STRUCTURE AND FUNCTION OF CELL CYCLE REGULATORY PROTEINS
SEPARASE
Separase
Scc1
"Cell division is studied both for its beauty and for the danger that it represents. When all goes well, new healthy cells are born"Â (Silke Hauf, Nature 2021). Aberrant cell division, however, causes the transformation of normal growing cells into cancer cells. To maintain genome stability during cell division, each emerging daughter cell needs to receive an identical set of sister chromatids. This requires precision during two key processes: DNA replication in S phase and segregation of sister chromatids during mitosis (M phase).
In early mitosis, the duplicated chromosomes are held together by the ring-shaped cohesin complex. Separation of chromosomes during anaphase is triggered by separase-a large cysteine endopeptidase that cleaves the cohesin subunit SCC1 (also known as RAD21). Separase is activated by degradation of its inhibitors, securin and cyclin B, but the molecular mechanisms of separase regulation are not clear. We show that both, securin and the Cdk1-cyclin B1-Cks1 complex, inhibit separase by pseudosubstrate motifs that block substrate binding at the catalytic site and at nearby docking sites. As in Caenorhabditis elegans (Boland et al., NSMB, 2017) and yeast, human securin contains its own pseudosubstrate motifs. By contrast, CDK1-cyclin B1 inhibits separase by deploying pseudosubstrate motifs from intrinsically disordered loops in separase itself. One autoinhibitory loop is oriented by CDK1-cyclin B1 to block the catalytic sites of both separase and CDK1. Another autoinhibitory loop blocks substrate docking in a cleft adjacent to the separase catalytic site. A third separase loop contains a phosphoserine that promotes complex assembly by binding to a conserved phosphate-binding pocket in cyclin B1. Our study reveals the diverse array of mechanisms by which securin and CDK1-cyclin B1 bind and inhibit separase, providing the molecular basis for the robust control of chromosome segregation.
Overall structure of inhibitory separase complexes. This video shows the overall architecture of the human separase-securin and the separase- Cdk1-cyclin B1-Cks1 complexes as electron density and ribbon representation while rotating around the X-axis.
Autoinhibitory loops mimic securin binding. This video shows a superposition of the autoinhibitory loop 1 and 3 with securin and highlights common binding motifs that recognise structural elements in separase adjacent to the catalytic site.
Separase and Cdk1-cyclin B1-Cks1 complex assembly. This video describes the structural assembly of the separase-Cdk1-cyclin B1-Cks1 complex. The phosphate-binding pocket in cyclin B1 and the Cdc6-like domain are highlighted. A superposition between a Cdk1 structure bound to a substrate peptide and the separase Cdc6- like domain bound to the catalytic site of Cdk1 illustrates the structural similarities between these two binding partners.
SUBSTRATE RECOGNITION BY HUMAN SEPARASE
The protein complex cohesin encircles the sister chromatids in early mitosis. At anaphase onset, sister separation is triggered by cleavage of the cohesin subunit SCC1/RAD21 by the cysteine protease separase. SCC1 contains two cleavage sites, where cleavage is stimulated by SCC1 phosphorylation. The molecular mechanisms of substrate recognition and cleavage are only partly understood. 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 sites 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.
This Video illustrates how separase recognizes substrates. Specific sequence motifs are highlighted including the recognition of substrates by phosphorylation. Four phosphate-binding sites have been identified in human separase that explain the enhanced cleavage efficiency of substrates upon substrate phosphorylation.
Binding of SCC1 to separase is enhanced by the cohesin subunit STAG2/SA2. This effect can be explained by a stabilization of the second cleavage site next to the catalytic site through a direct interaction between SA2 and separase.
A phosphate-binding pocket in B-type cyclins
Phosphorylation of substrates by cyclin-dependent kinases (CDKs) is the driving force of cell cycle progression. Several CDK-activating cyclins are involved, yet how they contribute to substrate specificity is still poorly understood. Together with the Thomas Mayer group at the University of Konstanz, we recently discovered that a positively charged pocket in cyclin B1, which is exclusively conserved within B-type cyclins and binds phosphorylated serine- or threonine-residues, is essential for correct execution of mitosis.
Cyclin B1
Phosphate-binding pocket
HeLa cells expressing pocket mutant cyclin B1 are strongly delayed in anaphase onset due to multiple defects in mitotic spindle function and timely activation of the E3 ligase APC/C. Pocket integrity is essential for APC/C phosphorylation particularly at non-consensus CDK1 sites and full in vitro ubiquitylation activity. Our results support a model in which cyclin B1’s pocket serves as a specificity site factor for sequential substrate phosphorylations involving initial priming events that facilitate subsequent pocket-dependent phosphorylations even at non-consensus CDK1 motifs.
New structural features of the APC/C revealed by high resolution cryo-EM structures
The multi-subunit anaphase-promoting complex/cyclosome (APC/C) is a master regulator of cell division. It controls progression through the cell cycle by timely marking mitotic cyclins, securin and other cell cycle regulatory proteins for degradation via the ubiquitin-proteasome pathway. The APC/C itself is regulated by the sequential action of its coactivator subunits CDC20 and CDH1, post-translational modifications (see our work on CDK1/cyclin B1), and its inhibitory binding partners EMI1 and the mitotic checkpoint complex (MCC). We determined the structures of human APC/C bound to CDH1 and EMI1 and apo-APC/C at 2.9 Å and 3.2 Å, respectively, providing novel insights into the regulation of APC/C activity. The high resolution maps allowed the unambigious assignment of a previously unassigned α-helix to the N-terminus of CDH1 (CDH1α1) in the ternary complex. CDH1α1 binds at the APC1-APC8 interface, thereby interacting with a loop segment of APC1 through electrostatic interactions only provided by CDH1 but not CDC20. We also indentified a novel zinc-binding module in APC2, and confirmed the presence of zinc ions experimentally. Finally, due to the higher resolution and well defined density of these maps we were able to build, aided by AlphaFold predictions, several intrinsically disordered regions in different APC/C subunits that play a fundamental role in proper complex assembly. The work is summarised in the video, generated by Anna Höfler.
