Phosphate-binding pocket on cyclin B governs CDK substrate phosphorylation and mitotic timing.

Cell cycle progression is governed by complexes of the cyclin-dependent kinases (CDKs) and their regulatory subunits cyclin and Cks1. CDKs phosphorylate hundreds of substrates, often at multiple sites. Multisite phosphorylation depends on Cks1, which binds initial priming phosphorylation sites to promote secondary phosphorylation at other sites. Here, we describe a similar role for a recently discovered phosphate-binding pocket (PP) on B-type cyclins. Mutation of the PP in Clb2, the major mitotic cyclin of budding yeast, alters bud morphology and delays the onset of anaphase. Using phosphoproteomics in vivo and kinase reactions in vitro, we find that mutation of the PP reduces phosphorylation of several CDK substrates, including the Bud6 subunit of the polarisome and the Cdc16 and Cdc27 subunits of the anaphase-promoting complex/cyclosome. We conclude that the cyclin PP, like Cks1, controls the timing of multisite phosphorylation on CDK substrates, thereby helping to establish the robust timing of cell-cycle events.

Assembly of SARS-CoV-2 ribonucleosomes by truncated N∗ variant of the nucleocapsid protein.

The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) compacts the RNA genome into viral ribonucleoprotein (vRNP) complexes within virions. Assembly of vRNPs is inhibited by phosphorylation of the N protein serine/arginine (SR) region. Several SARS-CoV-2 variants of concern carry N protein mutations that reduce phosphorylation and enhance the efficiency of viral packaging. Variants of the dominant B.1.1 viral lineage also encode a truncated N protein, termed N∗ or Δ(1-209), that mediates genome packaging despite lacking the N-terminal RNA-binding domain and SR region. Here, we use mass photometry and negative stain electron microscopy to show that purified Δ(1-209) and viral RNA assemble into vRNPs that are remarkably similar in size and shape to those formed with full-length N protein. We show that assembly of Δ(1-209) vRNPs requires the leucine-rich helix of the central disordered region and that this helix promotes N protein oligomerization. We also find that fusion of a phosphomimetic SR region to Δ(1-209) inhibits RNA binding and vRNP assembly. Our results provide new insights into the mechanisms by which RNA binding promotes N protein self-association and vRNP assembly, and how this process is modulated by phosphorylation.

The molecular mechanisms of human separase regulation.

Sister chromatid segregation is the final irreversible step of mitosis. It is initiated by a complex regulatory system that ultimately triggers the timely activation of a conserved cysteine protease named separase. Separase cleaves the cohesin protein ring that links the sister chromatids and thus facilitates their separation and segregation to the opposite poles of the dividing cell. Due to the irreversible nature of this process, separase activity is tightly controlled in all eukaryotic cells. In this mini-review, we summarize the latest structural and functional findings on the regulation of separase, with an emphasis on the regulation of the human enzyme by two inhibitors, the universal inhibitor securin and the vertebrate-specific inhibitor CDK1-cyclin B. We discuss the two fundamentally different inhibitory mechanisms by which these inhibitors block separase activity by occluding substrate binding. We also describe conserved mechanisms that facilitate substrate recognition and point out open research questions that will guide studies of this fascinating enzyme for years to come.

Reconstitution of the SARS-CoV-2 ribonucleosome provides insights into genomic RNA packaging and regulation by phosphorylation.

The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 is responsible for compaction of the ∼30-kb RNA genome in the ∼90-nm virion. Previous studies suggest that each virion contains 35 to 40 viral ribonucleoprotein (vRNP) complexes, or ribonucleosomes, arrayed along the genome. There is, however, little mechanistic understanding of the vRNP complex. Here, we show that N protein, when combined in vitro with short fragments of the viral genome, forms 15-nm particles similar to the vRNP structures observed within virions. These vRNPs depend on regions of N protein that promote protein-RNA and protein-protein interactions. Phosphorylation of N protein in its disordered serine/arginine region weakens these interactions to generate less compact vRNPs. We propose that unmodified N protein binds structurally diverse regions in genomic RNA to form compact vRNPs within the nucleocapsid, while phosphorylation alters vRNP structure to support other N protein functions in viral transcription.

Reconstitution of the SARS-CoV-2 ribonucleosome provides insights into genomic RNA packaging and regulation by phosphorylation.

The nucleocapsid (N) protein of coronaviruses is responsible for compaction of the ∼30-kb RNA genome in the ∼100-nm virion. Cryo-electron tomography suggests that each virion contains 35-40 viral ribonucleoprotein (vRNP) complexes, or ribonucleosomes, arrayed along the genome. There is, however, little mechanistic understanding of the vRNP complex. Here, we show that N protein, when combined with viral RNA fragments in vitro, forms cylindrical 15-nm particles similar to the vRNP structures observed within coronavirus virions. These vRNPs form in the presence of stem-loop-containing RNA and depend on regions of N protein that promote protein-RNA and protein-protein interactions. Phosphorylation of N protein in its disordered serine/arginine (SR) region weakens these interactions and disrupts vRNP assembly. We propose that unmodified N binds stem-loop-rich regions in genomic RNA to form compact vRNP complexes within the nucleocapsid, while phosphorylated N maintains uncompacted viral RNA to promote the protein's transcriptional function.

Single-molecule analysis of specificity and multivalency in binding of short linear substrate motifs to the APC/C.

Robust regulatory signals in the cell often depend on interactions between short linear motifs (SLiMs) and globular proteins. Many of these interactions are poorly characterized because the binding proteins cannot be produced in the amounts needed for traditional methods. To address this problem, we developed a single-molecule off-rate (SMOR) assay based on microscopy of fluorescent ligand binding to immobilized protein partners. We used it to characterize substrate binding to the Anaphase-Promoting Complex/Cyclosome (APC/C), a ubiquitin ligase that triggers chromosome segregation. We find that SLiMs in APC/C substrates (the D box and KEN box) display distinct affinities and specificities for the substrate-binding subunits of the APC/C, and we show that multiple SLiMs in a substrate generate a high-affinity multivalent interaction. The remarkably adaptable substrate-binding mechanisms of the APC/C have the potential to govern the order of substrate destruction in mitosis.

Multisite phosphorylation by Cdk1 initiates delayed negative feedback to control mitotic transcription.

Cell-cycle progression is driven by the phosphorylation of cyclin-dependent kinase (Cdk) substrates.1-3 The order of substrate phosphorylation depends in part on the general rise in Cdk activity during the cell cycle,4-7 together with variations in substrate docking to sites on associated cyclin and Cks subunits.3,6,8-10 Many substrates are modified at multiple sites to provide more complex regulation.10-14 Here, we describe an elegant regulatory circuit based on multisite phosphorylation of Ndd1, a transcriptional co-activator of budding yeast genes required for mitotic progression.11,12 As cells enter mitosis, Ndd1 phosphorylation by Cdk1 is known to promote mitotic cyclin (CLB2) gene transcription, resulting in positive feedback.13-16 Consistent with these findings, we show that low Cdk1 activity promotes CLB2 expression at mitotic entry. We also find, however, that when high Cdk1 activity accumulates in a mitotic arrest, CLB2 expression is inhibited. Inhibition is accompanied by Ndd1 degradation, and we present evidence that degradation is triggered by multisite Ndd1 phosphorylation by high mitotic Cdk1-Clb2 activity. Complete Ndd1 phosphorylation by Clb2-Cdk1-Cks1 requires the phosphothreonine-binding site of Cks1, as well as a recently identified phosphate-binding pocket on the cyclin Clb2.17 We therefore propose that initial phosphorylation by Cdk1 primes Ndd1 for delayed secondary phosphorylation at suboptimal sites that promote degradation. Together, our results suggest that rising levels of mitotic Cdk1 activity act at multiple phosphorylation sites on Ndd1, first triggering rapid positive feedback and then promoting delayed negative feedback, resulting in a pulse of mitotic gene expression.

Structural basis of human separase regulation by securin and CDK1-cyclin B1.

In early mitosis, the duplicated chromosomes are held together by the ring-shaped cohesin complex1. Separation of chromosomes during anaphase is triggered by separase-a large cysteine endopeptidase that cleaves the cohesin subunit SCC1 (also known as RAD212-4). Separase is activated by degradation of its inhibitors, securin5 and cyclin B6, but the molecular mechanisms of separase regulation are not clear. Here we used cryogenic electron microscopy to determine the structures of human separase in complex with either securin or CDK1-cyclin B1-CKS1. In both complexes, separase is inhibited by pseudosubstrate motifs that block substrate binding at the catalytic site and at nearby docking sites. As in Caenorhabditis elegans7 and yeast8, 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 CDK19,10. Another autoinhibitory loop blocks substrate docking in a cleft adjacent to the separase catalytic site. A third separase loop contains a phosphoserine6 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.

Phosphoregulation of Phase Separation by the SARS-CoV-2 N Protein Suggests a Biophysical Basis for its Dual Functions.

The nucleocapsid (N) protein of coronaviruses serves two major functions: compaction of the RNA genome in the virion and regulation of viral gene transcription. It is not clear how the N protein mediates such distinct functions. The N protein contains two RNA-binding domains surrounded by regions of intrinsic disorder. Phosphorylation of the central disordered region promotes the protein's transcriptional function, but the underlying mechanism is not known. Here, we show that the N protein of SARS-CoV-2, together with viral RNA, forms biomolecular condensates. Unmodified N protein forms partially ordered gel-like condensates and discrete 15-nm particles based on multivalent RNA-protein and protein-protein interactions. Phosphorylation reduces these interactions, generating a more liquid-like droplet. We propose that distinct oligomeric states support the two functions of the N protein: unmodified protein forms a structured oligomer that is suited for nucleocapsid assembly, and phosphorylated protein forms a liquid-like compartment for viral genome processing.

Phosphorylation modulates liquid-liquid phase separation of the SARS-CoV-2 N protein.

The nucleocapsid (N) protein of coronaviruses serves two major functions: compaction of the RNA genome in the virion and regulation of viral gene transcription in the infected cell 1-3 . The N protein contains two globular RNA-binding domains surrounded by regions of intrinsic disorder 4 . Phosphorylation of the central disordered region is required for normal viral genome transcription 5,6 , which occurs in a cytoplasmic structure called the replication transcription complex (RTC) 7-11 . It is not known how phosphorylation controls N protein function. Here we show that the N protein of SARS-CoV-2, together with viral RNA, forms biomolecular condensates 12-15 . Unmodified N protein forms partially ordered gel-like structures that depend on multivalent RNA-protein and protein-protein interactions. Phosphorylation reduces a subset of these interactions, generating a more liquid-like droplet. We speculate that distinct oligomeric states support the two functions of the N protein: unmodified protein forms a structured oligomer that is suited for nucleocapsid assembly, and phosphorylated protein forms a liquid-like compartment for viral genome processing. Inhibitors of N protein phosphorylation could therefore serve as antiviral therapy.

Polyanions provide selective control of APC/C interactions with the activator subunit.

Transient interactions between the anaphase-promoting complex/cyclosome (APC/C) and its activator subunit Cdc20 or Cdh1 generate oscillations in ubiquitylation activity necessary to maintain the order of cell cycle events. Activator binds the APC/C with high affinity and exhibits negligible dissociation kinetics in vitro, and it is not clear how the rapid turnover of APC/C-activator complexes is achieved in vivo. Here, we describe a mechanism that controls APC/C-activator interactions based on the availability of substrates. We find that APC/C-activator dissociation is stimulated by abundant cellular polyanions such as nucleic acids and polyphosphate. Polyanions also interfere with substrate ubiquitylation. However, engagement with high-affinity substrate blocks the inhibitory effects of polyanions on activator binding and APC/C activity. We propose that this mechanism amplifies the effects of substrate affinity on APC/C function, stimulating processive ubiquitylation of high-affinity substrates and suppressing ubiquitylation of low-affinity substrates.

Cohesin cleavage by separase is enhanced by a substrate motif distinct from the cleavage site.

Chromosome segregation begins when the cysteine protease, separase, cleaves the Scc1 subunit of cohesin at the metaphase-to-anaphase transition. Separase is inhibited prior to metaphase by the tightly bound securin protein, which contains a pseudosubstrate motif that blocks the separase active site. To investigate separase substrate specificity and regulation, here we develop a system for producing recombinant, securin-free human separase. Using this enzyme, we identify an LPE motif on the Scc1 substrate that is distinct from the cleavage site and is required for rapid and specific substrate cleavage. Securin also contains a conserved LPE motif, and we provide evidence that this sequence blocks separase engagement of the Scc1 LPE motif. Our results suggest that rapid cohesin cleavage by separase requires a substrate docking interaction outside the active site. This interaction is blocked by securin, providing a second mechanism by which securin inhibits cohesin cleavage.

The pseudosubstrate inhibitor Acm1 inhibits the anaphase-promoting complex/cyclosome by combining high-affinity activator binding with disruption of Doc1/Apc10 function.

The anaphase-promoting complex/cyclosome (APC/C) is a large, multisubunit ubiquitin ligase involved in regulation of cell division. APC/C substrate specificity arises from binding of short degron motifs in its substrates to transient activator subunits, Cdc20 and Cdh1. The destruction box (D-box) is the most common APC/C degron and plays a crucial role in substrate degradation by linking the activator to the Doc1/Apc10 subunit of core APC/C to stabilize the active holoenzyme and promote processive ubiquitylation. Degrons are also employed as pseudosubstrate motifs by APC/C inhibitors, and pseudosubstrates must bind their cognate activators tightly to outcompete substrate binding while blocking their own ubiquitylation. Here we examined how APC/C activity is suppressed by the small pseudosubstrate inhibitor Acm1 from budding yeast (Saccharomyces cerevisiae). Mutation of a conserved D-box converted Acm1 into an efficient ABBA (cyclin A, BubR1, Bub1, Acm1) motif-dependent APC/CCdh1 substrate in vivo, suggesting that this D-box somehow inhibits APC/C. We then identified a short conserved sequence at the C terminus of the Acm1 D-box that was necessary and sufficient for APC/C inhibition. In several APC/C substrates, the corresponding D-box region proved to be important for their degradation despite poor sequence conservation, redefining the D-box as a 12-amino acid motif. Biochemical analysis suggested that the Acm1 D-box extension inhibits reaction processivity by perturbing the normal interaction with Doc1/Apc10. Our results reveal a simple, elegant mode of pseudosubstrate inhibition that combines high-affinity activator binding with specific disruption of Doc1/Apc10 function in processive ubiquitylation.

Firing of Replication Origins Frees Dbf4-Cdc7 to Target Eco1 for Destruction.

Robust progression through the cell-division cycle depends on the precisely ordered phosphorylation of hundreds of different proteins by cyclin-dependent kinases (CDKs) and other kinases. The order of CDK substrate phosphorylation depends on rising CDK activity, coupled with variations in substrate affinities for different CDK-cyclin complexes and the opposing phosphatases [1-4]. Here, we address the ordering of substrate phosphorylation by a second major cell-cycle kinase, Cdc7-Dbf4 or Dbf4-dependent kinase (DDK). The primary function of DDK is to initiate DNA replication by phosphorylating the Mcm2-7 replicative helicase [5-7]. DDK also phosphorylates the cohesin acetyltransferase Eco1 [8]. Sequential phosphorylations of Eco1 by CDK, DDK, and Mck1 create a phosphodegron that is recognized by the ubiquitin ligase SCFCdc4. DDK, despite being activated in early S phase, does not phosphorylate Eco1 to trigger its degradation until late S phase [8]. DDK associates with docking sites on loaded Mcm double hexamers at unfired replication origins [9, 10]. We hypothesized that these docking interactions sequester limiting amounts of DDK, delaying Eco1 phosphorylation by DDK until replication is complete. Consistent with this hypothesis, we find that overproduction of DDK leads to premature Eco1 degradation. Eco1 degradation also occurs prematurely if Mcm complex loading at origins is prevented by depletion of Cdc6, and Eco1 is stabilized if loaded Mcm complexes are prevented from firing by a Cdc45 mutant. We propose that the timing of Eco1 phosphorylation, and potentially that of other DDK substrates, is determined in part by sequestration of DDK at unfired replication origins during S phase.

Building a Regulatory Network with Short Linear Sequence Motifs: Lessons from the Degrons of the Anaphase-Promoting Complex.

The anaphase-promoting complex or cyclosome (APC/C) is a ubiquitin ligase that polyubiquitinates specific substrates at precise times in the cell cycle, thereby triggering the events of late mitosis in a strict order. The robust substrate specificity of the APC/C prevents the potentially deleterious degradation of non-APC/C substrates and also averts the cell-cycle errors and genomic instability that could result from mistimed degradation of APC/C targets. The APC/C recognizes short linear sequence motifs, or degrons, on its substrates. The specific and timely modification and degradation of APC/C substrates is likely to be modulated by variations in degron sequence and context. We discuss the extensive affinity, specificity, and selectivity determinants encoded in APC/C degrons, and we describe some of the extrinsic mechanisms that control APC/C-substrate recognition. As an archetype for protein motif-driven regulation of cell function, the APC/C-substrate interaction provides insights into the general properties of post-translational regulatory systems.

Cell Size Determines the Strength of the Spindle Assembly Checkpoint during Embryonic Development.

The spindle assembly checkpoint (SAC) delays mitotic progression when chromosomes are not properly attached to microtubules of the mitotic spindle. Cells vary widely in the extent to which they delay mitotic progression upon SAC activation. To explore the mechanisms that determine checkpoint strength in different cells, we systematically measured the mitotic delay induced by microtubule disruption at different stages of embryogenesis in Caenorhabditis elegans. Strikingly, we observed a gradual increase in SAC strength after each round of division. Analysis of mutants that alter cell size or ploidy revealed that SAC strength is determined primarily by cell size and the number of kinetochores. These findings provide clear evidence in vivo that the kinetochore-to-cytoplasm ratio determines the strength of the SAC, providing new insights into why cells exhibit such large variations in their SAC responses.

Quantitative framework for ordered degradation of APC/C substrates.

BACKGROUND: During cell-cycle progression, substrates of a single master regulatory enzyme can be modified in a specific order. Here, we used experimental and computational approaches to dissect the quantitative mechanisms underlying the ordered degradation of the substrates of the ubiquitin ligase APC/C(Cdc20), a key regulator of chromosome segregation in mitosis. RESULTS: We show experimentally that the rate of catalysis varies with different substrates of APC/C(Cdc20). Using a computational model based on multi-step ubiquitination, we then show how changes in the interaction between a single substrate and APC/C(Cdc20) can alter the timing of degradation onset relative to APC/C(Cdc20) activation, while ensuring a fast degradation rate. Degradation timing and dynamics depend on substrate affinity for the enzyme as well as the catalytic rate at which the substrate is modified. When two substrates share the same pool of APC/C(Cdc20), their relative enzyme affinities and rates of catalysis influence the partitioning of APC/C(Cdc20) among substrates, resulting in substrate competition. Depending on how APC/C(Cdc20) is partitioned among its substrates, competition can have minor or major effects on the degradation of certain substrates. We show experimentally that increased expression of the early APC/C(Cdc20) substrate Clb5 does not delay the degradation of the later substrate securin, arguing against a role for competition with Clb5 in establishing securin degradation timing. CONCLUSIONS: The degradation timing of APC/C(Cdc20) substrates depends on the multi-step nature of ubiquitination, differences in substrate-APC/C(Cdc20) interactions, and competition among substrates. Our studies provide a conceptual framework for understanding how ordered modification can be established among substrates of the same regulatory enzyme, and facilitate our understanding of how precise temporal control is achieved by a small number of master regulators to ensure a successful cell division cycle.

An E2 accessory domain increases affinity for the anaphase-promoting complex and ensures E2 competition.

The anaphase-promoting complex/cyclosome (APC/C) is a member of the RING family of E3 ubiquitin ligases, which promote ubiquitin transfer from an E2 ubiquitin-conjugating enzyme to a substrate. In budding yeast, the APC/C collaborates with two E2s, Ubc4 and Ubc1, to promote the initiation and elongation, respectively, of polyubiquitin chains on the substrate. Ubc4 and Ubc1 are thought to compete for the same site on the APC/C, but it is not clear how their affinities are balanced. Here, we demonstrate that a C-terminal ubiquitin-associated (UBA) domain enhances the affinity of Ubc1 for the APC/C. Deletion of the UBA domain reduced apparent APC/C affinity for Ubc1 and decreased polyubiquitin chain length. Surprisingly, the positive effect of the UBA domain was not due to an interaction with the acceptor ubiquitin attached to the APC/C substrate or the donor ubiquitin attached to Ubc1 itself. Instead, our evidence suggests that the UBA domain binds to a site on the APC/C core, thereby increasing Ubc1 affinity and enhancing its ability to compete with Ubc4. The UBA domain is required for normal Ubc1 function and E2 competition in vivo. Thus, the UBA domain of Ubc1 ensures efficient polyubiquitination of substrate by balancing Ubc1 affinity with that of Ubc4.

Multiple mechanisms determine the order of APC/C substrate degradation in mitosis.

The ubiquitin protein ligase anaphase-promoting complex or cyclosome (APC/C) controls mitosis by promoting ordered degradation of securin, cyclins, and other proteins. The mechanisms underlying the timing of APC/C substrate degradation are poorly understood. We explored these mechanisms using quantitative fluorescence microscopy of GFP-tagged APC/C(Cdc20) substrates in living budding yeast cells. Degradation of the S cyclin, Clb5, begins early in mitosis, followed 6 min later by the degradation of securin and Dbf4. Anaphase begins when less than half of securin is degraded. The spindle assembly checkpoint delays the onset of Clb5 degradation but does not influence securin degradation. Early Clb5 degradation depends on its interaction with the Cdk1-Cks1 complex and the presence of a Cdc20-binding "ABBA motif" in its N-terminal region. The degradation of securin and Dbf4 is delayed by Cdk1-dependent phosphorylation near their Cdc20-binding sites. Thus, a remarkably diverse array of mechanisms generates robust ordering of APC/C(Cdc20) substrate destruction.