans structure, CtSPD fits poorly, especially Gly2082 and His2083, due to the narrower P1-binding pocket of CtSPD.
The three-dimensional classification was conducted using 665,331 motion-corrected particles that were divided into six classes. The resultant classes did not reveal any major structural rearrangements or classes of free separase. The best-defined and most complete densities were selected for future reconstructions to further improve map quality.
Supplementary Figures 1–9 (PDF 2502 kb)
Conformational differences at the P1 site of separase with substrate (Scc1) and inhibitor (securin).
This video shows the conformational differences at the P1 site of the catalytic sites of C. elegans separase in complex with securin and the Chaetomium thermophilum separase protease domain (CtSPD) in complex with the Scc1 peptide 13. In the C. elegans separase–securin structure, Met126 of securin (the P1 residue) engages the P1-binding site, whereas in the CtSPD–Scc1 complex the critical Arg residue of the substrate engages the P1 site. In the CtSPD–Scc1 complex, the guanidinium side chain of Arg(P1) restructures the catalytic site by donating hydrogen bonds to the carboxylate side chain of Asp2151 and the main chain carbonyl of Gly2082. The latter contact results in a shift of the imidazole side chain of His2083 (a component of the catalytic dyad with Cys2110) toward Arg(P1). The resultant hydrogen bond between the His2083 imidazole ring and the main chain carbonyl of Arg(P1) generates the oxyanion hole that stabilizes the oxyanion transition state intermediate. In the C. elegans separase–securin complex, the nonpolar Met126(P1) side chain acts to widen the P1-binding pocket. The Met126(P1) side chain forms a van der Waals contact with the carbonyl oxygen atom of Gly1013 (equivalent to Gly2082 of CtSPD), shifting the carbonyl group by 2 Å relative to Gly2082. The imidazole side chain of His1014 (equivalent to Hsi2083 of CtSDP) is displaced away from the main chain carbonyl of Met126(P1) by 1.7 Å, and the oxyanion hole is not created in the C. elegans separase–securin structure. In addition, Met126(P1) is unable to form hydrogen bonds to Asp1082 (equivalent to Asp2151 of CtSPD). The CtSPD in complex with the Scc1 peptide is from ref. 13 (PDB 5FC2). (MP4 940 kb)
About this article
Cite this article
Boland, A., Martin, T., Zhang, Z. et al. Cryo-EM structure of a metazoan separase–securin complex at near-atomic resolution. Nat Struct Mol Biol 24, 414–418 (2017). https://doi.org/10.1038/nsmb.3386
This article is cited by
A prometaphase mechanism of securin destruction is essential for meiotic progression in mouse oocytes
Nature Communications (2021)
Nature Communications (2019)