title: A closed conformation of the Caenorhabditis elegans separase-securin complex.
abstract: The protease separase plays a key role in sister chromatid disjunction and centriole disengagement. To maintain genomic stability, separase activity is strictly regulated by binding of an inhibitory protein, securin. Despite its central role in cell division, the separase and securin complex is poorly understood at the structural level. This is partly owing to the difficulty of generating a sufficient quantity of homogeneous, stable protein. Here, we report the production of Caenorhabditis elegans separase-securin complex, and its characterization using biochemical methods and by negative staining electron microscopy. Single particle analysis generated a density map at a resolution of 21-24 Å that reveals a close, globular structure of complex connectivity harbouring two lobes. One lobe matches closely a homology model of the N-terminal HEAT repeat domain of separase, whereas the second lobe readily accommodates homology models of the separase C-terminal death and caspase-like domains. The globular structure of the C. elegans separase-securin complex contrasts with the more elongated structure previously described for the Homo sapiens complex, which could represent a different functional state of the complex, suggesting a mechanism for the regulation of separase activity through conformational change.
authors.affiliation: Division of Structural Biology, The Institute of Cancer Research, London SW7 3RP, UK.
authors.forename: Gudrun
authors.initials: G
authors.lastname: Bachmann
authors.affiliation: Division of Structural Biology, The Institute of Cancer Research, London SW7 3RP, UK Department of Molecular and Cell Biology, University of Leicester, Leicester LE2 9HN, UK Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
authors.forename: Mark W
authors.initials: MW
authors.lastname: Richards
authors.affiliation: Department of Molecular and Cell Biology, University of Leicester, Leicester LE2 9HN, UK.
authors.forename: Anja
authors.initials: A
authors.lastname: Winter
authors.affiliation: Division of Structural Biology, The Institute of Cancer Research, London SW7 3RP, UK.
authors.forename: Fabienne
authors.initials: F
authors.lastname: Beuron
authors.affiliation: Division of Structural Biology, The Institute of Cancer Research, London SW7 3RP, UK edward.morris@icr.ac.uk.
authors.forename: Edward
authors.initials: E
authors.lastname: Morris
authors.affiliation: Division of Structural Biology, The Institute of Cancer Research, London SW7 3RP, UK Department of Molecular and Cell Biology, University of Leicester, Leicester LE2 9HN, UK Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK r.w.bayliss@leeds.ac.uk.
authors.forename: Richard
authors.identifier: http://orcid.org/0000-0003-0604-2773
authors.initials: R
authors.lastname: Bayliss
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D019295:Computational Biology; D064267:Intrinsically Disordered Proteins / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000378:metabolism; D000072417:Protein Domains; D055550:Protein Stability; D064249:Securin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism / Q000648:ultrastructure; D064247:Separase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism / Q000648:ultrastructure
chemical_list: D029742:Caenorhabditis elegans Proteins; D064267:Intrinsically Disordered Proteins; D046912:Multiprotein Complexes; D064249:Securin; D064247:Separase
keywords: chromosome segregation; electron microscopy; mitosis; protein purification