ABSTRACT
The anaphase-promoting complex/cyclosome (APC/C) regulates sister chromatid segregation and the exit from mitosis. Selection of most APC/C substrates is controlled by coactivator subunits (either Cdc20 or Cdh1) that interact with substrate destruction motifs--predominantly the destruction (D) box and KEN box degrons. How coactivators recognize D box degrons and how this is inhibited by APC/C regulatory proteins is not defined at the atomic level. Here, from the crystal structure of S. cerevisiae Cdh1 in complex with its specific inhibitor Acm1, which incorporates D and KEN box pseudosubstrate motifs, we describe the molecular basis for D box recognition. Additional interactions between Acm1 and Cdh1 identify a third protein-binding site on Cdh1 that is likely to confer coactivator-specific protein functions including substrate association. We provide a structural rationalization for D box and KEN box recognition by coactivators and demonstrate that many noncanonical APC/C degrons bind APC/C coactivators at the D box coreceptor.
Subject(s)
Repressor Proteins/chemistry , Repressor Proteins/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Ubiquitin-Protein Ligase Complexes/metabolism , Amino Acid Motifs , Anaphase-Promoting Complex-Cyclosome , Animals , Binding Sites , Cdh1 Proteins , Cell Cycle Proteins , Crystallography, X-Ray , Models, Molecular , Protein Conformation , Protein Multimerization , Repressor Proteins/genetics , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
In mitosis, the spindle assembly checkpoint (SAC) ensures genome stability by delaying chromosome segregation until all sister chromatids have achieved bipolar attachment to the mitotic spindle. The SAC is imposed by the mitotic checkpoint complex (MCC), whose assembly is catalysed by unattached chromosomes and which binds and inhibits the anaphase-promoting complex/cyclosome (APC/C), the E3 ubiquitin ligase that initiates chromosome segregation. Here, using the crystal structure of Schizosaccharomyces pombe MCC (a complex of mitotic spindle assembly checkpoint proteins Mad2, Mad3 and APC/C co-activator protein Cdc20), we reveal the molecular basis of MCC-mediated APC/C inhibition and the regulation of MCC assembly. The MCC inhibits the APC/C by obstructing degron recognition sites on Cdc20 (the substrate recruitment subunit of the APC/C) and displacing Cdc20 to disrupt formation of a bipartite D-box receptor with the APC/C subunit Apc10. Mad2, in the closed conformation (C-Mad2), stabilizes the complex by optimally positioning the Mad3 KEN-box degron to bind Cdc20. Mad3 and p31(comet) (also known as MAD2L1-binding protein) compete for the same C-Mad2 interface, which explains how p31(comet) disrupts MCC assembly to antagonize the SAC. This study shows how APC/C inhibition is coupled to degron recognition by co-activators.
Subject(s)
Cell Cycle Proteins/chemistry , M Phase Cell Cycle Checkpoints , Multiprotein Complexes/chemistry , Nuclear Proteins/chemistry , Schizosaccharomyces pombe Proteins/chemistry , Schizosaccharomyces/chemistry , Amino Acid Motifs , Anaphase-Promoting Complex-Cyclosome , Cdc20 Proteins , Cdh1 Proteins , Cell Cycle Proteins/metabolism , Conserved Sequence , Crystallography, X-Ray , Humans , Mad2 Proteins , Models, Molecular , Multiprotein Complexes/metabolism , Nuclear Proteins/metabolism , Protein Structure, Quaternary , Protein Structure, Tertiary , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Spindle Apparatus , Structure-Activity Relationship , Substrate Specificity , Ubiquitin-Protein Ligase Complexes/antagonists & inhibitors , Ubiquitin-Protein Ligase Complexes/chemistry , Ubiquitin-Protein Ligase Complexes/metabolism , Ubiquitin-Protein Ligase Complexes/ultrastructureABSTRACT
In metazoans, the Ras-Raf-MEK (mitogen-activated protein-kinase kinase)-ERK (extracellular signal-regulated kinase) signalling pathway relays extracellular stimuli to elicit changes in cellular function and gene expression. Aberrant activation of this pathway through oncogenic mutations is responsible for a large proportion of human cancer. Kinase suppressor of Ras (KSR) functions as an essential scaffolding protein to coordinate the assembly of Raf-MEK-ERK complexes. Here we integrate structural and biochemical studies to understand how KSR promotes stimulatory Raf phosphorylation of MEK (refs 6, 7). We show, from the crystal structure of the kinase domain of human KSR2 (KSR2(KD)) in complex with rabbit MEK1, that interactions between KSR2(KD) and MEK1 are mediated by their respective activation segments and C-lobe αG helices. Analogous to BRAF (refs 8, 9), KSR2 self-associates through a side-to-side interface involving Arg 718, a residue identified in a genetic screen as a suppressor of Ras signalling. ATP is bound to the KSR2(KD) catalytic site, and we demonstrate KSR2 kinase activity towards MEK1 by in vitro assays and chemical genetics. In the KSR2(KD)-MEK1 complex, the activation segments of both kinases are mutually constrained, and KSR2 adopts an inactive conformation. BRAF allosterically stimulates the kinase activity of KSR2, which is dependent on formation of a side-to-side KSR2-BRAF heterodimer. Furthermore, KSR2-BRAF heterodimerization results in an increase of BRAF-induced MEK phosphorylation via the KSR2-mediated relay of a signal from BRAF to release the activation segment of MEK for phosphorylation. We propose that KSR interacts with a regulatory Raf molecule in cis to induce a conformational switch of MEK, facilitating MEK's phosphorylation by a separate catalytic Raf molecule in trans.
Subject(s)
MAP Kinase Kinase 1/chemistry , MAP Kinase Kinase 1/metabolism , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins B-raf/metabolism , Adenosine Triphosphate/metabolism , Allosteric Regulation/physiology , Animals , Biocatalysis , Catalytic Domain , Crystallography, X-Ray , Enzyme Activation , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , Models, Molecular , Phosphorylation , Protein Multimerization , Protein Structure, Quaternary , Proto-Oncogene Proteins B-raf/chemistry , Proto-Oncogene Proteins B-raf/genetics , Rabbits , Signal TransductionABSTRACT
Mechanistic and structural studies of large multi-subunit assemblies are greatly facilitated by their reconstitution in heterologous recombinant systems. In the present paper, we describe the generation of recombinant human APC/C (anaphase-promoting complex/cyclosome), an E3 ubiquitin ligase that regulates cell-cycle progression. Human APC/C is composed of 14 distinct proteins that assemble into a complex of at least 19 subunits with a combined molecular mass of ~1.2 MDa. We show that recombinant human APC/C is correctly assembled, as judged by its capacity to ubiquitinate the budding yeast APC/C substrate Hsl1 (histone synthetic lethal 1) dependent on the APC/C co-activator Cdh1 [Cdc (cell division cycle) 20 homologue 1], and its three-dimensional reconstruction by electron microscopy and single-particle analysis. Successful reconstitution validates the subunit composition of human APC/C. The structure of human APC/C is compatible with the Saccharomyces cerevisiae APC/C homology model, and in contrast with endogenous human APC/C, no evidence for conformational flexibility of the TPR (tetratricopeptide repeat) lobe is observed. Additional density present in the human APC/C structure, proximal to Apc3/Cdc27 of the TPR lobe, is assigned to the TPR subunit Apc7, a subunit specific to vertebrate APC/C.
Subject(s)
Multiprotein Complexes/metabolism , Recombinant Proteins/metabolism , Ubiquitin-Protein Ligase Complexes/metabolism , Ubiquitin-Protein Ligases/metabolism , Anaphase-Promoting Complex-Cyclosome , Animals , Apc3 Subunit, Anaphase-Promoting Complex-Cyclosome , Apc7 Subunit, Anaphase-Promoting Complex-Cyclosome , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line , Humans , Microscopy, Electron , Models, Molecular , Multiprotein Complexes/chemistry , Multiprotein Complexes/genetics , Protein Multimerization , Protein Structure, Quaternary , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/ultrastructure , Substrate Specificity , Ubiquitin-Protein Ligase Complexes/chemistry , Ubiquitin-Protein Ligase Complexes/genetics , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/genetics , UbiquitinationABSTRACT
CDC20 is a coactivator of the anaphase promoting complex/cyclosome (APC/C) and is essential for mitotic progression. APC/CCDC20 is inhibited by the spindle assembly checkpoint (SAC), which prevents premature separation of sister chromatids and aneuploidy in daughter cells. Although overexpression of CDC20 is common in many cancers, oncogenic mutations have never been identified in humans. Using whole-exome sequencing, we identified heterozygous missense CDC20 variants (L151R and N331K) that segregate with ovarian germ cell tumors in two families. Functional characterization showed these mutants retain APC/C activation activity but have impaired binding to BUBR1, a component of the SAC. Expression of L151R and N331K variants promoted mitotic slippage in HeLa cells and primary skin fibroblasts derived from carriers. Generation of mice carrying the N331K variant using CRISPR-Cas9 showed that, although homozygous N331K mice were nonviable, heterozygotes displayed accelerated oncogenicity of Myc-driven cancers. These findings highlight an unappreciated role for CDC20 variants as tumor-promoting genes. SIGNIFICANCE: Two germline CDC20 missense variants that segregate with cancer in two families compromise the spindle assembly checkpoint and lead to aberrant mitotic progression, which could predispose cells to transformation. See related commentary by Villarroya-Beltri and Malumbres, p. 3432.
Subject(s)
Neoplasms , Spindle Apparatus , Anaphase-Promoting Complex-Cyclosome/genetics , Animals , Cdc20 Proteins/genetics , Cdc20 Proteins/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Germ Cells/metabolism , HeLa Cells , Humans , Mice , Mitosis/genetics , Neoplasms/metabolism , Protein Binding , Spindle Apparatus/metabolismABSTRACT
Sister-chromatid cohesion is established by Eco1-mediated acetylation on two conserved tandem lysines in the cohesin Smc3 subunit. However, the molecular basis of Eco1 substrate recognition and acetylation in cohesion is not fully understood. Here, we discover and rationalize the substrate specificity of Eco1 using mass spectrometry coupled with in-vitro acetylation assays and crystallography. Our structures of the X. laevis Eco2 (xEco2) bound to its primary and secondary Smc3 substrates demonstrate the plasticity of the substrate-binding site, which confers substrate specificity by concerted conformational changes of the central ß hairpin and the C-terminal extension.
Subject(s)
Acetyltransferases/chemistry , Cell Cycle Proteins/chemistry , Chromosomal Proteins, Non-Histone/chemistry , Chromosome Segregation , Nuclear Proteins/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Xenopus Proteins/chemistry , Acetylation , Acetyltransferases/genetics , Acetyltransferases/metabolism , Amino Acid Sequence , Animals , Binding Sites , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Crystallography, X-Ray , Gene Expression , Models, Molecular , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/ultrastructure , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Substrate Specificity , Xenopus Proteins/genetics , Xenopus Proteins/metabolism , Xenopus laevis/genetics , Xenopus laevis/metabolismABSTRACT
The functions of cohesin are central to genome integrity, chromosome organization and transcription regulation through its prevention of premature sister-chromatid separation and the formation of DNA loops. The loading of cohesin onto chromatin depends on the Scc2-Scc4 complex; however, little is known about how it stimulates the cohesion-loading activity. Here we determine the large 'hook' structure of Scc2 responsible for catalysing cohesin loading. We identify key Scc2 surfaces that are crucial for cohesin loading in vivo. With the aid of previously determined structures and homology modelling, we derive a pseudo-atomic structure of the full-length Scc2-Scc4 complex. Finally, using recombinantly purified Scc2-Scc4 and cohesin, we performed crosslinking mass spectrometry and interaction assays that suggest Scc2-Scc4 uses its modular structure to make multiple contacts with cohesin.
Subject(s)
Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/metabolism , Conserved Sequence , Models, Molecular , Protein Binding , Protein Subunits/chemistry , Saccharomyces cerevisiae Proteins/metabolism , CohesinsABSTRACT
The remarkable accuracy of eukaryotic cell division is partly maintained by the cohesin complex acting as a molecular glue to prevent premature sister chromatid separation. The loading of cohesin onto chromosomes is catalyzed by the Scc2-Scc4 loader complex. Here, we report the crystal structure of Scc4 bound to the N terminus of Scc2 and show that Scc4 is a tetratricopeptide repeat (TPR) superhelix. The Scc2 N terminus adopts an extended conformation and is entrapped by the core of the Scc4 superhelix. Electron microscopy (EM) analysis reveals that the Scc2-Scc4 loader complex comprises three domains: a head, body, and hook. Deletion studies unambiguously assign the Scc2N-Scc4 as the globular head domain, whereas in vitro cohesin loading assays show that the central body and the hook domains are sufficient to catalyze cohesin loading onto circular DNA, but not chromatinized DNA in vivo, suggesting a possible role for Scc4 as a chromatin adaptor.
Subject(s)
Ascomycota/chemistry , Chromosomal Proteins, Non-Histone/chemistry , Fungal Proteins/chemistry , Protein Structure, Quaternary , Protein Structure, Secondary , Protein Structure, TertiaryABSTRACT
Centrosome amplification has been extensively associated with cancer. Cancer cells with extra centrosomes have the ability to cluster the extra centrosomes and divide in a bipolar fashion. Although a number of proteins have been shown to be involved in centrosome clustering, a mechanistic understanding of how this process is coordinated is not yet well defined. Here, to reveal regulators of centrosome clustering, we perform small interfering RNA (siRNA) screens with multiple assay readouts in a human isogenic cellular model. We find that APC/C activity is essential for centrosome clustering. We show that the motor kinesin Eg5 is a substrate of APC/C-CDH1, and that inhibition of APC/C results in stabilization of Eg5. Increased Eg5 protein levels disturb the balance of forces on the spindle and prevent centrosome clustering. This process is completely reversed after a short treatment with the Eg5 inhibitor, monastrol. These data advance our understanding of the regulation of centrosome clustering.