RESUMEN
Endoplasmic reticulum-associated degradation (ERAD) is a protein quality control pathway of fundamental importance to cellular homeostasis. Although multiple ERAD pathways exist for targeting topologically distinct substrates, all pathways require substrate ubiquitination. Here, we characterize a key role for the UBE2G2 Binding Region (G2BR) of the ERAD accessory protein ancient ubiquitous protein 1 (AUP1) in ERAD pathways. This 27-amino acid (aa) region of AUP1 binds with high specificity and low nanomolar affinity to the backside of the ERAD ubiquitin-conjugating enzyme (E2) UBE2G2. The structure of the AUP1 G2BR (G2BRAUP1) in complex with UBE2G2 reveals an interface that includes a network of salt bridges, hydrogen bonds, and hydrophobic interactions essential for AUP1 function in cells. The G2BRAUP1 shares significant structural conservation with the G2BR found in the E3 ubiquitin ligase gp78 and in vitro can similarly allosterically activate ubiquitination in conjunction with ERAD E3s. In cells, AUP1 is uniquely required to maintain normal levels of UBE2G2; this is due to G2BRAUP1 binding to the E2 and preventing its rapid degradation. In addition, the G2BRAUP1 is required for both ER membrane recruitment of UBE2G2 and for its activation at the ER membrane. Thus, by binding to the backside of a critical ERAD E2, G2BRAUP1 plays multiple critical roles in ERAD.
Asunto(s)
Degradación Asociada con el Retículo Endoplásmico/genética , Proteínas de la Membrana/fisiología , Enzimas Ubiquitina-Conjugadoras/fisiología , Secuencia de Aminoácidos/genética , Línea Celular Tumoral , Retículo Endoplásmico/metabolismo , Degradación Asociada con el Retículo Endoplásmico/fisiología , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/ultraestructura , Unión Proteica/genética , Dominios Proteicos/genética , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Enzimas Ubiquitina-Conjugadoras/ultraestructura , UbiquitinaciónRESUMEN
Ufmylation is a post-translational modification essential for regulating key cellular processes. A three-enzyme cascade involving E1, E2 and E3 is required for UFM1 attachment to target proteins. How UBA5 (E1) and UFC1 (E2) cooperatively activate and transfer UFM1 is still unclear. Here, we present the crystal structure of UFC1 bound to the C-terminus of UBA5, revealing how UBA5 interacts with UFC1 via a short linear sequence, not observed in other E1-E2 complexes. We find that UBA5 has a region outside the adenylation domain that is dispensable for UFC1 binding but critical for UFM1 transfer. This region moves next to UFC1's active site Cys and compensates for a missing loop in UFC1, which exists in other E2s and is needed for the transfer. Overall, our findings advance the understanding of UFM1's conjugation machinery and may serve as a basis for the development of ufmylation inhibitors.
Asunto(s)
Procesamiento Proteico-Postraduccional , Proteínas/metabolismo , Enzimas Activadoras de Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Dominio Catalítico/genética , Humanos , Simulación del Acoplamiento Molecular , Resonancia Magnética Nuclear Biomolecular , Unión Proteica/genética , Proteínas/genética , Proteínas/aislamiento & purificación , Proteínas/ultraestructura , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura , Enzimas Activadoras de Ubiquitina/genética , Enzimas Activadoras de Ubiquitina/aislamiento & purificación , Enzimas Activadoras de Ubiquitina/ultraestructura , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/aislamiento & purificación , Enzimas Ubiquitina-Conjugadoras/ultraestructura , Difracción de Rayos XRESUMEN
The BRCA1-BARD1 tumour suppressor is an E3 ubiquitin ligase necessary for the repair of DNA double-strand breaks by homologous recombination1-10. The BRCA1-BARD1 complex localizes to damaged chromatin after DNA replication and catalyses the ubiquitylation of histone H2A and other cellular targets11-14. The molecular bases for the recruitment to double-strand breaks and target recognition of BRCA1-BARD1 remain unknown. Here we use cryo-electron microscopy to show that the ankyrin repeat and tandem BRCT domains in BARD1 adopt a compact fold and bind to nucleosomal histones, DNA and monoubiquitin attached to H2A amino-terminal K13 or K15, two signals known to be specific for double-strand breaks15,16. We further show that RING domains17 in BRCA1-BARD1 orient an E2 ubiquitin-conjugating enzyme atop the nucleosome in a dynamic conformation, primed for ubiquitin transfer to the flexible carboxy-terminal tails of H2A and variant H2AX. Our work reveals a regulatory crosstalk in which recognition of monoubiquitin by BRCA1-BARD1 at the N terminus of H2A blocks the formation of polyubiquitin chains and cooperatively promotes ubiquitylation at the C terminus of H2A. These findings elucidate the mechanisms of BRCA1-BARD1 chromatin recruitment and ubiquitylation specificity, highlight key functions of BARD1 in both processes and explain how BRCA1-BARD1 promotes homologous recombination by opposing the DNA repair protein 53BP1 in post-replicative chromatin18-22. These data provide a structural framework to evaluate BARD1 variants and help to identify mutations that drive the development of cancer.
Asunto(s)
Proteína BRCA1/metabolismo , Nucleosomas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Ubiquitinación , Microscopía por Crioelectrón , Reparación del ADN , Histonas/química , Histonas/metabolismo , Recombinación Homóloga , Humanos , Modelos Moleculares , Mutación , Neoplasias/genética , Nucleosomas/química , Nucleosomas/genética , Nucleosomas/ultraestructura , Dominios Proteicos , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/ultraestructura , Proteína 1 de Unión al Supresor Tumoral P53/antagonistas & inhibidores , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Enzimas Ubiquitina-Conjugadoras/química , Enzimas Ubiquitina-Conjugadoras/metabolismo , Enzimas Ubiquitina-Conjugadoras/ultraestructura , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/ultraestructuraRESUMEN
Mutations in the E3 ubiquitin ligase RING domains of BRCA1/BARD1 predispose carriers to breast and ovarian cancers. We present the structure of the BRCA1/BARD1 RING heterodimer with the E2 enzyme UbcH5c bound to its cellular target, the nucleosome, along with biochemical data that explain how the complex selectively ubiquitylates lysines 125, 127 and 129 in the flexible C-terminal tail of H2A in a fully human system. The structure reveals that a novel BARD1-histone interface couples to a repositioning of UbcH5c compared to the structurally similar PRC1 E3 ligase Ring1b/Bmi1 that ubiquitylates H2A Lys119 in nucleosomes. This interface is sensitive to both H3 Lys79 methylation status and mutations found in individuals with cancer. Furthermore, NMR reveals an unexpected mode of E3-mediated substrate regulation through modulation of dynamics in the C-terminal tail of H2A. Our findings provide insight into how E3 ligases preferentially target nearby lysine residues in nucleosomes by a steric occlusion and distancing mechanism.
Asunto(s)
Proteína BRCA1/química , Proteína BRCA1/metabolismo , Histonas/metabolismo , Nucleosomas/química , Nucleosomas/metabolismo , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Proteína BRCA1/ultraestructura , Sitios de Unión , Dominio Catalítico , Microscopía por Crioelectrón , Histonas/química , Histonas/ultraestructura , Humanos , Lisina/química , Lisina/metabolismo , Modelos Moleculares , Unión Proteica , Reproducibilidad de los Resultados , Proteínas Supresoras de Tumor/ultraestructura , Enzimas Ubiquitina-Conjugadoras/química , Enzimas Ubiquitina-Conjugadoras/metabolismo , Enzimas Ubiquitina-Conjugadoras/ultraestructura , Ubiquitina-Proteína Ligasas/ultraestructuraRESUMEN
The bacterial effector MavC modulates the host immune response by blocking Ube2N activity employing an E1-independent ubiquitin ligation, catalyzing formation of a γ-glutamyl-ε-Lys (Gln40Ub-Lys92Ube2N) isopeptide crosslink using a transglutaminase mechanism. Here we provide biochemical evidence in support of MavC targeting the activated, thioester-linked Ube2N~ubiquitin conjugate, catalyzing an intramolecular transglutamination reaction, covalently crosslinking the Ube2N and Ub subunits effectively inactivating the E2~Ub conjugate. Ubiquitin exhibits weak binding to MavC alone, but shows an increase in affinity when tethered to Ube2N in a disulfide-linked substrate that mimics the charged E2~Ub conjugate. Crystal structures of MavC in complex with the substrate mimic and crosslinked product provide insights into the reaction mechanism and underlying protein dynamics that favor transamidation over deamidation, while revealing a crucial role for the structurally unique insertion domain in substrate recognition. This work provides a structural basis of ubiquitination by transglutamination and identifies this enzyme's true physiological substrate.
Asunto(s)
Proteínas Bacterianas/metabolismo , Legionella pneumophila/enzimología , Transglutaminasas/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/ultraestructura , Dominio Catalítico/genética , Clonación Molecular , Cristalografía por Rayos X , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura , Especificidad por Sustrato , Transglutaminasas/genética , Transglutaminasas/aislamiento & purificación , Transglutaminasas/ultraestructura , Ubiquitina/aislamiento & purificación , Ubiquitina/ultraestructura , Enzimas Ubiquitina-Conjugadoras/aislamiento & purificación , Enzimas Ubiquitina-Conjugadoras/ultraestructura , UbiquitinaciónRESUMEN
Eukaryotic cell biology depends on cullin-RING E3 ligase (CRL)-catalysed protein ubiquitylation1, which is tightly controlled by the modification of cullin with the ubiquitin-like protein NEDD82-6. However, how CRLs catalyse ubiquitylation, and the basis of NEDD8 activation, remain unknown. Here we report the cryo-electron microscopy structure of a chemically trapped complex that represents the ubiquitylation intermediate, in which the neddylated CRL1ß-TRCP promotes the transfer of ubiquitin from the E2 ubiquitin-conjugating enzyme UBE2D to its recruited substrate, phosphorylated IκBα. NEDD8 acts as a nexus that binds disparate cullin elements and the RING-activated ubiquitin-linked UBE2D. Local structural remodelling of NEDD8 and large-scale movements of CRL domains converge to juxtapose the substrate and the ubiquitylation active site. These findings explain how a distinctive ubiquitin-like protein alters the functions of its targets, and show how numerous NEDD8-dependent interprotein interactions and conformational changes synergistically configure a catalytic CRL architecture that is both robust, to enable rapid ubiquitylation of the substrate, and fragile, to enable the subsequent functions of cullin-RING proteins.
Asunto(s)
Microscopía por Crioelectrón , Proteína NEDD8/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Biocatálisis , Humanos , Modelos Moleculares , Proteína NEDD8/química , Proteína NEDD8/ultraestructura , Inhibidor NF-kappaB alfa/química , Inhibidor NF-kappaB alfa/metabolismo , Inhibidor NF-kappaB alfa/ultraestructura , Fosforilación , Conformación Proteica , Especificidad por Sustrato , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/química , Enzimas Ubiquitina-Conjugadoras/ultraestructura , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/ultraestructura , UbiquitinaciónRESUMEN
Aggregation of amyloidogenic proteins is an abnormal biological process implicated in neurodegenerative disorders. Whereas the aggregation process of amyloid-forming proteins has been studied extensively, the mechanism of aggregate removal is poorly understood. We recently demonstrated that proteasomes could fragment filamentous aggregates into smaller entities, restricting aggregate size [1]. Here, we show in vitro that UBE2W can modify the N-terminus of both α-synuclein and a tau tetra-repeat domain with a single ubiquitin. We demonstrate that an engineered N-terminal ubiquitin modification changes the aggregation process of both proteins, resulting in the formation of structurally distinct aggregates. Single-molecule approaches further reveal that the proteasome can target soluble oligomers assembled from ubiquitin-modified proteins independently of its peptidase activity, consistent with our recently reported fibril-fragmenting activity. Based on these results, we propose that proteasomes are able to target oligomers assembled from N-terminally ubiquitinated proteins. Our data suggest a possible disassembly mechanism by which N-terminal ubiquitination and the proteasome may together impede aggregate formation.
Asunto(s)
Proteínas Amiloidogénicas/genética , Enfermedades Neurodegenerativas/genética , Enzimas Ubiquitina-Conjugadoras/genética , alfa-Sinucleína/genética , Proteínas tau/genética , Proteínas Amiloidogénicas/ultraestructura , Citoplasma/genética , Citoplasma/ultraestructura , Holoenzimas/genética , Holoenzimas/ultraestructura , Humanos , Enfermedades Neurodegenerativas/patología , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/ultraestructura , Agregación Patológica de Proteínas/genética , Dominios Proteicos , Multimerización de Proteína , Ubiquitina/genética , Enzimas Ubiquitina-Conjugadoras/ultraestructura , Ubiquitinación/genética , alfa-Sinucleína/ultraestructura , Proteínas tau/ultraestructuraRESUMEN
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
The mitotic checkpoint complex (MCC) coordinates proper chromosome biorientation on the spindle with ubiquitination activities of CDC20-activated anaphase-promoting complex/cyclosome (APC/C(CDC20)). APC/C(CDC20) and two E2s, UBE2C and UBE2S, catalyze ubiquitination through distinct architectures for linking ubiquitin (UB) to substrates and elongating polyUB chains, respectively. MCC, which contains a second molecule of CDC20, blocks APC/C(CDC20)-UBE2C-dependent ubiquitination of Securin and Cyclins, while differentially determining or inhibiting CDC20 ubiquitination to regulate spindle surveillance, checkpoint activation, and checkpoint termination. Here electron microscopy reveals conformational variation of APC/C(CDC20)-MCC underlying this multifaceted regulation. MCC binds APC/C-bound CDC20 to inhibit substrate access. However, rotation about the CDC20-MCC assembly and conformational variability of APC/C modulate UBE2C-catalyzed ubiquitination of MCC's CDC20 molecule. Access of UBE2C is limiting for subsequent polyubiquitination by UBE2S. We propose that conformational dynamics of APC/C(CDC20)-MCC modulate E2 activation and determine distinctive ubiquitination activities as part of a response mechanism ensuring accurate sister chromatid segregation.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Ciclosoma-Complejo Promotor de la Anafase/ultraestructura , Segregación Cromosómica , Microscopía por Crioelectrón , Puntos de Control de la Fase M del Ciclo Celular , Huso Acromático/metabolismo , Huso Acromático/ultraestructura , Ubiquitina/metabolismo , Sitios de Unión , Proteínas Cdc20/metabolismo , Proteínas Cdc20/ultraestructura , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad , Enzimas Activadoras de Ubiquitina/metabolismo , Enzimas Activadoras de Ubiquitina/ultraestructura , Enzimas Ubiquitina-Conjugadoras/metabolismo , Enzimas Ubiquitina-Conjugadoras/ultraestructura , UbiquitinaciónRESUMEN
Protein phosphorylation is a modification that offers a dynamic and reversible mechanism to regulate the majority of cellular processes. Numerous diseases are associated with aberrant regulation of phosphorylation-induced switches. Phosphorylation is emerging as a mechanism to modulate ubiquitination by regulating key enzymes in this pathway. The molecular mechanisms underpinning how phosphorylation regulates ubiquitinating enzymes, however, are elusive. Here, we show the high conservation of a functional site in E2 ubiquitin-conjugating enzymes. In catalytically active E2s, this site contains aspartate or a phosphorylatable serine and we refer to it as the conserved E2 serine/aspartate (CES/D) site. Molecular simulations of substrate-bound and -unbound forms of wild type, mutant and phosphorylated E2s, provide atomistic insight into the role of the CES/D residue for optimal E2 activity. Both the size and charge of the side group at the site play a central role in aligning the substrate lysine toward E2 catalytic cysteine to control ubiquitination efficiency. The CES/D site contributes to the fingerprint of the E2 superfamily. We propose that E2 enzymes can be divided into constitutively active or regulated families. E2s characterized by an aspartate at the CES/D site signify constitutively active E2s, whereas those containing a serine can be regulated by phosphorylation.
Asunto(s)
Modelos Químicos , Simulación del Acoplamiento Molecular , Enzimas Ubiquitina-Conjugadoras/química , Enzimas Ubiquitina-Conjugadoras/ultraestructura , Ubiquitina/química , Ubiquitina/ultraestructura , Sitios de Unión , Catálisis , Activación Enzimática , Fosforilación , Unión Proteica , UbiquitinaciónRESUMEN
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
Importin13 (Imp13) is an unusual ß-karyopherin that is able to both import and export cargoes in and out of the nucleus. In the cytoplasm, Imp13 associates with different cargoes such as Mago-Y14 and Ubc9, and facilitates their import into the nucleus where RanGTP binding promotes the release of the cargo. In this study, we present the 2.8 Å resolution crystal structure of Imp13 in complex with the SUMO E2-conjugating enzyme, Ubc9. The structure shows an uncommon mode of cargo-karyopherin recognition with Ubc9 binding at the N-terminal portion of Imp13, occupying the entire RanGTP-binding site. Comparison of the Imp13-Ubc9 complex with Imp13-Mago-Y14 shows the remarkable plasticity of Imp13, whose conformation changes from a closed ring to an open superhelix when bound to the two different cargoes. The structure also shows that the binding mode is compatible with the sumoylated states of Ubc9. Indeed, we find that Imp13 is able to bind sumoylated Ubc9 in vitro and suppresses autosumoylation activity in the complex.
Asunto(s)
Transporte Activo de Núcleo Celular/genética , Carioferinas/ultraestructura , Modelos Moleculares , Complejos Multiproteicos/ultraestructura , Enzimas Ubiquitina-Conjugadoras/ultraestructura , Sitios de Unión/genética , Western Blotting , Cristalografía , Humanos , Carioferinas/genética , Carioferinas/metabolismo , Complejos Multiproteicos/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/ultraestructura , Sumoilación , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismoRESUMEN
Ubiquitin and ubiquitin-like protein-conjugating enzymes play central roles in posttranslational modification processes. The ubiquitin-fold modifier 1 (Ufm1), one of a variety of ubiquitin-like modifiers, is covalently attached to target proteins via Uba5 and Ufm1-conjugating enzyme 1 (Ufc1), which are analogous to the E1 and E2 ubiquitylation enzymes. As Ufm1-related proteins are conserved in metazoa and plants, the Ufm1 system likely plays important roles in various multicellular organisms. Herein, we report the X-ray structure of human Ufc1 determined at 1.6 A resolution. The Ufc1 structure comprises a canonical E2 domain and an additional N-terminal domain. The Uba5 binding site on Ufc1 was assigned by structural comparison of Ufc1 and Ubc12 and related mutational analyses. In addition, we show that the N-terminal unique domain of Ufc1 contributes to thermal stability.