RESUMEN
Kinase suppressor of Ras 2 (KSR2) is an intracellular scaffolding protein involved in multiple signaling pathways. Targeted deletion of Ksr2 leads to obesity in mice, suggesting a role in energy homeostasis. We explored the role of KSR2 in humans by sequencing 2,101 individuals with severe early-onset obesity and 1,536 controls. We identified multiple rare variants in KSR2 that disrupt signaling through the Raf-MEKERK pathway and impair cellular fatty acid oxidation and glucose oxidation in transfected cells; effects that can be ameliorated by the commonly prescribed antidiabetic drug, metformin. Mutation carriers exhibit hyperphagia in childhood, low heart rate, reduced basal metabolic rate and severe insulin resistance. These data establish KSR2 as an important regulator of energy intake, energy expenditure, and substrate utilization in humans. Modulation of KSR2-mediated effects may represent a novel therapeutic strategy for obesity and type 2 diabetes.
Asunto(s)
Resistencia a la Insulina , Obesidad/genética , Proteínas Serina-Treonina Quinasas/genética , Factores de Edad , Edad de Inicio , Secuencia de Aminoácidos , Animales , Niño , Metabolismo Energético , Ácidos Grasos/metabolismo , Femenino , Glucosa/metabolismo , Humanos , Hiperfagia/genética , Hiperfagia/metabolismo , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Obesidad/epidemiología , Obesidad/metabolismo , Oxidación-Reducción , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas B-raf/química , Proteínas Proto-Oncogénicas B-raf/metabolismo , Alineación de SecuenciaRESUMEN
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.
Asunto(s)
Proteína Quinasa CDC2/química , Proteína Quinasa CDC2/metabolismo , Ciclina B1/química , Ciclina B1/metabolismo , Securina/química , Securina/metabolismo , Separasa/química , Separasa/metabolismo , Secuencias de Aminoácidos , Proteína Quinasa CDC2/antagonistas & inhibidores , Proteína Quinasa CDC2/ultraestructura , Quinasas CDC2-CDC28/química , Quinasas CDC2-CDC28/metabolismo , Quinasas CDC2-CDC28/ultraestructura , Proteínas de Ciclo Celular/metabolismo , Segregación Cromosómica , Microscopía por Crioelectrón , Ciclina B1/ultraestructura , Proteínas de Unión al ADN/metabolismo , Humanos , Modelos Moleculares , Fosfoserina/metabolismo , Unión Proteica , Dominios Proteicos , Securina/ultraestructura , Separasa/antagonistas & inhibidores , Separasa/ultraestructura , Especificidad por SustratoRESUMEN
Protein tyrosine phosphatase 1B (PTP1B) plays important roles in downregulation of insulin and leptin signaling and is an established therapeutic target for diabetes and obesity. PTP1B is regulated by reactive oxygen species (ROS) produced in response to various stimuli, including insulin. The reversibly oxidized form of the enzyme (PTP1B-OX) is inactive and undergoes profound conformational changes at the active site. We generated conformation-sensor antibodies, in the form of single-chain variable fragments (scFvs), that stabilize PTP1B-OX and thereby inhibit its phosphatase function. Expression of conformation-sensor scFvs as intracellular antibodies (intrabodies) enhanced insulin-induced tyrosyl phosphorylation of the ß subunit of the insulin receptor and its substrate IRS-1 and increased insulin-induced phosphorylation of PKB/AKT. Our data suggest that stabilization of the oxidized, inactive form of PTP1B with appropriate therapeutic molecules may offer a paradigm for phosphatase drug development.
Asunto(s)
Proteína Tirosina Fosfatasa no Receptora Tipo 1/química , Proteína Tirosina Fosfatasa no Receptora Tipo 1/metabolismo , Anticuerpos , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Oxidación-Reducción , Biblioteca de Péptidos , Proteína Tirosina Fosfatasa no Receptora Tipo 1/antagonistas & inhibidores , Anticuerpos de Cadena Única/químicaRESUMEN
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.
Asunto(s)
Proteína A Centromérica/metabolismo , Cinetocoros/química , Cinetocoros/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Nucleosomas/química , Nucleosomas/metabolismo , Proteína A Centromérica/química , Proteína A Centromérica/ultraestructura , Microscopía por Crioelectrón , ADN/química , ADN/metabolismo , ADN/ultraestructura , Cinetocoros/ultraestructura , Modelos Moleculares , Complejos Multiproteicos/ultraestructura , Nucleosomas/ultraestructura , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestructuraRESUMEN
The control of protein abundance is a fundamental regulatory mechanism during mitosis. The anaphase-promoting complex/cyclosome (APC/C) is the main protein ubiquitin ligase responsible for the temporal regulation of mitotic progression. It has been proposed that the APC/C might fulfil other functions, including assembly of the mitotic spindle. Here, we show that the APC/C localizes to centrosomes, the organizers of the eukaryotic microtubule cytoskeleton, specifically during mitosis. Recruitment of the APC/C to spindle poles requires the centrosomal protein Cep152, and we identified Cep152 as both an APC/C interaction partner and an APC/C substrate. Previous studies have shown that Cep152 forms a complex with Cep57 and Cep63. The APC/C-mediated ubiquitylation of Cep152 at the centrosome releases Cep57 from this inhibitory complex and enables its interaction with pericentrin, a critical step in promoting microtubule nucleation. Thus, our study extends the function of the APC/C from being a regulator of mitosis to also acting as a positive governor of spindle assembly. The APC/C thereby integrates control of these two important processes in a temporal manner.
Asunto(s)
Centrosoma , Huso Acromático , Ciclosoma-Complejo Promotor de la Anafase/genética , Proteínas de Ciclo Celular/genética , Células HeLa , Humanos , Proteínas Asociadas a Microtúbulos/genética , Mitosis , Proteínas NuclearesRESUMEN
The maintenance of genome stability during mitosis is coordinated by the spindle assembly checkpoint (SAC) through its effector the mitotic checkpoint complex (MCC), an inhibitor of the anaphase-promoting complex (APC/C, also known as the cyclosome)1,2. Unattached kinetochores control MCC assembly by catalysing a change in the topology of the ß-sheet of MAD2 (an MCC subunit), thereby generating the active closed MAD2 (C-MAD2) conformer3-5. Disassembly of free MCC, which is required for SAC inactivation and chromosome segregation, is an ATP-dependent process driven by the AAA+ ATPase TRIP13. In combination with p31comet, an SAC antagonist6, TRIP13 remodels C-MAD2 into inactive open MAD2 (O-MAD2)7-10. Here, we present a mechanism that explains how TRIP13-p31comet disassembles the MCC. Cryo-electron microscopy structures of the TRIP13-p31comet-C-MAD2-CDC20 complex reveal that p31comet recruits C-MAD2 to a defined site on the TRIP13 hexameric ring, positioning the N terminus of C-MAD2 (MAD2NT) to insert into the axial pore of TRIP13 and distorting the TRIP13 ring to initiate remodelling. Molecular modelling suggests that by gripping MAD2NT within its axial pore, TRIP13 couples sequential ATP-driven translocation of its hexameric ring along MAD2NT to push upwards on, and simultaneously rotate, the globular domains of the p31comet-C-MAD2 complex. This unwinds a region of the αA helix of C-MAD2 that is required to stabilize the C-MAD2 ß-sheet, thus destabilizing C-MAD2 in favour of O-MAD2 and dissociating MAD2 from p31comet. Our study provides insights into how specific substrates are recruited to AAA+ ATPases through adaptor proteins and suggests a model of how translocation through the axial pore of AAA+ ATPases is coupled to protein remodelling.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Mad2/química , Proteínas Mad2/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/química , ATPasas Asociadas con Actividades Celulares Diversas/ultraestructura , Apoproteínas/química , Apoproteínas/metabolismo , Apoproteínas/ultraestructura , Sitios de Unión , Biocatálisis/efectos de los fármacos , Proteínas Cdc20/química , Proteínas Cdc20/metabolismo , Proteínas Cdc20/ultraestructura , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/ultraestructura , Microscopía por Crioelectrón , Humanos , Puntos de Control de la Fase M del Ciclo Celular/efectos de los fármacos , Proteínas Mad2/ultraestructura , Modelos Moleculares , Conformación Proteica , Huso Acromático/efectos de los fármacos , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Amidas/farmacología , ARN Polimerasa Dependiente de ARN de Coronavirus/metabolismo , Inhibidores Enzimáticos/farmacología , Pirazinas/farmacología , SARS-CoV-2/ultraestructura , Amidas/química , ARN Polimerasa Dependiente de ARN de Coronavirus/antagonistas & inhibidores , ARN Polimerasa Dependiente de ARN de Coronavirus/química , Microscopía por Crioelectrón/métodos , Inhibidores Enzimáticos/química , Pirazinas/química , Ribonucleótidos/química , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/enzimología , Imagen Individual de Molécula/métodosRESUMEN
During metaphase, in response to improper kinetochore-microtubule attachments, the spindle assembly checkpoint (SAC) activates the mitotic checkpoint complex (MCC), an inhibitor of the anaphase-promoting complex/cyclosome (APC/C). This process is orchestrated by the kinase Mps1, which initiates the assembly of the MCC onto kinetochores through a sequential phosphorylation-dependent signalling cascade. The Mad1-Mad2 complex, which is required to catalyse MCC formation, is targeted to kinetochores through a direct interaction with the phosphorylated conserved domain 1 (CD1) of Bub1. Here, we present the crystal structure of the C-terminal domain of Mad1 (Mad1CTD ) bound to two phosphorylated Bub1CD1 peptides at 1.75 Å resolution. This interaction is mediated by phosphorylated Bub1 Thr461, which not only directly interacts with Arg617 of the Mad1 RLK (Arg-Leu-Lys) motif, but also directly acts as an N-terminal cap to the CD1 α-helix dipole. Surprisingly, only one Bub1CD1 peptide binds to the Mad1 homodimer in solution. We suggest that this stoichiometry is due to inherent asymmetry in the coiled-coil of Mad1CTD and has implications for how the Mad1-Bub1 complex at kinetochores promotes efficient MCC assembly.
Asunto(s)
Proteínas de Ciclo Celular , Cinetocoros , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Segregación Cromosómica , Cinetocoros/metabolismo , Proteínas Mad2/genética , Proteínas Mad2/metabolismo , Fosforilación , Transducción de Señal , Huso Acromático/metabolismoRESUMEN
The anaphase-promoting complex (APC/C) is the key E3 ubiquitin ligase which directs mitotic progression and exit by catalysing the sequential ubiquitination of specific substrates. The activity of the APC/C in mitosis is restrained by the spindle assembly checkpoint (SAC), which coordinates chromosome segregation with the assembly of the mitotic spindle. The SAC effector is the mitotic checkpoint complex (MCC), which binds and inhibits the APC/C. It is incompletely understood how the APC/C switches substrate specificity in a cell cycle-specific manner. For instance, it is unclear how in prometaphase, when APC/C activity towards cyclin B and securin is repressed by the MCC, the kinase Nek2A is ubiquitinated. Here, we combine biochemical and structural analysis with functional studies in cells to show that Nek2A is a conformational-specific binder of the APC/C-MCC complex (APC/CMCC ) and that, in contrast to cyclin A, Nek2A can be ubiquitinated efficiently by the APC/C in conjunction with both the E2 enzymes UbcH10 and UbcH5. We propose that these special features of Nek2A allow its prometaphase-specific ubiquitination.
Asunto(s)
Puntos de Control de la Fase M del Ciclo Celular , Prometafase , Ciclosoma-Complejo Promotor de la Anafase/genética , Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Proteínas Cdc20/metabolismo , Proteínas de Ciclo Celular/metabolismo , Células HeLa , Humanos , Mitosis , Huso Acromático/metabolismo , UbiquitinaciónRESUMEN
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.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Mitosis , Fosfoproteínas/metabolismo , Secuencias de Aminoácidos , Ciclosoma-Complejo Promotor de la Anafase/química , Ciclosoma-Complejo Promotor de la Anafase/ultraestructura , Antígenos CD , Subunidad Apc1 del Ciclosoma-Complejo Promotor de la Anafase/química , Subunidad Apc1 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Subunidad Apc3 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Apoenzimas/metabolismo , Sitios de Unión , Cadherinas/química , Cadherinas/metabolismo , Cadherinas/ultraestructura , Proteínas Cdc20/antagonistas & inhibidores , Proteínas Cdc20/química , Proteínas Cdc20/metabolismo , Proteínas Cdc20/ultraestructura , Microscopía por Crioelectrón , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Activación Enzimática , Humanos , Modelos Moleculares , Fosfoproteínas/química , Fosfoproteínas/ultraestructura , Fosforilación , Unión Proteica , Conformación Proteica , Tosilarginina Metil Éster/farmacologíaRESUMEN
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.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/antagonistas & inhibidores , Ciclosoma-Complejo Promotor de la Anafase/ultraestructura , Microscopía por Crioelectrón , Puntos de Control de la Fase M del Ciclo Celular/fisiología , Huso Acromático/metabolismo , Huso Acromático/ultraestructura , Ciclosoma-Complejo Promotor de la Anafase/química , Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Biocatálisis , Proteínas Cdc20/química , Proteínas Cdc20/metabolismo , Proteínas Cdc20/ultraestructura , Proteínas de Ciclo Celular/metabolismo , Segregación Cromosómica , Humanos , Cinetocoros/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/ultraestructura , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Huso Acromático/química , Relación Estructura-Actividad , Enzimas Ubiquitina-Conjugadoras/química , Enzimas Ubiquitina-Conjugadoras/metabolismo , Enzimas Ubiquitina-Conjugadoras/ultraestructura , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
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.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas Cromosómicas no Histona , Proteínas de Unión al ADN , Cinetocoros , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/química , Proteínas de Ciclo Celular/química , Proteínas Cromosómicas no Histona/química , Segregación Cromosómica , Proteínas de Unión al ADN/química , Cinetocoros/química , Nucleosomas , Unión Proteica , Dominios Proteicos , Proteínas de Saccharomyces cerevisiae/química , Huso AcromáticoRESUMEN
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.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Ciclosoma-Complejo Promotor de la Anafase/ultraestructura , Ubiquitinación , Ciclosoma-Complejo Promotor de la Anafase/química , Antígenos CD , Subunidad Apc1 del Ciclosoma-Complejo Promotor de la Anafase/química , Subunidad Apc1 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Subunidad Apc1 del Ciclosoma-Complejo Promotor de la Anafase/ultraestructura , Subunidad Apc10 del Ciclosoma-Complejo Promotor de la Anafase/química , Subunidad Apc10 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Subunidad Apc10 del Ciclosoma-Complejo Promotor de la Anafase/ultraestructura , Subunidad Apc11 del Ciclosoma-Complejo Promotor de la Anafase/química , Subunidad Apc11 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Subunidad Apc3 del Ciclosoma-Complejo Promotor de la Anafase/química , Subunidad Apc3 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Subunidad Apc8 del Ciclosoma-Complejo Promotor de la Anafase/química , Subunidad Apc8 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Subunidad Apc8 del Ciclosoma-Complejo Promotor de la Anafase/ultraestructura , Cadherinas/química , Cadherinas/metabolismo , Cadherinas/ultraestructura , Dominio Catalítico , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/ultraestructura , Microscopía por Crioelectrón , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/metabolismo , Proteínas F-Box/química , Proteínas F-Box/metabolismo , Proteínas F-Box/ultraestructura , Humanos , Lisina/metabolismo , Modelos Moleculares , Fosforilación , Unión Proteica , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Relación Estructura-Actividad , Especificidad por Sustrato , Ubiquitina/química , Ubiquitina/metabolismo , Ubiquitina/ultraestructura , Enzimas Ubiquitina-Conjugadoras/química , Enzimas Ubiquitina-Conjugadoras/metabolismo , Enzimas Ubiquitina-Conjugadoras/ultraestructuraRESUMEN
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.
Asunto(s)
Proteínas Represoras/química , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Secuencias de Aminoácidos , Ciclosoma-Complejo Promotor de la Anafase , Animales , Sitios de Unión , Proteínas Cdh1 , Proteínas de Ciclo Celular , Cristalografía por Rayos X , Modelos Moleculares , Conformación Proteica , Multimerización de Proteína , Proteínas Represoras/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
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.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Ciclosoma-Complejo Promotor de la Anafase/ultraestructura , Regulación Alostérica , Ciclosoma-Complejo Promotor de la Anafase/química , Subunidad Apc10 del Ciclosoma-Complejo Promotor de la Anafase/química , Subunidad Apc10 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Dominio Catalítico , Proteínas Cdh1/química , Proteínas Cdh1/metabolismo , Proteínas Cdh1/ultraestructura , Microscopía por Crioelectrón , Humanos , Modelos Moleculares , Docilidad , Pliegue de Proteína , Estructura Secundaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , UbiquitinaciónRESUMEN
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.
Asunto(s)
Biocatálisis , Proteínas de la Membrana/química , Methanococcus/enzimología , Péptido Hidrolasas/química , Péptido Hidrolasas/metabolismo , Prenilación , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Proteínas Arqueales/química , Proteínas Arqueales/metabolismo , Secuencia Conservada , Cristalografía por Rayos X , Cisteína/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Endopeptidasas/química , Endopeptidasas/metabolismo , Retículo Endoplásmico/enzimología , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Ácido Glutámico/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Metaloendopeptidasas/química , Metaloendopeptidasas/metabolismo , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Péptido Hidrolasas/clasificación , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas p21(ras)/química , Transducción de Señal , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Proteína Metiltransferasas/química , Proteína Metiltransferasas/metabolismo , Membrana Celular/metabolismo , Cristalografía por Rayos X , Citosol/metabolismo , Metabolismo de los Lípidos , Methanosarcina/enzimología , Metilación , Modelos Moleculares , Mutación , Proteína Metiltransferasas/genética , Estructura Terciaria de Proteína , S-Adenosilmetionina/metabolismo , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/química , Subunidad Apc1 del Ciclosoma-Complejo Promotor de la Anafase/química , Cadherinas/química , Proteínas de Ciclo Celular/química , Proteínas Mutantes/química , Regulación Alostérica/genética , Ciclosoma-Complejo Promotor de la Anafase/genética , Antígenos CD , Subunidad Apc1 del Ciclosoma-Complejo Promotor de la Anafase/genética , Sitios de Unión , Cadherinas/genética , Proteínas de Ciclo Celular/genética , Cristalografía por Rayos X , Humanos , Proteínas Mutantes/genética , Unión Proteica , Conformación Proteica , Dominios Proteicos , Ubiquitina/química , Ubiquitina/genética , Enzimas Ubiquitina-Conjugadoras/química , Enzimas Ubiquitina-Conjugadoras/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación/genética , Repeticiones WD40/genéticaRESUMEN
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.