A closed conformation of the Caenorhabditis elegans separase-securin complex.

Gudrun Bachmann,Richard Bayliss,Fabienne Beuron,Edward Morris,Mark W Richards,Anja Winter

doi:10.1098/rsob.160032

Gudrun Bachmann, Richard Bayliss + Show 4 more

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https://doi.org/10.1098/rsob.160032

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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.

Highlights

The stages of the eukaryotic cell cycle are defined on the basis of chromosomal events and are referred to as G1, S, G2 and M phase
Fold recognition predictions carried out using HHpred [34] and Phyre2 [35] matched the N-terminal regions of separase from H. sapiens, S. cerevisiae and C. elegans to helical and super-helical structures such as Tpr repeats, and, with less confidence, ARM or HEAT repeats
The core region of the C. elegans separase homologue, comprising the a-helical repeat region and the caspaselike region, is smaller than that of other separase proteins, and so we investigated its suitability as a model system for structural studies

Summary

Introduction

The stages of the eukaryotic cell cycle are defined on the basis of chromosomal events and are referred to as G1, S, G2 and M phase. A cell in G1 phase commits to divide in the presence of favourable growth conditions, or growth signals, and enters S phase, the period when DNA synthesis takes place. Connections between the newly replicated DNA molecules, called sister chromatids, are established [1,2,3], allowing the dividing cell to unambiguously identify chromatids as sisters. Once the chromosomes have been successfully duplicated the cell enters G2 phase. The highly conserved cohesin complex holds the sister chromatid together and contains four core subunits: the kleisin family protein Scc, two subunits of the structural maintenance of chromosomes Smc and Smc, and the accessory subunit Scc3 [4]. The core subunits form a ring-like structure that is thought to topologically encircle the DNA helices of the two sister chromatids [5,6,7]. A protease named separase dissolves the cohesion between the sister chromatids by cleaving Scc at the onset of anaphase [2,8,9,10]

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