ABSTRACT
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.
Subject(s)
COVID-19 , Coronavirus Nucleocapsid Proteins/chemistry , Protein Multimerization , RNA, Viral/chemistry , SARS-CoV-2/chemistry , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus Nucleocapsid Proteins/metabolism , Humans , Phosphoproteins/chemistry , Phosphoproteins/genetics , Phosphoproteins/metabolism , Phosphorylation , Protein Domains , RNA, Viral/genetics , RNA, Viral/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolismABSTRACT
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.
Subject(s)
CDC2 Protein Kinase/chemistry , CDC2 Protein Kinase/metabolism , Cyclin B1/chemistry , Cyclin B1/metabolism , Securin/chemistry , Securin/metabolism , Separase/chemistry , Separase/metabolism , Amino Acid Motifs , CDC2 Protein Kinase/antagonists & inhibitors , CDC2 Protein Kinase/ultrastructure , CDC2-CDC28 Kinases/chemistry , CDC2-CDC28 Kinases/metabolism , CDC2-CDC28 Kinases/ultrastructure , Cell Cycle Proteins/metabolism , Chromosome Segregation , Cryoelectron Microscopy , Cyclin B1/ultrastructure , DNA-Binding Proteins/metabolism , Humans , Models, Molecular , Phosphoserine/metabolism , Protein Binding , Protein Domains , Securin/ultrastructure , Separase/antagonists & inhibitors , Separase/ultrastructure , Substrate SpecificityABSTRACT
The cell divides in a series of discrete steps that occur in a specific order. Lu and Cross (2010) now propose that cell-cycle events are ordered by a regulatory system in which a master oscillator, based on cyclin-dependent kinases, entrains a series of peripheral oscillators controlling individual events.
ABSTRACT
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.
Subject(s)
Nucleocapsid Proteins , SARS-CoV-2 , Humans , COVID-19/virology , Nucleocapsid Proteins/genetics , Nucleocapsid Proteins/metabolism , Nucleocapsid Proteins/ultrastructure , RNA, Viral/metabolism , RNA, Viral/ultrastructure , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , SARS-CoV-2/ultrastructure , Phosphorylation , Virus Assembly/geneticsABSTRACT
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.
Subject(s)
Anaphase-Promoting Complex-Cyclosome/metabolism , Anaphase , Cdc20 Proteins/metabolism , Protein Processing, Post-Translational , Amino Acid Motifs , Anaphase-Promoting Complex-Cyclosome/chemistry , Anaphase-Promoting Complex-Cyclosome/genetics , Animals , Binding Sites , Cdc20 Proteins/chemistry , Cdc20 Proteins/genetics , Humans , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Proteolysis , Sequence Alignment , Sequence Homology, Amino Acid , Substrate Specificity , UbiquitinationABSTRACT
The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 is responsible for compaction of the â¼30-kb RNA genome in the â¼90-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.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , Phosphorylation , RNA, Viral/metabolism , COVID-19/genetics , Nucleocapsid Proteins/metabolism , Ribonucleoproteins/metabolism , GenomicsABSTRACT
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.
Subject(s)
Cell Cycle Proteins , Mitosis , Humans , Separase/chemistry , Separase/genetics , Separase/metabolism , Cell Cycle Proteins/metabolism , Endopeptidases/geneticsABSTRACT
In this issue of Molecular Cell, He and colleagues (2013) unveil a high-resolution structure of a key regulatory interface in cell-cycle control: the destruction box sequence bound to the anaphase-promoting complex.
Subject(s)
Repressor Proteins/chemistry , Repressor Proteins/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Ubiquitin-Protein Ligase Complexes/metabolism , Anaphase-Promoting Complex-Cyclosome , Animals , Cdh1 Proteins , Cell Cycle ProteinsABSTRACT
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.
Subject(s)
Apc10 Subunit, Anaphase-Promoting Complex-Cyclosome/metabolism , Cell Cycle Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Amino Acid Motifs , Cell Cycle , Cell Cycle Proteins/chemistry , Protein Interaction Maps , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae Proteins/chemistry , Substrate Specificity , UbiquitinationABSTRACT
The fidelity of chromosome segregation depends on the spindle assembly checkpoint (SAC). In the presence of unattached kinetochores, anaphase is delayed when three SAC components (Mad2, Mad3/BubR1, and Bub3) inhibit Cdc20, the activating subunit of the anaphase-promoting complex (APC/C). We analyzed the role of Cdc20 autoubiquitination in the SAC of budding yeast. Reconstitution with purified components revealed that a Mad3-Bub3 complex synergizes with Mad2 to lock Cdc20 on the APC/C and stimulate Cdc20 autoubiquitination, while inhibiting ubiquitination of substrates. SAC-dependent Cdc20 autoubiquitination required the Mnd2/Apc15 subunit of the APC/C. General inhibition of Cdc20 ubiquitination in vivo resulted in high Cdc20 levels and a failure to establish a SAC arrest, suggesting that SAC establishment depends on low Cdc20 levels. Specific inhibition of SAC-dependent ubiquitination, by deletion of Mnd2, allowed establishment of a SAC arrest but delayed release from the arrest, suggesting that Cdc20 ubiquitination is also required for SAC inactivation.
Subject(s)
Chromosome Segregation , M Phase Cell Cycle Checkpoints , Spindle Apparatus/metabolism , Ubiquitin-Protein Ligase Complexes/metabolism , Anaphase-Promoting Complex-Cyclosome , Cdc20 Proteins , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Mad2 Proteins , Nuclear Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Spindle Apparatus/genetics , UbiquitinationABSTRACT
The final stages of mitosis begin in anaphase, when the mitotic spindle segregates the duplicated chromosomes. Mitotic exit is then completed by disassembly of the spindle and packaging of chromosomes into daughter nuclei. The successful completion of mitosis requires that these events occur in a strict order. Two main mechanisms govern progression through late mitosis: dephosphorylation of cyclin-dependent kinase (Cdk) substrates and destruction of the substrates of the anaphase-promoting complex (APC). Here, we discuss the hypothesis that the order of late mitotic events depends, at least in part, on the order in which different Cdk and APC substrates are dephosphorylated or destroyed, respectively.
Subject(s)
Mitosis , Anaphase-Promoting Complex-Cyclosome , Animals , Cyclins/metabolism , Humans , Phosphoprotein Phosphatases/metabolism , Phosphorylation , Substrate Specificity , Ubiquitin-Protein Ligase Complexes/metabolismABSTRACT
Accurate genome segregation depends on cohesion mechanisms that link duplicated sister chromatids, thereby allowing their tension-dependent biorientation in metaphase. In Saccharomyces cerevisiae, cohesion is established during DNA replication when Eco1 acetylates the cohesin subunit Smc3. Cohesion establishment is restricted to S phase of the cell cycle, but the molecular basis of this regulation is unknown. Here, we show that Eco1 is negatively regulated by the protein kinase Cdk1. Phosphorylation of Eco1 after S phase targets it to SCF(Cdc4) for ubiquitination and subsequent degradation. A nonphosphorylatable mutant of Eco1 establishes cohesion after DNA replication, suggesting that Cdk1-dependent phosphorylation of Eco1 is a key factor limiting establishment to S phase. We also show that deregulation of Eco1 results in chromosome separation defects in anaphase. We conclude that this regulatory mechanism helps optimize the level of sister chromatid cohesion, ensuring a robust and efficient anaphase.
Subject(s)
Acetyltransferases/metabolism , CDC2 Protein Kinase/metabolism , Nuclear Proteins/metabolism , S Phase , Saccharomyces cerevisiae Proteins/metabolism , Acetylation , Acetyltransferases/genetics , Anaphase , Blotting, Western , CDC2 Protein Kinase/genetics , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Chromosome Segregation , DNA Replication , F-Box Proteins/metabolism , Mutation , Nuclear Proteins/genetics , Phosphorylation , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Time Factors , Ubiquitin-Protein Ligases/metabolism , Ubiquitination , CohesinsABSTRACT
Cdk specificity is determined by the intrinsic selectivity of the active site and by substrate docking sites on the cyclin subunit. There is a long-standing debate about the relative importance of these factors in the timing of Cdk1 substrate phosphorylation. We analyzed major budding yeast cyclins (the G1/S-cyclin Cln2, S-cyclin Clb5, G2/M-cyclin Clb3, and M-cyclin Clb2) and found that the activity of Cdk1 toward the consensus motif increased gradually in the sequence Cln2-Clb5-Clb3-Clb2, in parallel with cell cycle progression. Further, we identified a docking element that compensates for the weak intrinsic specificity of Cln2 toward G1-specific targets. In addition, Cln2-Cdk1 showed distinct consensus site specificity, suggesting that cyclins do not merely activate Cdk1 but also modulate its active-site specificity. Finally, we identified several Cln2-, Clb3-, and Clb2-specific Cdk1 targets. We propose that robust timing and ordering of cell cycle events depend on gradual changes in the substrate specificity of Cdk1.
Subject(s)
CDC2 Protein Kinase/metabolism , Cell Cycle , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Amino Acid Motifs , Binding Sites , CDC2 Protein Kinase/chemistry , Consensus Sequence , Hydrophobic and Hydrophilic Interactions , Models, Biological , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae Proteins/chemistry , Substrate SpecificitySubject(s)
Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Animals , Cell Cycle , HumansSubject(s)
Cell Cycle , Drosophila melanogaster/physiology , Saccharomyces cerevisiae/physiology , Schizosaccharomyces/physiology , Xenopus laevis/physiology , Anaphase-Promoting Complex-Cyclosome , Animals , Cyclin-Dependent Kinases/metabolism , Cyclins/metabolism , Humans , Models, Biological , Ubiquitin-Protein Ligase Complexes/metabolism , Cyclin-Dependent Kinase-Activating KinaseABSTRACT
Protein ubiquitination is catalyzed by ubiquitin-conjugating enzymes (E2s) in collaboration with ubiquitin-protein ligases (E3s). This process depends on nucleophilic attack by a substrate lysine on a thioester bond linking the C terminus of ubiquitin to a cysteine in the E2 active site. Different E2 family members display specificity for lysines in distinct contexts. We addressed the mechanistic basis for this lysine selectivity in Ubc1, an E2 that catalyzes the ubiquitination of lysine 48 (K48) in ubiquitin, leading to the formation of K48-linked polyubiquitin chains. We identified a cluster of polar residues near the Ubc1 active site, as well as a residue in ubiquitin itself, that are required for catalysis of K48-specific ubiquitin ligation, but not for general activity toward other lysines. Our results suggest that the active site of Ubc1, as well as the surface of ubiquitin, contains specificity determinants that channel specific lysines to the central residues involved directly in catalysis.
Subject(s)
Protein Processing, Post-Translational , Saccharomyces cerevisiae Proteins/metabolism , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitin/metabolism , Alanine , Binding Sites , Catalysis , Catalytic Domain , Glutamine , Hydrogen-Ion Concentration , Kinetics , Leucine , Lysine , Models, Molecular , Polyubiquitin/metabolism , Protein Conformation , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Structure-Activity Relationship , Threonine , Tyrosine , Ubiquitin-Conjugating Enzymes/chemistry , Ubiquitin-Conjugating Enzymes/genetics , UbiquitinationABSTRACT
Multisite phosphorylation of proteins has been proposed to transform a graded protein kinase signal into an ultrasensitive switch-like response. Although many multiphosphorylated targets have been identified, the dynamics and sequence of individual phosphorylation events within the multisite phosphorylation process have never been thoroughly studied. In Saccharomyces cerevisiae, the initiation of S phase is thought to be governed by complexes of Cdk1 and Cln cyclins that phosphorylate six or more sites on the Clb5-Cdk1 inhibitor Sic1, directing it to SCF-mediated destruction. The resulting Sic1-free Clb5-Cdk1 complex triggers S phase. Here, we demonstrate that Sic1 destruction depends on a more complex process in which both Cln2-Cdk1 and Clb5-Cdk1 act in processive multiphosphorylation cascades leading to the phosphorylation of a small number of specific phosphodegrons. The routes of these phosphorylation cascades are shaped by precisely oriented docking interactions mediated by cyclin-specific docking motifs in Sic1 and by Cks1, the phospho-adaptor subunit of Cdk1. Our results indicate that Clb5-Cdk1-dependent phosphorylation generates positive feedback that is required for switch-like Sic1 destruction. Our evidence for a docking network within clusters of phosphorylation sites uncovers a new level of complexity in Cdk1-dependent regulation of cell cycle transitions, and has general implications for the regulation of cellular processes by multisite phosphorylation.
Subject(s)
Cyclin-Dependent Kinase Inhibitor Proteins/metabolism , S Phase/physiology , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Binding Sites , CDC2 Protein Kinase/metabolism , Cell Cycle Proteins/metabolism , Cyclin B/metabolism , Cyclins/metabolism , Phosphorylation , ProteolysisABSTRACT
The anaphase-promoting complex or cyclosome (APC/C) is a ubiquitin ligase essential for the completion of mitosis in all eukaryotic cells. Substrates are recruited to the APC/C by activator proteins (Cdc20 or Cdh1), but it is not known where substrates are bound during catalysis. We explored this problem by analyzing mutations in the tetratricopeptide-repeat-containing APC/C subunits. We identified residues in Cdc23 and Cdc27 that are required for APC/C binding to Cdc20 and Cdh1 and for APC/C function in vivo. Mutation of these sites increased the rate of activator dissociation from the APC/C but did not affect reaction processivity, suggesting that the mutations have little effect on substrate dissociation from the active site. Further studies revealed that activator dissociation from the APC/C is inhibited by substrate, and that substrates are not bound solely to activator during catalysis but interact bivalently with an additional binding site on the APC/C core.
Subject(s)
Saccharomyces cerevisiae/enzymology , Ubiquitin-Protein Ligase Complexes/metabolism , Amino Acid Motifs , Amino Acid Sequence , Anaphase-Promoting Complex-Cyclosome , Apc8 Subunit, Anaphase-Promoting Complex-Cyclosome , Binding Sites , Cdc20 Proteins , Cdh1 Proteins , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Enzyme Activation , Models, Biological , Molecular Sequence Data , Mutation , Protein Binding/physiology , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Sequence Analysis, Protein , Ubiquitin-Protein Ligase Complexes/chemistryABSTRACT
Cik1, in association with the kinesin Kar3, controls both the mitotic spindle and nuclear fusion during mating. Here, we show that there are two Cik1 isoforms, and that the mitotic form includes an N-terminal domain required for ubiquitination by the Anaphase-Promoting Complex/Cyclosome (APC/C). During vegetative growth, Cik1 is expressed during mitosis and regulates the mitotic spindle, allowing for accurate chromosome segregation. After mitosis, APC/C(Cdh1) targets Cik1 for ubiquitin-mediated proteolysis. Upon exposure to the mating pheromone alpha factor, a smaller APC/C-resistant Cik1 isoform is expressed from an alternate transcriptional start site. This shorter Cik1 isoform is stable and cannot be ubiquitinated by APC/C(Cdh1). Moreover, the two Cik1 isoforms are functionally distinct. Cells that express only the long isoform have defects in nuclear fusion, whereas cells expressing only the short isoform have an increased rate of chromosome loss. These results demonstrate a coupling of transcriptional regulation and APC/C-mediated proteolysis.