RESUMEN
Protein ubiquitination involves E1, E2, and E3 trienzyme cascades. E2 and RING E3 enzymes often collaborate to first prime a substrate with a single ubiquitin (UB) and then achieve different forms of polyubiquitination: multiubiquitination of several sites and elongation of linkage-specific UB chains. Here, cryo-EM and biochemistry show that the human E3 anaphase-promoting complex/cyclosome (APC/C) and its two partner E2s, UBE2C (aka UBCH10) and UBE2S, adopt specialized catalytic architectures for these two distinct forms of polyubiquitination. The APC/C RING constrains UBE2C proximal to a substrate and simultaneously binds a substrate-linked UB to drive processive multiubiquitination. Alternatively, during UB chain elongation, the RING does not bind UBE2S but rather lures an evolving substrate-linked UB to UBE2S positioned through a cullin interaction to generate a Lys11-linked chain. Our findings define mechanisms of APC/C regulation, and establish principles by which specialized E3-E2-substrate-UB architectures control different forms of polyubiquitination.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/química , Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina/metabolismo , Secuencia de Aminoácidos , Biocatálisis , Microscopía por Crioelectrón , Humanos , Modelos Moleculares , Proteínas de Saccharomyces cerevisiae/química , Relación Estructura-Actividad , UbiquitinaciónRESUMEN
Co-opting Cullin4 RING ubiquitin ligases (CRL4s) to inducibly degrade pathogenic proteins is emerging as a promising therapeutic strategy. Despite intense efforts to rationally design degrader molecules that co-opt CRL4s, much about the organization and regulation of these ligases remains elusive. Here, we establish protein interaction kinetics and estimation of stoichiometries (PIKES) analysis, a systematic proteomic profiling platform that integrates cellular engineering, affinity purification, chemical stabilization, and quantitative mass spectrometry to investigate the dynamics of interchangeable multiprotein complexes. Using PIKES, we show that ligase assemblies of Cullin4 with individual substrate receptors differ in abundance by up to 200-fold and that Cand1/2 act as substrate receptor exchange factors. Furthermore, degrader molecules can induce the assembly of their cognate CRL4, and higher expression of the associated substrate receptor enhances degrader potency. Beyond the CRL4 network, we show how PIKES can reveal systems level biochemistry for cellular protein networks important to drug development.
Asunto(s)
Cromatografía Líquida de Alta Presión , Proteómica/métodos , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en Tándem , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Cullin/genética , Proteínas Cullin/metabolismo , Células HEK293 , Humanos , Cinética , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Proteína NEDD8/genética , Proteína NEDD8/metabolismo , Mapas de Interacción de Proteínas , Proteolisis , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Molecular glue degraders are an effective therapeutic modality, but their design principles are not well understood. Recently, several unexpectedly diverse compounds were reported to deplete cyclin K by linking CDK12-cyclin K to the DDB1-CUL4-RBX1 E3 ligase. Here, to investigate how chemically dissimilar small molecules trigger cyclin K degradation, we evaluated 91 candidate degraders in structural, biophysical and cellular studies and reveal all compounds acquire glue activity via simultaneous CDK12 binding and engagement of DDB1 interfacial residues, in particular Arg928. While we identify multiple published kinase inhibitors as cryptic degraders, we also show that these glues do not require pronounced inhibitory properties for activity and that the relative degree of CDK12 inhibition versus cyclin K degradation is tuneable. We further demonstrate cyclin K degraders have transcriptional signatures distinct from CDK12 inhibitors, thereby offering unique therapeutic opportunities. The systematic structure-activity relationship analysis presented herein provides a conceptual framework for rational molecular glue design.
Asunto(s)
Ciclinas , Ubiquitina-Proteína Ligasas , Ciclinas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteolisis , Relación Estructura-ActividadRESUMEN
Molecular glue compounds induce protein-protein interactions that, in the context of a ubiquitin ligase, lead to protein degradation1. Unlike traditional enzyme inhibitors, these molecular glue degraders act substoichiometrically to catalyse the rapid depletion of previously inaccessible targets2. They are clinically effective and highly sought-after, but have thus far only been discovered serendipitously. Here, through systematically mining databases for correlations between the cytotoxicity of 4,518 clinical and preclinical small molecules and the expression levels of E3 ligase components across hundreds of human cancer cell lines3-5, we identify CR8-a cyclin-dependent kinase (CDK) inhibitor6-as a compound that acts as a molecular glue degrader. The CDK-bound form of CR8 has a solvent-exposed pyridyl moiety that induces the formation of a complex between CDK12-cyclin K and the CUL4 adaptor protein DDB1, bypassing the requirement for a substrate receptor and presenting cyclin K for ubiquitination and degradation. Our studies demonstrate that chemical alteration of surface-exposed moieties can confer gain-of-function glue properties to an inhibitor, and we propose this as a broader strategy through which target-binding molecules could be converted into molecular glues.
Asunto(s)
Ciclinas/deficiencia , Ciclinas/metabolismo , Proteolisis/efectos de los fármacos , Purinas/química , Purinas/farmacología , Piridinas/química , Piridinas/farmacología , Línea Celular Tumoral , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/química , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/química , Proteínas de Unión al ADN/metabolismo , Humanos , Modelos Moleculares , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica/efectos de los fármacos , Purinas/toxicidad , Piridinas/toxicidad , Bibliotecas de Moléculas Pequeñas/análisis , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Ubiquitinación/efectos de los fármacosRESUMEN
Targeted protein degradation is a new therapeutic modality based on drugs that destabilize proteins by inducing their proximity to E3 ubiquitin ligases. Of particular interest are molecular glues that can degrade otherwise unligandable proteins by orchestrating direct interactions between target and ligase. However, their discovery has so far been serendipitous, thus hampering broad translational efforts. Here, we describe a scalable strategy toward glue degrader discovery that is based on chemical screening in hyponeddylated cells coupled to a multi-omics target deconvolution campaign. This approach led us to identify compounds that induce ubiquitination and degradation of cyclin K by prompting an interaction of CDK12-cyclin K with a CRL4B ligase complex. Notably, this interaction is independent of a dedicated substrate receptor, thus functionally segregating this mechanism from all described degraders. Collectively, our data outline a versatile and broadly applicable strategy to identify degraders with nonobvious mechanisms and thus empower future drug discovery efforts.
Asunto(s)
Acetamidas/química , Antibacterianos/farmacología , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Doxiciclina/farmacología , Hidrazinas/química , Indoles/química , Proteolisis/efectos de los fármacos , Proteína 7 de Unión a Retinoblastoma/metabolismo , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Regulación de la Expresión Génica , Humanos , Estructura Molecular , Unión Proteica , Conformación Proteica , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/efectos de los fármacosRESUMEN
Thalidomide and its derivatives, lenalidomide and pomalidomide, are immune modulatory drugs (IMiDs) used in the treatment of haematologic malignancies. IMiDs bind CRBN, the substrate receptor of the CUL4-RBX1-DDB1-CRBN (also known as CRL4(CRBN)) E3 ubiquitin ligase, and inhibit ubiquitination of endogenous CRL4(CRBN) substrates. Unexpectedly, IMiDs also repurpose the ligase to target new proteins for degradation. Lenalidomide induces degradation of the lymphoid transcription factors Ikaros and Aiolos (also known as IKZF1 and IKZF3), and casein kinase 1α (CK1α), which contributes to its clinical efficacy in the treatment of multiple myeloma and 5q-deletion associated myelodysplastic syndrome (del(5q) MDS), respectively. How lenalidomide alters the specificity of the ligase to degrade these proteins remains elusive. Here we present the 2.45 Å crystal structure of DDB1-CRBN bound to lenalidomide and CK1α. CRBN and lenalidomide jointly provide the binding interface for a CK1α ß-hairpin-loop located in the kinase N-lobe. We show that CK1α binding to CRL4(CRBN) is strictly dependent on the presence of an IMiD. Binding of IKZF1 to CRBN similarly requires the compound and both, IKZF1 and CK1α, use a related binding mode. Our study provides a mechanistic explanation for the selective efficacy of lenalidomide in del(5q) MDS therapy. We anticipate that high-affinity protein-protein interactions induced by small molecules will provide opportunities for drug development, particularly for targeted protein degradation.
Asunto(s)
Caseína Quinasa Ialfa/metabolismo , Talidomida/análogos & derivados , Ubiquitina-Proteína Ligasas/metabolismo , Sitios de Unión/efectos de los fármacos , Caseína Quinasa Ialfa/química , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Factor de Transcripción Ikaros/química , Factor de Transcripción Ikaros/metabolismo , Lenalidomida , Modelos Moleculares , Unión Proteica/efectos de los fármacos , Proteolisis/efectos de los fármacos , Relación Estructura-Actividad , Especificidad por Sustrato/efectos de los fármacos , Talidomida/química , Talidomida/metabolismo , Talidomida/farmacología , Ubiquitina-Proteína Ligasas/química , Ubiquitinación/efectos de los fármacosRESUMEN
The cullin-RING ubiquitin E3 ligase (CRL) family comprises over 200 members in humans. The COP9 signalosome complex (CSN) regulates CRLs by removing their ubiquitin-like activator NEDD8. The CUL4A-RBX1-DDB1-DDB2 complex (CRL4A(DDB2)) monitors the genome for ultraviolet-light-induced DNA damage. CRL4A(DBB2) is inactive in the absence of damaged DNA and requires CSN to regulate the repair process. The structural basis of CSN binding to CRL4A(DDB2) and the principles of CSN activation are poorly understood. Here we present cryo-electron microscopy structures for CSN in complex with neddylated CRL4A ligases to 6.4 Å resolution. The CSN conformers defined by cryo-electron microscopy and a novel apo-CSN crystal structure indicate an induced-fit mechanism that drives CSN activation by neddylated CRLs. We find that CSN and a substrate cannot bind simultaneously to CRL4A, favouring a deneddylated, inactive state for substrate-free CRL4 complexes. These architectural and regulatory principles appear conserved across CRL families, allowing global regulation by CSN.
Asunto(s)
Biocatálisis , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructura , Péptido Hidrolasas/metabolismo , Péptido Hidrolasas/ultraestructura , Regulación Alostérica , Apoproteínas/química , Apoproteínas/metabolismo , Apoproteínas/ultraestructura , Sitios de Unión , Complejo del Señalosoma COP9 , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas Portadoras/ultraestructura , Microscopía por Crioelectrón , Cristalografía por Rayos X , Proteínas Cullin/química , Proteínas Cullin/metabolismo , Proteínas Cullin/ultraestructura , Daño del ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/ultraestructura , Humanos , Cinética , Modelos Moleculares , Complejos Multiproteicos/química , Péptido Hidrolasas/química , Unión Proteica , Ubiquitinación , Ubiquitinas/metabolismoRESUMEN
Polyubiquitination by E2 and E3 enzymes is a predominant mechanism regulating protein function. Some RING E3s, including anaphase-promoting complex/cyclosome (APC), catalyze polyubiquitination by sequential reactions with two different E2s. An initiating E2 ligates ubiquitin to an E3-bound substrate. Another E2 grows a polyubiquitin chain on the ubiquitin-primed substrate through poorly defined mechanisms. Here we show that human APC's RING domain is repurposed for dual functions in polyubiquitination. The canonical RING surface activates an initiating E2-ubiquitin intermediate for substrate modification. However, APC engages and activates its specialized ubiquitin chain-elongating E2 UBE2S in ways that differ from current paradigms. During chain assembly, a distinct APC11 RING surface helps deliver a substrate-linked ubiquitin to accept another ubiquitin from UBE2S. Our data define mechanisms of APC/UBE2S-mediated polyubiquitination, reveal diverse functions of RING E3s and E2s, and provide a framework for understanding distinctive RING E3 features specifying ubiquitin chain elongation.
Asunto(s)
Subunidad Apc11 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Subunidad Apc2 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Biosíntesis de Péptidos Independientes de Ácidos Nucleicos , Poliubiquitina/biosíntesis , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitinación/fisiología , Secuencia de Aminoácidos , Subunidad Apc4 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Puntos de Control del Ciclo Celular , Células HeLa , Humanos , Datos de Secuencia Molecular , Poliubiquitina/genética , Estructura Terciaria de ProteínaRESUMEN
Analyses of protein complexes are facilitated by methods that enable the generation of recombinant complexes via coexpression of their subunits from multigene DNA constructs. However, low experimental throughput limits the generation of such constructs in parallel. Here we describe a method that allows up to 25 cDNAs to be assembled into a single baculoviral expression vector in only two steps. This method, called biGBac, uses computationally optimized DNA linker sequences that enable the efficient assembly of linear DNA fragments, using reactions developed by Gibson for the generation of synthetic genomes. The biGBac method uses a flexible and modular "mix and match" approach and enables the generation of baculoviruses from DNA constructs at any assembly stage. Importantly, it is simple, efficient, and fast enough to allow the manual generation of many multigene expression constructs in parallel. We have used this method to generate and characterize recombinant forms of the anaphase-promoting complex/cyclosome, cohesin, and kinetochore complexes.
Asunto(s)
Baculoviridae/genética , Vectores Genéticos/genética , Familia de Multigenes/genética , Complejos Multiproteicos/genética , Ingeniería de Proteínas/métodos , Transfección/métodos , Clonación Molecular/métodos , Complejos Multiproteicos/metabolismo , Mutagénesis Sitio-Dirigida/métodos , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Although splicing is essential for the expression of most eukaryotic genes, inactivation of splicing factors causes specific defects in mitosis. The molecular cause of this defect is unknown. Here, we show that the spliceosome subunits SNW1 and PRPF8 are essential for sister chromatid cohesion in human cells. A transcriptome-wide analysis revealed that SNW1 or PRPF8 depletion affects the splicing of specific introns in a subset of pre-mRNAs, including pre-mRNAs encoding the cohesion protein sororin and the APC/C subunit APC2. SNW1 depletion causes cohesion defects predominantly by reducing sororin levels, which causes destabilisation of cohesin on DNA. SNW1 depletion also reduces APC/C activity and contributes to cohesion defects indirectly by delaying mitosis and causing "cohesion fatigue". Simultaneous expression of sororin and APC2 from intron-less cDNAs restores cohesion in SNW1-depleted cells. These results indicate that the spliceosome is required for mitosis because it enables expression of genes essential for cohesion. Our transcriptome-wide identification of retained introns in SNW1- and PRPF8-depleted cells may help to understand the aetiology of diseases associated with splicing defects, such as retinosa pigmentosum and cancer.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Proteínas del Citoesqueleto/metabolismo , Coactivadores de Receptor Nuclear/metabolismo , Precursores del ARN/metabolismo , Empalme del ARN/fisiología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas de Ciclo Celular/genética , Cromátides/genética , Proteínas del Citoesqueleto/genética , Eliminación de Gen , Células HeLa , Humanos , Coactivadores de Receptor Nuclear/genética , Precursores del ARN/genética , Transcriptoma/fisiologíaRESUMEN
Molecular machines or macromolecular complexes are supramolecular assemblies of biomolecules with a variety of functions. Structure determination of these complexes in a purified state is often tedious owing to their compositional complexity and the associated relative structural instability. To improve the stability of macromolecular complexes in vitro, we present a generic method that optimizes the stability, homogeneity and solubility of macromolecular complexes by sparse-matrix screening of their thermal unfolding behavior in the presence of various buffers and small molecules. The method includes the automated analysis of thermal unfolding curves based on a biophysical unfolding model for complexes. We found that under stabilizing conditions, even large multicomponent complexes reveal an almost ideal two-state unfolding behavior. We envisage an improved biochemical understanding of purified macromolecules as well as a substantial boost in successful macromolecular complex structure determination by both X-ray crystallography and cryo-electron microscopy.
Asunto(s)
Algoritmos , Modelos Químicos , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/ultraestructura , Programas Informáticos , Sitios de Unión , Simulación por Computador , Cristalización , Unión Proteica , Conformación Proteica , Pliegue de ProteínaRESUMEN
We successfully repurpose the DNA repair protein methylguanine methyltransferase (MGMT) as an inducible degron for protein fusions. MGMT is a suicide protein that removes alkyl groups from the O6 position of guanine (O6G) and is thereafter quickly degraded by the ubiquitin proteasome pathway (UPP). Starting with MGMT pseudosubstrates (benzylguanine and lomeguatrib), we first demonstrate that these lead to potent MGMT depletion while affecting little else in the proteome. We then show that fusion proteins of MGMT undergo rapid UPP-dependent degradation in response to pseudosubstrates. Mechanistic studies confirm the involvement of the UPP, while revealing that at least two E3 ligase classes can degrade MGMT depending on cell-line and expression type (native or ectopic). We also demonstrate the technique's versatility with two clinically relevant examples: degradation of KRASG12C and a chimeric antigen receptor.
Asunto(s)
Metilasas de Modificación del ADN/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Reparación del ADN , Proteínas Supresoras de Tumor/metabolismo , Sistemas CRISPR-Cas , Línea Celular , Daño del ADN , Metilasas de Modificación del ADN/antagonistas & inhibidores , Metilasas de Modificación del ADN/genética , Enzimas Reparadoras del ADN/antagonistas & inhibidores , Enzimas Reparadoras del ADN/genética , Humanos , Ligandos , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genéticaRESUMEN
PCSK9 heightens LDL cholesterol levels by chaperoning the liver LDL receptor to lysosomes for degradation. In this issue of Cell Chemical Biology, Petrilli et al. (2020) identify novel PCSK9 ligands and convert them into a proof-of-concept degrader, offering a unique way to modulate this key protein-protein interaction.
Asunto(s)
Proproteína Convertasa 9 , LDL-Colesterol , Ligandos , ProteolisisRESUMEN
The small molecules thalidomide, lenalidomide, and pomalidomide induce the ubiquitination and proteasomal degradation of the transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) by recruiting a Cys2-His2 (C2H2) zinc finger domain to Cereblon (CRBN), the substrate receptor of the CRL4CRBN E3 ubiquitin ligase. We screened the human C2H2 zinc finger proteome for degradation in the presence of thalidomide analogs, identifying 11 zinc finger degrons. Structural and functional characterization of the C2H2 zinc finger degrons demonstrates how diverse zinc finger domains bind the permissive drug-CRBN interface. Computational zinc finger docking and biochemical analysis predict that more than 150 zinc fingers bind the drug-CRBN complex in vitro, and we show that selective zinc finger degradation can be achieved through compound modifications. Our results provide a rationale for therapeutically targeting transcription factors that were previously considered undruggable.
Asunto(s)
Dedos de Zinc CYS2-HIS2 , Lenalidomida/farmacología , Péptido Hidrolasas/metabolismo , Proteolisis/efectos de los fármacos , Talidomida/análogos & derivados , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Células HEK293 , Humanos , Factor de Transcripción Ikaros/metabolismo , Proteoma/metabolismo , Talidomida/farmacologíaRESUMEN
The gram-positive bacterium Bacillus subtilis contains three proteins belonging to the signal recognition particle (SRP) type GTPase family. The well characterized signal sequence-binding protein SRP54 and the SRP receptor protein FtsY are universally conserved components of the SRP system of protein transport. The third member, FlhF, has been implicated in the placement and assembly of polar flagella. This article describes the overexpression and preliminary X-ray crystallographic analysis of an FlhF fragment that corresponds to the well characterized GTPase domains in SRP54 and FtsY. Three crystal forms are reported with either GDP or GMPPNP and diffract to a resolution of about 3 A.
Asunto(s)
Bacillus subtilis/química , Proteínas Bacterianas/química , Proteínas de Unión al GTP Monoméricas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Clonación Molecular , Cristalización , Cristalografía por Rayos X , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/aislamiento & purificación , Conformación ProteicaRESUMEN
BACKGROUND: Cohesin mediates sister chromatid cohesion by topologically entrapping sister DNA molecules inside its ring structure. Cohesin is loaded onto DNA by the Scc2/NIPBL-Scc4/MAU2-loading complex in a manner that depends on the adenosine triphosphatase (ATPase) activity of cohesin's Smc1 and Smc3 subunits. Subsequent cohesion establishment during DNA replication depends on Smc3 acetylation by Esco1 and Esco2 and on recruitment of sororin, which "locks" cohesin on DNA by inactivating the cohesin release factor Wapl. RESULTS: Human cohesin ATPase mutants associate transiently with DNA in a manner that depends on the loading complex but cannot be stabilized on chromatin by depletion of Wapl. These mutants cannot be acetylated, fail to interact with sororin, and do not mediate cohesion. The absence of Smc3 acetylation in the ATPase mutants is not a consequence of their transient association with DNA but is directly caused by their inability to hydrolyze ATP because acetylation of wild-type cohesin also depends on ATP hydrolysis. CONCLUSIONS: Our data indicate that cohesion establishment involves the following steps. First, cohesin transiently associates with DNA in a manner that depends on the loading complex. Subsequently, ATP hydrolysis by cohesin leads to entrapment of DNA and converts Smc3 into a state that can be acetylated. Finally, Smc3 acetylation leads to recruitment of sororin, inhibition of Wapl, and stabilization of cohesin on DNA. Our finding that cohesin's ATPase activity is required for both cohesin loading and Smc3 acetylation raises the possibility that cohesion establishment is directly coupled to the reaction in which cohesin entraps DNA.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Regulación de la Expresión Génica , Adenosina Trifosfatasas/genética , Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN , Humanos , Hidrólisis , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Mutación , Proteínas/genética , Proteínas/metabolismo , Intercambio de Cromátides Hermanas , CohesinasRESUMEN
The anaphase-promoting complex/cyclosome (APC/C) is a ~1.5-MDa multiprotein E3 ligase enzyme that regulates cell division by promoting timely ubiquitin-mediated proteolysis of key cell-cycle regulatory proteins. Inhibition of human APC/C(CDH1) during interphase by early mitotic inhibitor 1 (EMI1) is essential for accurate coordination of DNA synthesis and mitosis. Here, we report a hybrid structural approach involving NMR, electron microscopy and enzymology, which reveal that EMI1's 143-residue C-terminal domain inhibits multiple APC/C(CDH1) functions. The intrinsically disordered D-box, linker and tail elements, together with a structured zinc-binding domain, bind distinct regions of APC/C(CDH1) to synergistically both block the substrate-binding site and inhibit ubiquitin-chain elongation. The functional importance of intrinsic structural disorder is explained by enabling a small inhibitory domain to bind multiple sites to shut down various functions of a 'molecular machine' nearly 100 times its size.
Asunto(s)
Cadherinas/química , Proteínas de Ciclo Celular/química , Proteínas F-Box/química , Complejos de Ubiquitina-Proteína Ligasa/química , Secuencia de Aminoácidos , Ciclosoma-Complejo Promotor de la Anafase , Antígenos CD , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/farmacología , Proteínas de Ciclo Celular/ultraestructura , Proteínas F-Box/metabolismo , Proteínas F-Box/farmacología , Proteínas F-Box/ultraestructura , Humanos , Microscopía Electrónica , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Especificidad por Sustrato , Complejos de Ubiquitina-Proteína Ligasa/antagonistas & inhibidores , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/ultraestructura , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Proteínas Ubiquitinadas/metabolismo , Ubiquitinación , UltracentrifugaciónRESUMEN
The anaphase-promoting complex/cyclosome (APC/C) bound to CDC20 (APC/C(CDC20)) initiates anaphase by ubiquitylating B-type cyclins and securin. During chromosome bi-orientation, CDC20 assembles with MAD2, BUBR1 and BUB3 into a mitotic checkpoint complex (MCC) that inhibits substrate recruitment to the APC/C. APC/C activation depends on MCC disassembly, which was proposed to require CDC20 autoubiquitylation. Here we characterize APC15, a human APC/C subunit related to yeast Mnd2. APC15 is located near APC/C's MCC binding site; it is required for APC/C-bound MCC (APC/C(MCC))-dependent CDC20 autoubiquitylation and degradation and for timely anaphase initiation but is dispensable for substrate ubiquitylation by APC/C(CDC20) and APC/C(CDH1). Our results support the model wherein MCC is continuously assembled and disassembled to enable rapid activation of APC/C(CDC20) and CDC20 autoubiquitylation promotes MCC disassembly. We propose that APC15 and Mnd2 negatively regulate APC/C coactivators and report generation of recombinant human APC/C.