32 33 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 1 / 2 2 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 1 / 2 2 Understandingmolecular scissors to prevent cancer cellsmultiplying Related publication: Yu, J., Raia, P., Ghent, C. M., Raisch, T., Sadian, Y., Cavadini, S., Sabale, P. M., Barford, D., Raunser, S., Morgan, D. O., & Boland, A. Structural basis of human separase regulation by securin and CDK1–cyclin B1. Nature 596 , 138–142 (2021). DOI: 10.1038/s41586-021-03764-0 Publication keywords: Cell cycle regulation; Cohesin; Aneuploidy; Oncogene; Mitosis; Caspase N ew cells formthrough cell division (mitosis), with two genetically identical daughter cells created froma singlemother cell. Aberrant cell division can transformnormal growing cells into cancer cells. Before mitosis, the chromosomes inside the cell are duplicated. The resulting sister chromatids (identical chromosomes) are glued together through a ring-like structure named cohesin. Dissolution of the cohesin complex during cell division allows equal distribution of the sister chromatids into two daughter cells, a process called chromosome segregation. A protein named separase acts as 'molecular scissors' andmediates the cleavage of the cohesin ring. Separase activity is tightly regulated by binding partners that inhibit cleavage activity and ensure a faithful and timely separation. As separase is often hyperactive in cancer cells, understanding themolecularmechanisms of separase regulation at an atomic level will informfuture drugdesign to target separase activity in certain cancer types. Researchers used Cryo-EM at the electron Bio-Imaging Centre (eBIC) to determine structures of human separase with its inhibitory binding partners - securin or the CDK1-cyclin B1-CKS1 (CCC) complex. Although the molecular mechanisms of separase inhibition are fundamentally different, both inhibitors prevent cleavage activity of the molecular scissors by occluding binding of the cohesin subunit SCC1. The teamhas identified multiple binding motifs present in the substrate and inhibitor that are crucial for binding to separase. These motifs mediate binding to specific separase sites andfindingnewdrugs to block these sites could prevent premature chromosome separation, often observed in cancer cells. Arguably, the most fundamental task in the life of a eukaryotic cell is the ordered progression through cell division, in which one parental cell divides into two daughter cells. This process is known as mitosis. In the metaphase of mitosis sister chromatids align at the equator of themitotic spindle before being segregated in anaphase. The transition frommetaphase to anaphase is initiated by the dissolution of sister chromatid cohesion through cleavage of the cohesin complex component SCC1. Untimely and erroneous chromosome segregation leads to genomic instability and causes aneuploidy and tumorigenesis. Separase is the protease responsible for cleavage of the cohesin ring 1 . Human separase is regulated through binding to three different inhibitors 2 . Securin, the first inhibitor identified, is conserved in all eukaryotic cells. In contrast, the CDK1-cyclin B1-CKS1 (CCC) complex seems to be vertebrate specific and interestingly, binding of the CCC complex leads to a complex in which both the kinase activity of CDK1 and the protease activity of separase are inhibited. The third inhibitory complex contains shugoshin 2-MAD2 and has only recently been identified. While the structures of yeast 3 and worm separase 4 in complex with securin were solved in 2007, structural information on human separase remained enigmatic. By using cryogenic Electron Microscopy, the structures of the human separase-securin and separase-CDK1 cyclin B1-CKS1 complexes were determined to high resolution 5 , with the use of the infrastructure at the Diamond Light Source and the Max Planck Institute of Molecular Physiology. The structures provided insights into the regulation of separase activity by Biological Cryo-Imaging Group eBIC these two inhibitory partners.While both complexes abolish substrate cleavage through blocking multiple SCC1-binding sites, the molecular mechanisms underlying substrate-occlusion are fundamentally different. These novel insights into substrate- and inhibitor-binding to separase will guide future structure-based drug design studies. During cell division the DNA content of a cell, the chromosomes, are duplicated so that each emerging daughter cell receives an identical set of sister chromatids. After duplication the sister chromatids arephysically joined together through the ring-shaped cohesin complex. Subsequently, the duplicated and joint chromosomes align at the cell equator and attach to the mitotic spindle. In a final step, sister chromatid segregation and subsequent migration towards the opposite poles of the dividing cell occurs. This process needs to be tightly regulated as aberrant chromosome segregation causes aneuploidy and genomic instability, characteristics of most malignant tumour cells. Timely separation of sister chromatids is initialised through cleavage of the cohesin subunit SCC1 by the cellular protease separase. Human separase is a large enzyme with a molecular weight of roughly 233 kDa. It consists of an N-terminal HEAT-repeat domain, a central TPR-like domain and a highly conserved C-terminal protease domain. Cleavage activity of separase is tightly regulated by different inhibitors in mammalian cells. Recent structural studies showed that securin binds to all three domains of separase and inserts itself into the active site of the protease domain using pseudosubstrate sequence, to serve as a non-cleavable substrate. Human separase is also inhibited by the heterotrimeric complex of cyclin-dependent kinase 1 (CDK1), cyclin B1 or cyclin B2, and the regulatory subunit CKS1 (CCC complex). Interestingly, binding of the CCC complex to separase was shown to be dependent on the phosphorylation of a specific serine residue in separase and results in an inactive kinase- protease complex with both enzymes inhibited. In addition, the observed pseudosubstrate sequences present in securin are absent in the CCC complex, which prompted the question how separase inhibition is mediated by this inhibitory complex. To elucidate the molecular mechanisms of separase inhibition by securin and the CCC complex the authors used cryogenic Electron Microscopy and determined the structures of human separase bound to either securin or the CCC complex 5 (Fig. 1). The structure of separase bound to securin confirmed previous studies in which securin binds in an antiparallel extended orientation to all three domains of separase to occupy multiple binding sites and sterically block substrate recognition (Fig. 1a). The structure of the separase-CCC complex however revealed novel and fascinating aspects of separase regulation. Binding of the CCC complex to the periphery of the protease domain repositions and rigidifies several intrinsically disordered regions in separase itself. As a consequence, these loop regions now occupy binding sites used by securin or the substrate SCC1 to bind to separase and thereby occlude substrate binding. The authors therefore termed these loop segments Autoinhibitory Loops or AILs (Fig. 1b,c). A multiple sequence alignment between the inhibitor securin, the substrate SCC1, and the enzyme separase revealed that the sequence motifs present in these AILs can also be found in SCC1 and securin (Fig. 1d). In other words, these separase binding motifs have evolved multiple times and independently in evolution to occupy similar binding sites in separase. As aforementioned, complex formation between the CCC complex and separase depends on the phosphorylation of a specific serine residue (SepS1126) in separase. This phosphoserine is recognised by a newly identified phospho- binding pocket present in B-type cyclins (Fig. 2a). The authors propose that this binding pocket represents a novel specificity site that recognises and regulates many other cell cycle proteins. Finally, the structure of the separase-CCC complex also revealed the mutually inhibitory nature of this complex. While separase activity is abolished through autoinhibitory loop elements, a CDC6-like sequencemotifs upstreamof the inhibitory motif in AIL3 directly inserts into the active site of CDK1. However, the phospho-acceptor serine is replaced in this CDC6-like sequence by an alanine which prevents phosphorylation of this peptide sequence and thus subsequent dissociation (Fig. 2b). This binding results in a mutual inhibitory complex and converts the protease separase into a regulatory CDK1-inhibitor. This work highlights the multiple and important roles of separase in cell cycle progression and illustrates the power of cryo-EM to visualise regulatory, intrinsically disordered regions that provide crucial insights into the function of enzyme regulation. References: 1. Ciosk, R. et al. An ESP1/PDS1 complex regulates loss of sister chromatid cohesion at the metaphase to anaphase transition in yeast. Cell 93 , 1067–1076 (1998). DOI: 10.1016/S0092-8674(00)81211-8 2. Hauf, S. Two giants of cell division in an oppressive embrace. Nature 596 , 41–42 (2021). DOI : 10.1038/d41586-021-01944-6 3. Luo, S. et al. Molecular mechanism for the regulation of yeast separase by securin. Nature 542 , 255–259 (2017). DOI: 10.1038/nature21061 4. Boland, A. et al. Cryo-EM structure of a metazoan separase–securin complex at near-atomic resolution. Nature Structural &Molecular Biology 24 , 414–418 (2017). DOI: 10.1038/nsmb.3386 5. Yu, J. et al. Structural basis of human separase regulation by securin and CDK1–cyclin B1. Nature 596 , 138–142 (2021). DOI: 10.1038/s41586-021- 03764-0 Funding acknowledgement: This work was supported by the Swiss National Science Foundation (310030_185235) to A.B., the U.S. National Institute of General Medical Sciences (R35-GM118053) to D.O.M, and MRC (MC_UP_1201/6) to D.B. Corresponding author: Dr. Jun Yu, University of Geneva, Jun.Yu@unige.ch Dr. Pierre Raia, University of Geneva, Pierre.Raia@unige.ch Figure 1: Cryo-EM structures of human separase complexes bound to securin or CDK1-cyclin B1-CKS1 (CCC); (a) Separase is depicted in a surface representation in blue, with domains being indicated; Securin is shown in orange; (b) CDK1 (red), cyclin B1 (yellow) and CKS1 (pale yellow) are shown as ribbon representation. The autoinhibitory loops (AILs) of separase are shown in blue (AIL1), purple (AIL2) and cyan (AIL3). The cyclin B1-binding loop containing serine 1126 is shown in green. Of note, the separase N-terminal domain is flexible and excluded in this figure; (c) Overlay of separase AILs with securin which highlights similar binding modes to separase; (d) Multiple sequence alignment between separase, securin and SCC1 reveals common separase binding motifs. Figure 2: CDK1-cyclin B1-CKS1 (CCC) interactions with separase. Colour scheme as in Fig. 1 (a) The CCC complex binds in an interdomain cleft between the protease domain and the TPR-like domain (top). Close-up view of the phosphorylated serine 1126 (pS1126) and it’s recognition by residues lining the phosphate-binding pocket of B-type cyclins; (b) Inhibition of CDK1 by the CDC6-like domain of separase (top); Close-up view of the CDC6-like domain inserted into the active site. The structure of CDK1 bound to a CDC6 peptide was superimposed onto the separase-CCC structure, revealing a similar binding mode of the CDC6-like domain (separase) to the CDC6 peptide. Importantly, the phospho-acceptor serine is replaced with an alanine (SepA1380) rationalising the inhibitory function of separase towards CDK1.