Cryo-EM structure of a metazoan separase-securin complex at near-atomic resolution

Abstract

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.

Figure 1. Overview of the C. elegans separase-securin complex.

a, Schematic of separase and securin. b, Two views of the separase-securin complex. The catalytic dyad of His1014 and Cys1040, and L4 loop indicate the catalytic site. The positions of Insert-1 and Insert-2 relative to the catalytic site (corresponding to the Cα-atom of the P1 Met of securin) are indicated as spheres. Distances between the insert boundaries and the Cα-atom of the P1 residue of securin (Met126^Sec) are shown. c, Two views of the molecular surface of separase showing electrostatic potential, with securin in stick representation. The positively charged surface is indicated (left).

Figure 2. The α-solenoid domain stabilizes the L4 loop.

a, Overview of separase-securin showing the molecular surface of the separase α-solenoid domain. b, Details of the L4 loop interactions with the α-solenoid domain. c, View of the L4 loop docking into the L4-loop binding pocket of the α-solenoid domain shown as a hydrophobic surface colour-ramped from white to green with increasing hydrophobicity. d, The L4 loops of CeSPD and CtSPD adopt an active conformation, with differences confined to the tip of the loop. Securin contacts the L4 loop.

Figure 3. The interactions of the securin pseudo-substrate motif with separase resemble Scc1.

a, Securin pseudo-substrate motif at the CeSPD. b, Scc1-mimicking peptide at CtSPD . c, The molecular surface of Ce separase showing the securin pseudo-substrate sequence engaging the peptide-substrate binding site of separase. d, Details of the Met126(P1) residue at the P1-binding site and its corresponding EM density. e, P1 Arg of the Scc1-mimicking peptide at the P1-site of CtSPD showing the oxyanion hole formed from the catalytic His2083 (from ref. 25). The guanidinium side chain of the P1-Arg residue donates a hydrogen bond to the main-chain carbonyl of Gly2082, positioning the imidazole side chain of His2083 to create the oxyanion hole and donate a hydrogen bond to the main-chain carbonyl of Arg(P1). In contrast, Met126(P1) of C. elegans Scc1 widens the P1-binding pocket C. elegans separase such that the main-chain of Gly1012 and side chain of His1014 (equivalent to Gly2082 and His2083, respectively of CtSPD) are displaced by ~2 Å.

Figure 4. Cryo-EM reconstruction of human separase-securin complex.

a and b, Two views showing the cryo-EM density as a transparent molecular envelop with the coordinates of C. elegans separase-securin fitted to the map as a rigid body. Colour-code according to Fig. 1.