Substrate recognition by human separase

The protein complex cohesin encircles the sister chromatids in early mitosis1. At anaphase onset, sister separation is triggered by cleavage of the cohesin subunit SCC1/RAD21 by the cysteine protease separase2-5. SCC1 contains two cleavage sites, where cleavage is stimulated by SCC1 phosphorylation5,6. The molecular mechanisms of substrate recognition and cleavage are only partly understood7. Here, we determined a series of cryoEM structures of human separase in apo-or substrate-bound forms that, together with biochemical analysis, provide novel insights into the regulation of separase cleavage activity. We verify the first SCC1 cleavage site and reassign the second site. We show that multiple substrates, including separase autocleavage sites8,9 and the two SCC1 cleavage sites, interact with several docking sites in separase, including four phosphate-binding sites. We also describe the structural basis of the interaction between the cohesin subunit SA1/A2 and separase, which promotes cleavage at the second site in SCC1. Finally, using cross-linking mass spectrometry and cryoEM, we propose a model of how cohesin is targeted by human separase. Our work provides an extensive functional and structural framework that explains one of the most fundamental events in cell division.