RESUMO
In eukaryotes, accurate chromosome segregation in mitosis and meiosis maintains genome stability and prevents aneuploidy. Kinetochores are large protein complexes that, by assembling onto specialized Cenp-A nucleosomes1,2, function to connect centromeric chromatin to microtubules of the mitotic spindle3,4. Whereas the centromeres of vertebrate chromosomes comprise millions of DNA base pairs and attach to multiple microtubules, the simple point centromeres of budding yeast are connected to individual microtubules5,6. All 16 budding yeast chromosomes assemble complete kinetochores using a single Cenp-A nucleosome (Cenp-ANuc), each of which is perfectly centred on its cognate centromere7-9. The inner and outer kinetochore modules are responsible for interacting with centromeric chromatin and microtubules, respectively. Here we describe the cryo-electron microscopy structure of the Saccharomyces cerevisiae inner kinetochore module, the constitutive centromere associated network (CCAN) complex, assembled onto a Cenp-A nucleosome (CCAN-Cenp-ANuc). The structure explains the interdependency of the constituent subcomplexes of CCAN and shows how the Y-shaped opening of CCAN accommodates Cenp-ANuc to enable specific CCAN subunits to contact the nucleosomal DNA and histone subunits. Interactions with the unwrapped DNA duplex at the two termini of Cenp-ANuc are mediated predominantly by a DNA-binding groove in the Cenp-L-Cenp-N subcomplex. Disruption of these interactions impairs assembly of CCAN onto Cenp-ANuc. Our data indicate a mechanism of Cenp-A nucleosome recognition by CCAN and how CCAN acts as a platform for assembly of the outer kinetochore to link centromeres to the mitotic spindle for chromosome segregation.
Assuntos
Proteína Centromérica A/metabolismo , Cinetocoros/química , Cinetocoros/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Nucleossomos/química , Nucleossomos/metabolismo , Proteína Centromérica A/química , Proteína Centromérica A/ultraestrutura , Microscopia Crioeletrônica , DNA/química , DNA/metabolismo , DNA/ultraestrutura , Cinetocoros/ultraestrutura , Modelos Moleculares , Complexos Multiproteicos/ultraestrutura , Nucleossomos/ultraestrutura , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestruturaRESUMO
The RNA polymerase inhibitor favipiravir is currently in clinical trials as a treatment for infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), despite limited information about the molecular basis for its activity. Here we report the structure of favipiravir ribonucleoside triphosphate (favipiravir-RTP) in complex with the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) bound to a template:primer RNA duplex, determined by electron cryomicroscopy (cryoEM) to a resolution of 2.5 Å. The structure shows clear evidence for the inhibitor at the catalytic site of the enzyme, and resolves the conformation of key side chains and ions surrounding the binding pocket. Polymerase activity assays indicate that the inhibitor is weakly incorporated into the RNA primer strand, and suppresses RNA replication in the presence of natural nucleotides. The structure reveals an unusual, nonproductive binding mode of favipiravir-RTP at the catalytic site of SARS-CoV-2 RdRp, which explains its low rate of incorporation into the RNA primer strand. Together, these findings inform current and future efforts to develop polymerase inhibitors for SARS coronaviruses.
Assuntos
Amidas/farmacologia , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Inibidores Enzimáticos/farmacologia , Pirazinas/farmacologia , SARS-CoV-2/ultraestrutura , Amidas/química , RNA-Polimerase RNA-Dependente de Coronavírus/antagonistas & inibidores , RNA-Polimerase RNA-Dependente de Coronavírus/química , Microscopia Crioeletrônica/métodos , Inibidores Enzimáticos/química , Pirazinas/química , Ribonucleotídeos/química , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , Imagem Individual de Molécula/métodosRESUMO
In eukaryotes, the anaphase-promoting complex (APC/C, also known as the cyclosome) regulates the ubiquitin-dependent proteolysis of specific cell-cycle proteins to coordinate chromosome segregation in mitosis and entry into the G1 phase. The catalytic activity of the APC/C and its ability to specify the destruction of particular proteins at different phases of the cell cycle are controlled by its interaction with two structurally related coactivator subunits, Cdc20 and Cdh1. Coactivators recognize substrate degrons, and enhance the affinity of the APC/C for its cognate E2 (refs 4-6). During mitosis, cyclin-dependent kinase (Cdk) and polo-like kinase (Plk) control Cdc20- and Cdh1-mediated activation of the APC/C. Hyperphosphorylation of APC/C subunits, notably Apc1 and Apc3, is required for Cdc20 to activate the APC/C, whereas phosphorylation of Cdh1 prevents its association with the APC/C. Since both coactivators associate with the APC/C through their common C-box and Ile-Arg tail motifs, the mechanism underlying this differential regulation is unclear, as is the role of specific APC/C phosphorylation sites. Here, using cryo-electron microscopy and biochemical analysis, we define the molecular basis of how phosphorylation of human APC/C allows for its control by Cdc20. An auto-inhibitory segment of Apc1 acts as a molecular switch that in apo unphosphorylated APC/C interacts with the C-box binding site and obstructs engagement of Cdc20. Phosphorylation of the auto-inhibitory segment displaces it from the C-box-binding site. Efficient phosphorylation of the auto-inhibitory segment, and thus relief of auto-inhibition, requires the recruitment of Cdk-cyclin in complex with a Cdk regulatory subunit (Cks) to a hyperphosphorylated loop of Apc3. We also find that the small-molecule inhibitor, tosyl-l-arginine methyl ester, preferentially suppresses APC/C(Cdc20) rather than APC/C(Cdh1), and interacts with the binding sites of both the C-box and Ile-Arg tail motifs. Our results reveal the mechanism for the regulation of mitotic APC/C by phosphorylation and provide a rationale for the development of selective inhibitors of this state.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Mitose , Fosfoproteínas/metabolismo , Motivos de Aminoácidos , Ciclossomo-Complexo Promotor de Anáfase/química , Ciclossomo-Complexo Promotor de Anáfase/ultraestrutura , Antígenos CD , Subunidade Apc1 do Ciclossomo-Complexo Promotor de Anáfase/química , Subunidade Apc1 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Subunidade Apc3 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Apoenzimas/metabolismo , Sítios de Ligação , Caderinas/química , Caderinas/metabolismo , Caderinas/ultraestrutura , Proteínas Cdc20/antagonistas & inibidores , Proteínas Cdc20/química , Proteínas Cdc20/metabolismo , Proteínas Cdc20/ultraestrutura , Microscopia Crioeletrônica , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Ativação Enzimática , Humanos , Modelos Moleculares , Fosfoproteínas/química , Fosfoproteínas/ultraestrutura , Fosforilação , Ligação Proteica , Conformação Proteica , Tosilarginina Metil Éster/farmacologiaRESUMO
In the dividing eukaryotic cell, the spindle assembly checkpoint (SAC) ensures that each daughter cell inherits an identical set of chromosomes. The SAC coordinates the correct attachment of sister chromatid kinetochores to the mitotic spindle with activation of the anaphase-promoting complex (APC/C), the E3 ubiquitin ligase responsible for initiating chromosome separation. In response to unattached kinetochores, the SAC generates the mitotic checkpoint complex (MCC), which inhibits the APC/C and delays chromosome segregation. By cryo-electron microscopy, here we determine the near-atomic resolution structure of a human APC/CMCC complex (APC/C(MCC)). Degron-like sequences of the MCC subunit BubR1 block degron recognition sites on Cdc20, the APC/C coactivator subunit responsible for substrate interactions. BubR1 also obstructs binding of the initiating E2 enzyme UbcH10 to repress APC/C ubiquitination activity. Conformational variability of the complex enables UbcH10 association, and structural analysis shows how the Cdc20 subunit intrinsic to the MCC (Cdc20(MCC)) is ubiquitinated, a process that results in APC/C reactivation when the SAC is silenced.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase/antagonistas & inibidores , Ciclossomo-Complexo Promotor de Anáfase/ultraestrutura , Microscopia Crioeletrônica , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , Fuso Acromático/metabolismo , Fuso Acromático/ultraestrutura , Ciclossomo-Complexo Promotor de Anáfase/química , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Biocatálise , Proteínas Cdc20/química , Proteínas Cdc20/metabolismo , Proteínas Cdc20/ultraestrutura , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos , Humanos , Cinetocoros/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/ultraestrutura , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Fuso Acromático/química , Relação Estrutura-Atividade , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/ultraestrutura , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Kinetochores are large multi-subunit complexes that attach centromeric chromatin to microtubules of the mitotic spindle, enabling sister chromatid segregation in mitosis. The inner kinetochore constitutive centromere associated network (CCAN) complex assembles onto the centromere-specific Cenp-A nucleosome (Cenp-ANuc), thereby coupling the centromere to the microtubule-binding outer kinetochore. CCAN is a conserved 14-16 subunit complex composed of discrete modules. Here, we determined the crystal structure of the Saccharomyces cerevisiae Cenp-HIKHead-TW sub-module, revealing how Cenp-HIK and Cenp-TW interact at the conserved Cenp-HIKHead-Cenp-TW interface. A major interface is formed by the C-terminal anti-parallel α-helices of the histone fold extension (HFE) of the Cenp-T histone fold domain (HFD) combining with α-helix H3 of Cenp-K to create a compact three α-helical bundle. We fitted the Cenp-HIKHead-TW sub-module to the previously determined cryo-EM map of the S. cerevisiae CCAN-Cenp-ANuc complex. This showed that the HEAT repeat domain of Cenp-IHead and C-terminal HFD of Cenp-T of the Cenp-HIKHead-TW sub-module interact with the nucleosome DNA gyre at a site close to the Cenp-ANuc dyad axis. Our structure provides a framework for understanding how Cenp-T links centromeric Cenp-ANuc to the outer kinetochore through its HFD and N-terminal Ndc80-binding motif, respectively.
Assuntos
Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Proteínas de Ligação a DNA , Cinetocoros , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/química , Proteínas de Ciclo Celular/química , Proteínas Cromossômicas não Histona/química , Segregação de Cromossomos , Proteínas de Ligação a DNA/química , Cinetocoros/química , Nucleossomos , Ligação Proteica , Domínios Proteicos , Proteínas de Saccharomyces cerevisiae/química , Fuso AcromáticoRESUMO
The anaphase-promoting complex (APC/C) is a multimeric RING E3 ubiquitin ligase that controls chromosome segregation and mitotic exit. Its regulation by coactivator subunits, phosphorylation, the mitotic checkpoint complex and interphase early mitotic inhibitor 1 (Emi1) ensures the correct order and timing of distinct cell-cycle transitions. Here we use cryo-electron microscopy to determine atomic structures of APC/C-coactivator complexes with either Emi1 or a UbcH10-ubiquitin conjugate. These structures define the architecture of all APC/C subunits, the position of the catalytic module and explain how Emi1 mediates inhibition of the two E2s UbcH10 and Ube2S. Definition of Cdh1 interactions with the APC/C indicates how they are antagonized by Cdh1 phosphorylation. The structure of the APC/C with UbcH10-ubiquitin reveals insights into the initiating ubiquitination reaction. Our results provide a quantitative framework for the design of future experiments to investigate APC/C functions in vivo.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase/ultraestrutura , Ubiquitinação , Ciclossomo-Complexo Promotor de Anáfase/química , Antígenos CD , Subunidade Apc1 do Ciclossomo-Complexo Promotor de Anáfase/química , Subunidade Apc1 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Subunidade Apc1 do Ciclossomo-Complexo Promotor de Anáfase/ultraestrutura , Subunidade Apc10 do Ciclossomo-Complexo Promotor de Anáfase/química , Subunidade Apc10 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Subunidade Apc10 do Ciclossomo-Complexo Promotor de Anáfase/ultraestrutura , Subunidade Apc11 do Ciclossomo-Complexo Promotor de Anáfase/química , Subunidade Apc11 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Subunidade Apc3 do Ciclossomo-Complexo Promotor de Anáfase/química , Subunidade Apc3 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Subunidade Apc8 do Ciclossomo-Complexo Promotor de Anáfase/química , Subunidade Apc8 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Subunidade Apc8 do Ciclossomo-Complexo Promotor de Anáfase/ultraestrutura , Caderinas/química , Caderinas/metabolismo , Caderinas/ultraestrutura , Domínio Catalítico , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/ultraestrutura , Microscopia Crioeletrônica , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Proteínas F-Box/química , Proteínas F-Box/metabolismo , Proteínas F-Box/ultraestrutura , Humanos , Lisina/metabolismo , Modelos Moleculares , Fosforilação , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Ubiquitina/química , Ubiquitina/metabolismo , Ubiquitina/ultraestrutura , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/ultraestruturaRESUMO
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.
Assuntos
Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Motivos de Aminoácidos , Ciclossomo-Complexo Promotor de Anáfase , Animais , Sítios de Ligação , Proteínas Cdh1 , Proteínas de Ciclo Celular , Cristalografia por Raios X , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Proteínas Repressoras/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The ubiquitination of cell cycle regulatory proteins by the anaphase-promoting complex/cyclosome (APC/C) controls sister chromatid segregation, cytokinesis and the establishment of the G1 phase of the cell cycle. The APC/C is an unusually large multimeric cullin-RING ligase. Its activity is strictly dependent on regulatory coactivator subunits that promote APC/C-substrate interactions and stimulate its catalytic reaction. Because the structures of many APC/C subunits and their organization within the assembly are unknown, the molecular basis for these processes is poorly understood. Here, from a cryo-electron microscopy reconstruction of a human APC/C-coactivator-substrate complex at 7.4 Å resolution, we have determined the complete secondary structural architecture of the complex. With this information we identified protein folds for structurally uncharacterized subunits, and the definitive location of all 20 APC/C subunits within the 1.2 MDa assembly. Comparison with apo APC/C shows that the coactivator promotes a profound allosteric transition involving displacement of the cullin-RING catalytic subunits relative to the degron-recognition module of coactivator and APC10. This transition is accompanied by increased flexibility of the cullin-RING subunits and enhanced affinity for UBCH10-ubiquitin, changes which may contribute to coactivator-mediated stimulation of APC/C E3 ligase activity.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase/ultraestrutura , Regulação Alostérica , Ciclossomo-Complexo Promotor de Anáfase/química , Subunidade Apc10 do Ciclossomo-Complexo Promotor de Anáfase/química , Subunidade Apc10 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Domínio Catalítico , Proteínas Cdh1/química , Proteínas Cdh1/metabolismo , Proteínas Cdh1/ultraestrutura , Microscopia Crioeletrônica , Humanos , Modelos Moleculares , Maleabilidade , Dobramento de Proteína , Estrutura Secundária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
CAAX proteins have essential roles in multiple signalling pathways, controlling processes such as proliferation, differentiation and carcinogenesis. The â¼120 mammalian CAAX proteins function at cellular membranes and include the Ras superfamily of small GTPases, nuclear lamins, the γ-subunit of heterotrimeric GTPases, and several protein kinases and phosphatases. The proper localization of CAAX proteins to cell membranes is orchestrated by a series of post-translational modifications of the carboxy-terminal CAAX motifs (where C is cysteine, A is an aliphatic amino acid and X is any amino acid). These reactions involve prenylation of the cysteine residue, cleavage at the AAX tripeptide and methylation of the carboxyl-prenylated cysteine residue. The major CAAX protease activity is mediated by Rce1 (Ras and a-factor converting enzyme 1), an intramembrane protease (IMP) of the endoplasmic reticulum. Information on the architecture and proteolytic mechanism of Rce1 has been lacking. Here we report the crystal structure of a Methanococcus maripaludis homologue of Rce1, whose endopeptidase specificity for farnesylated peptides mimics that of eukaryotic Rce1. Its structure, comprising eight transmembrane α-helices, and catalytic site are distinct from those of other IMPs. The catalytic residues are located â¼10 Å into the membrane and are exposed to the cytoplasm and membrane through a conical cavity that accommodates the prenylated CAAX substrate. We propose that the farnesyl lipid binds to a site at the opening of two transmembrane α-helices, which results in the scissile bond being positioned adjacent to a glutamate-activated nucleophilic water molecule. This study suggests that Rce1 is the founding member of a novel IMP family, the glutamate IMPs.
Assuntos
Biocatálise , Proteínas de Membrana/química , Mathanococcus/enzimologia , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Prenilação , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Sequência Conservada , Cristalografia por Raios X , Cisteína/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Endopeptidases/química , Endopeptidases/metabolismo , Retículo Endoplasmático/enzimologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Ácido Glutâmico/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Metaloendopeptidases/química , Metaloendopeptidases/metabolismo , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Peptídeo Hidrolases/classificação , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas p21(ras)/química , Transdução de Sinais , Especificidade por SubstratoRESUMO
The posttranslational modification of C-terminal CAAX motifs in proteins such as Ras, most Rho GTPases, and G protein γ subunits, plays an essential role in determining their subcellular localization and correct biological function. An integral membrane methyltransferase, isoprenylcysteine carboxyl methyltransferase (ICMT), catalyzes the final step of CAAX processing after prenylation of the cysteine residue and endoproteolysis of the -AAX motif. We have determined the crystal structure of a prokaryotic ICMT ortholog, revealing a markedly different architecture from conventional methyltransferases that utilize S-adenosyl-L-methionine (SAM) as a cofactor. ICMT comprises a core of five transmembrane α helices and a cofactor-binding pocket enclosed within a highly conserved C-terminal catalytic subdomain. A tunnel linking the reactive methyl group of SAM to the inner membrane provides access for the prenyl lipid substrate. This study explains how an integral membrane methyltransferase achieves recognition of both a hydrophilic cofactor and a lipophilic prenyl group attached to a polar protein substrate.
Assuntos
Proteínas Metiltransferases/química , Proteínas Metiltransferases/metabolismo , Membrana Celular/metabolismo , Cristalografia por Raios X , Citosol/metabolismo , Metabolismo dos Lipídeos , Methanosarcina/enzimologia , Metilação , Modelos Moleculares , Mutação , Proteínas Metiltransferases/genética , Estrutura Terciária de Proteína , S-Adenosilmetionina/metabolismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
The anaphase-promoting complex/cyclosome (APC/C) is a large multimeric cullin-RING E3 ubiquitin ligase that orchestrates cell-cycle progression by targeting cell-cycle regulatory proteins for destruction via the ubiquitin proteasome system. The APC/C assembly comprises two scaffolding subcomplexes: the platform and the TPR lobe that together coordinate the juxtaposition of the catalytic and substrate-recognition modules. The platform comprises APC/C subunits Apc1, Apc4, Apc5, and Apc15. Although the role of Apc1 as an APC/C scaffolding subunit has been characterized, its specific functions in contributing toward APC/C catalytic activity are not fully understood. Here, we report the crystal structure of the N-terminal domain of human Apc1 (Apc1N) determined at 2.2-Å resolution and provide an atomic-resolution description of the architecture of its WD40 (WD40 repeat) domain (Apc1(WD40)). To understand how Apc1(WD40) contributes to APC/C activity, a mutant form of the APC/C with Apc1(WD40) deleted was generated and evaluated biochemically and structurally. We found that the deletion of Apc1(WD40) abolished the UbcH10-dependent ubiquitination of APC/C substrates without impairing the Ube2S-dependent ubiquitin chain elongation activity. A cryo-EM structure of an APC/C-Cdh1 complex with Apc1(WD40) deleted showed that the mutant APC/C is locked into an inactive conformation in which the UbcH10-binding site of the catalytic module is inaccessible. Additionally, an EM density for Apc15 is not visible. Our data show that Apc1(WD40) is required to mediate the coactivator-induced conformational change of the APC/C that is responsible for stimulating APC/C catalytic activity by promoting UbcH10 binding. In contrast, Ube2S activity toward APC/C substrates is not dependent on the initiation-competent conformation of the APC/C.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase/química , Subunidade Apc1 do Ciclossomo-Complexo Promotor de Anáfase/química , Caderinas/química , Proteínas de Ciclo Celular/química , Proteínas Mutantes/química , Regulação Alostérica/genética , Ciclossomo-Complexo Promotor de Anáfase/genética , Antígenos CD , Subunidade Apc1 do Ciclossomo-Complexo Promotor de Anáfase/genética , Sítios de Ligação , Caderinas/genética , Proteínas de Ciclo Celular/genética , Cristalografia por Raios X , Humanos , Proteínas Mutantes/genética , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Ubiquitina/química , Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genética , Repetições WD40/genéticaRESUMO
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.
Assuntos
Proteínas de Ciclo Celular/química , Pontos de Checagem da Fase M do Ciclo Celular , Complexos Multiproteicos/química , Proteínas Nucleares/química , Proteínas de Schizosaccharomyces pombe/química , Schizosaccharomyces/química , Motivos de Aminoácidos , Ciclossomo-Complexo Promotor de Anáfase , Proteínas Cdc20 , Proteínas Cdh1 , Proteínas de Ciclo Celular/metabolismo , Sequência Conservada , Cristalografia por Raios X , Humanos , Proteínas Mad2 , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Fuso Acromático , Relação Estrutura-Atividade , Especificidade por Substrato , Complexos Ubiquitina-Proteína Ligase/antagonistas & inibidores , Complexos Ubiquitina-Proteína Ligase/química , Complexos Ubiquitina-Proteína Ligase/metabolismo , Complexos Ubiquitina-Proteína Ligase/ultraestruturaRESUMO
The capacity to reconstitute complex biological processes in vitro is a crucial step in providing a quantitative understanding of these systems. It provides material for structural, biochemical and biophysical analyses and allows the testing of biological hypotheses and the introduction of chemical probes and tags for single molecule analysis. Reconstitution of these systems requires access to homogenous components, usually through their over-production in heterologous over-expression systems. Here we describe the application of the USER (Uracil-Specific Excision Reagent) ligation-free cloning method to assemble recombinant MultiBac transfer vectors for the generation of recombinant baculovirus suitable for the expression of multi-protein complexes in insect cells.
Assuntos
Baculoviridae/genética , Clonagem Molecular/métodos , Vetores Genéticos/química , Complexos Multiproteicos/genética , Plasmídeos/química , Transgenes , Animais , Baculoviridae/metabolismo , Sequência de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/metabolismo , Dados de Sequência Molecular , Complexos Multiproteicos/metabolismo , Plasmídeos/metabolismo , Engenharia de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Uracila/química , Uracila/metabolismoRESUMO
The anaphase-promoting complex or cyclosome (APC/C) is an unusually large E3 ubiquitin ligase responsible for regulating defined cell cycle transitions. Information on how its 13 constituent proteins are assembled, and how they interact with co-activators, substrates and regulatory proteins is limited. Here, we describe a recombinant expression system that allows the reconstitution of holo APC/C and its sub-complexes that, when combined with electron microscopy, mass spectrometry and docking of crystallographic and homology-derived coordinates, provides a precise definition of the organization and structure of all essential APC/C subunits, resulting in a pseudo-atomic model for 70% of the APC/C. A lattice-like appearance of the APC/C is generated by multiple repeat motifs of most APC/C subunits. Three conserved tetratricopeptide repeat (TPR) subunits (Cdc16, Cdc23 and Cdc27) share related superhelical homo-dimeric architectures that assemble to generate a quasi-symmetrical structure. Our structure explains how this TPR sub-complex, together with additional scaffolding subunits (Apc1, Apc4 and Apc5), coordinate the juxtaposition of the catalytic and substrate recognition module (Apc2, Apc11 and Apc10 (also known as Doc1)), and TPR-phosphorylation sites, relative to co-activator, regulatory proteins and substrates.
Assuntos
Complexos Ubiquitina-Proteína Ligase/química , Complexos Ubiquitina-Proteína Ligase/metabolismo , Motivos de Aminoácidos , Ciclossomo-Complexo Promotor de Anáfase , Animais , Subunidade Apc2 do Ciclossomo-Complexo Promotor de Anáfase , Subunidade Apc5 do Ciclossomo-Complexo Promotor de Anáfase , Subunidade Apc8 do Ciclossomo-Complexo Promotor de Anáfase , Biocatálise , Linhagem Celular , Holoenzimas/química , Holoenzimas/metabolismo , Holoenzimas/ultraestrutura , Espectrometria de Massas , Microscopia Eletrônica , Modelos Moleculares , Peso Molecular , Ligação Proteica , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/isolamento & purificação , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Espalhamento de Radiação , Schizosaccharomyces/química , Relação Estrutura-Atividade , Especificidade por Substrato , Complexos Ubiquitina-Proteína Ligase/ultraestrutura , UbiquitinaçãoRESUMO
The ubiquitylation of cell-cycle regulatory proteins by the large multimeric anaphase-promoting complex (APC/C) controls sister chromatid segregation and the exit from mitosis. Selection of APC/C targets is achieved through recognition of destruction motifs, predominantly the destruction (D)-box and KEN (Lys-Glu-Asn)-box. Although this process is known to involve a co-activator protein (either Cdc20 or Cdh1) together with core APC/C subunits, the structural basis for substrate recognition and ubiquitylation is not understood. Here we investigate budding yeast APC/C using single-particle electron microscopy and determine a cryo-electron microscopy map of APC/C in complex with the Cdh1 co-activator protein (APC/C(Cdh1)) bound to a D-box peptide at â¼10 Å resolution. We find that a combined catalytic and substrate-recognition module is located within the central cavity of the APC/C assembled from Cdh1, Apc10--a core APC/C subunit previously implicated in substrate recognition--and the cullin domain of Apc2. Cdh1 and Apc10, identified from difference maps, create a co-receptor for the D-box following repositioning of Cdh1 towards Apc10. Using NMR spectroscopy we demonstrate specific D-box-Apc10 interactions, consistent with a role for Apc10 in directly contributing towards D-box recognition by the APC/C(Cdh1) complex. Our results rationalize the contribution of both co-activator and core APC/C subunits to D-box recognition and provide a structural framework for understanding mechanisms of substrate recognition and catalysis by the APC/C.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/química , Complexos Ubiquitina-Proteína Ligase/química , Complexos Ubiquitina-Proteína Ligase/metabolismo , Motivos de Aminoácidos , Ciclossomo-Complexo Promotor de Anáfase , Subunidade Apc10 do Ciclossomo-Complexo Promotor de Anáfase , Subunidade Apc2 do Ciclossomo-Complexo Promotor de Anáfase , Biocatálise , Proteínas Cdh1 , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/ultraestrutura , Microscopia Crioeletrônica , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Especificidade por Substrato , Complexos Ubiquitina-Proteína Ligase/ultraestrutura , UbiquitinaçãoRESUMO
The anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase responsible for controlling cell cycle transitions, is a multisubunit complex assembled from 13 different proteins. Numerous APC/C subunits incorporate multiple copies of the tetratricopeptide repeat (TPR). Here, we report the crystal structure of Schizosaccharomyces pombe Cut9 (Cdc16/Apc6) in complex with Hcn1 (Cdc26), showing that Cdc16/Cut9 is a contiguous TPR superhelix of 14 TPR units. A C-terminal block of TPR motifs interacts with Hcn1, whereas an N-terminal TPR block mediates Cdc16/Cut9 self-association through a homotypic interface. This dimer interface is structurally related to the N-terminal dimerization domain of Cdc27, demonstrating that both Cdc16/Cut9 and Cdc27 form homo-dimers through a conserved mechanism. The acetylated N-terminal Met residue of Hcn1 is enclosed within a chamber created from the Cut9 TPR superhelix. Thus, in complex with Cdc16/Cut9, the N-acetyl-Met residue of Hcn1, a putative degron for the Doa10 E3 ubiquitin ligase, is inaccessible for Doa10 recognition, protecting Hcn1/Cdc26 from ubiquitin-dependent degradation. This finding may provide a structural explanation for a mechanism to control the stoichiometry of proteins participating in multisubunit complexes.
Assuntos
DNA Polimerase III/química , Proteínas Nucleares/química , Sequências Repetitivas de Aminoácidos , Proteínas Repressoras/química , Proteínas de Schizosaccharomyces pombe/química , Schizosaccharomyces/metabolismo , Sequência de Aminoácidos , Ciclossomo-Complexo Promotor de Anáfase , Subunidade Apc6 do Ciclossomo-Complexo Promotor de Anáfase , Cristalização , Cristalografia por Raios X , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , Evolução Molecular , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Conformação Proteica , Multimerização Proteica , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/crescimento & desenvolvimento , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
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.
Assuntos
Complexos Multiproteicos/metabolismo , Proteínas Recombinantes/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Animais , Subunidade Apc3 do Ciclossomo-Complexo Promotor de Anáfase , Subunidade Apc7 do Ciclossomo-Complexo Promotor de Anáfase , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Humanos , Microscopia Eletrônica , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Multimerização Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/ultraestrutura , Especificidade por Substrato , Complexos Ubiquitina-Proteína Ligase/química , Complexos Ubiquitina-Proteína Ligase/genética , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Faithful chromosome segregation requires robust, load-bearing attachments of chromosomes to the mitotic spindle, a function accomplished by large macromolecular complexes termed kinetochores. In most eukaryotes, the constitutive centromere-associated network (CCAN) complex of the inner kinetochore recruits to centromeres the ten-subunit outer kinetochore KMN network that comprises the KNL1C, MIS12C and NDC80C complexes. The KMN network directly attaches CCAN to microtubules through MIS12C and NDC80C. Here, we determined a high-resolution cryo-EM structure of the human KMN network. This showed an intricate and extensive assembly of KMN subunits, with the central MIS12C forming rigid interfaces with NDC80C and KNL1C, augmented by multiple peptidic inter-subunit connections. We also observed that unphosphorylated MIS12C exists in an auto-inhibited state that suppresses its capacity to interact with CCAN. Ser100 and Ser109 of the N-terminal segment of the MIS12C subunit Dsn1, two key targets of Aurora B kinase, directly stabilize this auto-inhibition. Our study indicates how selectively relieving this auto-inhibition through Ser100 and Ser109 phosphorylation might restrict outer kinetochore assembly to functional centromeres during cell division.
Assuntos
Microscopia Crioeletrônica , Cinetocoros , Proteínas Associadas aos Microtúbulos , Modelos Moleculares , Proteínas Nucleares , Humanos , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/química , Proteínas Nucleares/metabolismo , Proteínas Nucleares/química , Fosforilação , Aurora Quinase B/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas do Citoesqueleto/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/química , Conformação Proteica , Proteínas Cromossômicas não HistonaRESUMO
The anaphase-promoting complex (APC/C) is a large E3 ubiquitin ligase that regulates progression through specific stages of the cell cycle by coordinating the ubiquitin-dependent degradation of cell-cycle regulatory proteins. Depending on the species, the active form of the APC/C consists of 14-15 different proteins that assemble into a 20-subunit complex with a mass of approximately 1.3 MDa. A hybrid approach of single-particle electron microscopy and protein crystallography of individual APC/C subunits has been applied to generate pseudo-atomic models of various functional states of the complex. Three approaches for assigning regions of the EM-derived APC/C density map to specific APC/C subunits are described. This information was used to dock atomic models of APC/C subunits, determined either by protein crystallography or homology modelling, to specific regions of the APC/C EM map, allowing the generation of a pseudo-atomic model corresponding to 80% of the entire complex.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase/química , Subunidade Apc1 do Ciclossomo-Complexo Promotor de Anáfase/química , Modelos Moleculares , Proteínas de Saccharomyces cerevisiae/química , Ciclossomo-Complexo Promotor de Anáfase/ultraestrutura , Subunidade Apc1 do Ciclossomo-Complexo Promotor de Anáfase/ultraestrutura , Cristalografia por Raios X , Humanos , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Homologia de Sequência de AminoácidosRESUMO
Kinetochores couple chromosomes to the mitotic spindle to segregate the genome during cell division. An error correction mechanism drives the turnover of kinetochore-microtubule attachments until biorientation is achieved. The structural basis for how kinetochore-mediated chromosome segregation is accomplished and regulated remains an outstanding question. In this work, we describe the cryo-electron microscopy structure of the budding yeast outer kinetochore Ndc80 and Dam1 ring complexes assembled onto microtubules. Complex assembly occurs through multiple interfaces, and a staple within Dam1 aids ring assembly. Perturbation of key interfaces suppresses yeast viability. Force-rupture assays indicated that this is a consequence of impaired kinetochore-microtubule attachment. The presence of error correction phosphorylation sites at Ndc80-Dam1 ring complex interfaces and the Dam1 staple explains how kinetochore-microtubule attachments are destabilized and reset.