Molecular mechanism for the regulation of yeast separase by securin

Separase has a critical role in dissolving the cohesion among sister chromatids during chromosome segregation (1–7). Separase is over-expressed in human tumors, making it a potential target for drug discovery (8). The protease activity of separase is strictly regulated by the inhibitor securin, which forms a tight complex with separase and may also stabilize this enzyme (9–16). Separases are large, 140–250 kD enzymes, with an N-terminal α-helical region and a caspase-like catalytic domain (CD) at the C-terminus. While crystal structures of the C-terminal two domains of separase (17) and low-resolution electron microscopy reconstructions of the separase-securin complex (18,19) have been reported, the atomic structures of full-length separase and especially the complex with securin are not known. Here we report crystal structures at up to 2.6 Å resolution of the yeast Saccharomyces cerevisiae separase-securin complex. The α-helical region of separase (also known as Esp1) contains four domains (I–IV), and a substrate-binding domain (SD) immediately precedes the CD and has tight associations with it. The separase-securin complex assumes a highly elongated structure. Residues 258–373 of securin (Pds1), named the separase interaction segment (SIS), is primarily in an extended conformation and traverses the entire length of separase, having interactions with all of its domains. Most importantly, residues 258–269 of securin are located in the separase active site, illuminating its mechanism of inhibition. Biochemical studies confirm the structural observations and indicate that contacts outside the separase active site are crucial for stabilizing the complex, thereby defining an important function for the helical region of separase.