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STRUCTURE AND FUNCTION OF CELL CYCLE REGULATORY PROTEINS

SEPARASE

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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" (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.

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. 

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Cyclin B1
Phosphate-binding pocket

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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.

https://www.researchsquare.com/article/rs-4125014/v1

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