Molecular mechanism for the regulation of yeast separase by securin

Abstract

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.

PP2ACdc55dephosphorylates Pds1 to inhibit spindle elongation

“…PP2A Cdc55 regulates Pds1 phosphorylation status both in unperturbed and replication stress conditions specifically at CDK consensus sites necessary for its Esp1 interaction. Recent studies showed that the Pds1 C-terminal segment (residues 258-373) is required for the Esp1 interaction, which is consistent with the idea that phosphorylation at the C-terminal sites located at S277, S292, T304 are required for Esp1 binding (47). They propose a model that S277 should have favorable interactions with Esp1 due to a possible conformational change induced by S292 and T304 phosphorylation.…”

“…They propose a model that S277 should have favorable interactions with Esp1 due to a possible conformational change induced by S292 and T304 phosphorylation. When Pds1 and Esp1 are bound, the Pds1 C-terminal region is located in the Esp1 active site (47). While this explains how Pds1 may block Esp1 protease activity, it remains unclear if the interaction site affects Esp1 function in spindle elongation.…”

Yeast Securin is trafficked through the endomembrane system to regulate cell cycle arrest during the DNA damage response

“…Pds1 is highly unstructured, but when bound to Esp1 they together form an elongated shape. However a crystal structure of Pds1 bound to Esp1 only resolved residues 258-373 bound to Esp1's protease site, leaving open the possibility that the C-terminal of Pds1 may have flexibility to interact with other proteins while still inhibiting Esp1's activity (51).…”

MultiBac: from protein complex structures to synthetic viral nanosystems

“…MultiBac was first adopted by the structural biology community and has enabled structure elucidation of many multiprotein complexes, illuminating their cellular functions. Here, we show a selection of recent structures obtained with MultiBac-produced specimens [22][23][24][25][26][27][28][29][30][31] (Fig. 2).…”

“…Multiprotein complex structural biology. A selection of recent near-atomic structures of MultiBac-produced specimens, determined by X-ray crystallography or electron cryo-microscopy[22][23][24][25][26][27][28][29][30][31]. In the case of mTOR complex 1, heterologous Raptor was produced by using MultiBac and crystallized, and the crystal coordinates fitted into a cryo-EM map of endogenously purified holo-complex in a hybrid approach[31].…”