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1.
Nature ; 596(7870): 138-142, 2021 08.
Artículo en Inglés | MEDLINE | ID: mdl-34290405

RESUMEN

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 Sustrato
2.
Biochem Soc Trans ; 51(3): 1225-1233, 2023 06 28.
Artículo en Inglés | MEDLINE | ID: mdl-37140261

RESUMEN

Sister chromatid segregation is the final irreversible step of mitosis. It is initiated by a complex regulatory system that ultimately triggers the timely activation of a conserved cysteine protease named separase. Separase cleaves the cohesin protein ring that links the sister chromatids and thus facilitates their separation and segregation to the opposite poles of the dividing cell. Due to the irreversible nature of this process, separase activity is tightly controlled in all eukaryotic cells. In this mini-review, we summarize the latest structural and functional findings on the regulation of separase, with an emphasis on the regulation of the human enzyme by two inhibitors, the universal inhibitor securin and the vertebrate-specific inhibitor CDK1-cyclin B. We discuss the two fundamentally different inhibitory mechanisms by which these inhibitors block separase activity by occluding substrate binding. We also describe conserved mechanisms that facilitate substrate recognition and point out open research questions that will guide studies of this fascinating enzyme for years to come.


Asunto(s)
Proteínas de Ciclo Celular , Mitosis , Humanos , Separasa/química , Separasa/genética , Separasa/metabolismo , Proteínas de Ciclo Celular/metabolismo , Endopeptidasas/genética
3.
Mol Cell ; 54(5): 737-50, 2014 Jun 05.
Artículo en Inglés | MEDLINE | ID: mdl-24768540

RESUMEN

CCR4-NOT is a major effector complex in miRNA-mediated gene silencing. It is recruited to miRNA targets through interactions with tryptophan (W)-containing motifs in TNRC6/GW182 proteins and is required for both translational repression and degradation of miRNA targets. Here, we elucidate the structural basis for the repressive activity of CCR4-NOT and its interaction with TNRC6/GW182s. We show that the conserved CNOT9 subunit attaches to a domain of unknown function (DUF3819) in the CNOT1 scaffold. The resulting complex provides binding sites for TNRC6/GW182, and its crystal structure reveals tandem W-binding pockets located in CNOT9. We further show that the CNOT1 MIF4G domain interacts with the C-terminal RecA domain of DDX6, a translational repressor and decapping activator. The crystal structure of this complex demonstrates striking similarity to the eIF4G-eIF4A complex. Together, our data provide the missing physical links in a molecular pathway that connects miRNA target recognition with translational repression, deadenylation, and decapping.


Asunto(s)
ARN Helicasas DEAD-box/química , MicroARNs/genética , Proteínas Proto-Oncogénicas/química , Interferencia de ARN , Factores de Transcripción/química , Animales , Sitios de Unión , Cristalografía por Rayos X , ARN Helicasas DEAD-box/metabolismo , Drosophila melanogaster , Células HEK293 , Humanos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción/metabolismo
4.
Mol Cell ; 51(3): 360-73, 2013 Aug 08.
Artículo en Inglés | MEDLINE | ID: mdl-23932717

RESUMEN

The PAN2-PAN3 deadenylase complex functions in general and miRNA-mediated mRNA degradation and is specifically recruited to miRNA targets by GW182/TNRC6 proteins. We describe the PAN3 adaptor protein crystal structure that, unexpectedly, forms intertwined and asymmetric homodimers. Dimerization is mediated by a coiled coil that links an N-terminal pseudokinase to a C-terminal knob domain. The PAN3 pseudokinase binds ATP, and this function is required for mRNA degradation in vivo. We further identified conserved surfaces required for mRNA degradation, including the binding surface for the PAN2 deadenylase on the knob domain. The most remarkable structural feature is the presence of a tryptophan-binding pocket at the dimer interface, which mediates binding to TNRC6C in human cells. Together, our data reveal the structural basis for the interaction of PAN3 with PAN2 and the recruitment of the PAN2-PAN3 complex to miRNA targets by TNRC6 proteins.


Asunto(s)
Autoantígenos/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Exorribonucleasas/metabolismo , Proteínas de Unión al ARN/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Autoantígenos/química , Sitios de Unión , Cristalografía por Rayos X , Humanos , MicroARNs/genética , Datos de Secuencia Molecular , Multimerización de Proteína , Estructura Terciaria de Proteína , ARN/metabolismo , Estabilidad del ARN , Proteínas de Unión al ARN/química
5.
Nat Methods ; 12(2): 131-3, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25506719

RESUMEN

We describe a data collection method that uses a single crystal to solve X-ray structures by native SAD (single-wavelength anomalous diffraction). We solved the structures of 11 real-life examples, including a human membrane protein, a protein-DNA complex and a 266-kDa multiprotein-ligand complex, using this method. The data collection strategy is suitable for routine structure determination and can be implemented at most macromolecular crystallography synchrotron beamlines.


Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de la Membrana/química , Complejos Multiproteicos/química , Difracción de Rayos X/métodos , Animales , Humanos , Modelos Moleculares , Conformación Proteica , Programas Informáticos , Sincrotrones
6.
Proc Natl Acad Sci U S A ; 108(26): 10466-71, 2011 Jun 28.
Artículo en Inglés | MEDLINE | ID: mdl-21646546

RESUMEN

Argonaute proteins (AGOs) are essential effectors in RNA-mediated gene silencing pathways. They are characterized by a bilobal architecture, in which one lobe contains the N-terminal and PAZ domains and the other contains the MID and PIWI domains. Here, we present the first crystal structure of the MID-PIWI lobe from a eukaryotic AGO, the Neurospora crassa QDE-2 protein. Compared to prokaryotic AGOs, the domain orientation is conserved, indicating a conserved mode of nucleic acid binding. The PIWI domain shows an adaptable surface loop next to a eukaryote-specific α-helical insertion, which are both likely to contact the PAZ domain in a conformation-dependent manner to sense the functional state of the protein. The MID-PIWI interface is hydrophilic and buries residues that were previously thought to participate directly in the allosteric regulation of guide RNA binding. The interface includes the binding pocket for the guide RNA 5' end, and residues from both domains contribute to binding. Accordingly, micro-RNA (miRNA) binding is particularly sensitive to alteration in the MID-PIWI interface in Drosophila melanogaster AGO1 in vivo. The structure of the QDE-2 MID-PIWI lobe provides molecular and mechanistic insight into eukaryotic AGOs and has significant implications for understanding the role of these proteins in silencing.


Asunto(s)
Proteínas Fúngicas/química , Cristalografía por Rayos X , Proteínas Fúngicas/metabolismo , Modelos Moleculares , Neurospora crassa/química , Ácidos Nucleicos/metabolismo , Unión Proteica , Conformación Proteica
7.
Sci Adv ; 10(1): eadj4686, 2024 Jan 05.
Artículo en Inglés | MEDLINE | ID: mdl-38170783

RESUMEN

Type 1 diabetes mellitus (T1DM) is characterized by insulin deficiency leading to hyperglycemia and several metabolic defects. Insulin therapy remains the cornerstone of T1DM management, yet it increases the risk of life-threatening hypoglycemia and the development of major comorbidities. Here, we report an insulin signaling-independent pathway able to improve glycemic control in T1DM rodents. Co-treatment with recombinant S100 calcium-binding protein A9 (S100A9) enabled increased adherence to glycemic targets with half as much insulin and without causing hypoglycemia. Mechanistically, we demonstrate that the hyperglycemia-suppressing action of S100A9 is due to a Toll-like receptor 4-dependent increase in glucose uptake in specific skeletal muscles (i.e., soleus and diaphragm). In addition, we found that T1DM mice have abnormal systemic inflammation, which is resolved by S100A9 therapy alone (or in combination with low insulin), hence uncovering a potent anti-inflammatory action of S100A9 in T1DM. In summary, our findings reveal the S100A9-TLR4 skeletal muscle axis as a promising therapeutic target for improving T1DM treatment.


Asunto(s)
Diabetes Mellitus Tipo 1 , Hiperglucemia , Hipoglucemia , Animales , Ratones , Insulina/metabolismo , Hipoglucemiantes/farmacología , Hipoglucemiantes/uso terapéutico , Hipoglucemia/complicaciones , Hipoglucemia/tratamiento farmacológico , Hiperglucemia/tratamiento farmacológico , Calgranulina B
8.
FEBS Lett ; 597(6): 794-810, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36271211

RESUMEN

DOCK proteins are a family of multi-domain guanine nucleotide exchange factors (GEFs) that activate the RHO GTPases CDC42 and RAC1, thereby regulating several RHO GTPase-dependent cellular processes. DOCK proteins are characterized by the catalytic DHR2 domain (DOCKDHR2 ), and a phosphatidylinositol(3,4,5)P3 -binding DHR1 domain (DOCKDHR1 ) that targets DOCK proteins to plasma membranes. DOCK-family GEFs are divided into four subfamilies (A to D) differing in their specificities for CDC42 and RAC1, and the composition of accessory signalling domains. Additionally, the DOCK-A and DOCK-B subfamilies are constitutively associated with ELMO proteins that auto-inhibit DOCK GEF activity. We review structural studies that have provided mechanistic insights into DOCK-protein functions. These studies revealed how a conserved nucleotide sensor in DOCKDHR2 catalyses nucleotide exchange, the basis for how different DOCK proteins activate specifically CDC42 and RAC1, and sometimes both, and how up-stream regulators relieve the ELMO-mediated auto-inhibition. We conclude by presenting a model for full-length DOCK9 of the DOCK-D subfamily. The involvement of DOCK GEFs in a range of diseases highlights the importance of gaining structural insights into these proteins to better understand and specifically target them.


Asunto(s)
Factores de Intercambio de Guanina Nucleótido , Proteína de Unión al GTP cdc42 , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Proteínas de Unión al GTP rho/genética , Transducción de Señal , Nucleótidos/metabolismo , Biología , Proteína de Unión al GTP rac1/metabolismo
9.
FEBS J ; 290(16): 3946-3962, 2023 08.
Artículo en Inglés | MEDLINE | ID: mdl-35997767

RESUMEN

CEMIP (cell migration-inducing protein), also known as KIAA1199 or HYBID, is a protein involved in the depolymerisation of hyaluronic acid (HA), a major glycosaminoglycan component of the extracellular matrix. CEMIP was originally described in patients affected by nonsyndromic hearing loss and has subsequently been shown to play a key role in tumour initiation and progression, as well as arthritis, atherosclerosis and idiopathic pulmonary fibrosis. Despite the vast literature associating CEMIP with these diseases, its biology remains elusive. The present review article summarises all the major scientific evidence regarding its structure, function, role and expression, and attempts to cast light on a protein that modulates EMT, fibrosis and tissue inflammation, an unmet key aspect in several inflammatory disease conditions.


Asunto(s)
Hialuronoglucosaminidasa , Humanos , Movimiento Celular , Matriz Extracelular/metabolismo , Ácido Hialurónico/metabolismo , Hialuronoglucosaminidasa/genética , Hialuronoglucosaminidasa/metabolismo
10.
bioRxiv ; 2023 Dec 07.
Artículo en Inglés | MEDLINE | ID: mdl-38106026

RESUMEN

The µ-opioid receptor (µOR), a prototypical member of the G protein-coupled receptor (GPCR) family, is the molecular target of opioid analgesics such as morphine and fentanyl. Due to the limitations and severe side effects of currently available opioid drugs, there is considerable interest in developing novel modulators of µOR function. Most GPCR ligands today are small molecules, however biologics, including antibodies and nanobodies, are emerging as alternative therapeutics with clear advantages such as affinity and target selectivity. Here, we describe the nanobody NbE, which selectively binds to the µOR and acts as an antagonist. We functionally characterize NbE as an extracellular and genetically encoded µOR ligand and uncover the molecular basis for µOR antagonism by solving the cryo-EM structure of the NbE-µOR complex. NbE displays a unique ligand binding mode and achieves µOR selectivity by interactions with the orthosteric pocket and extracellular receptor loops. Based on a ß-hairpin loop formed by NbE that deeply inserts into the µOR and centers most binding contacts, we design short peptide analogues that retain µOR antagonism. The work illustrates the potential of nanobodies to uniquely engage with GPCRs and describes novel µOR ligands that can serve as a basis for therapeutic developments.

11.
EMBO Rep ; 11(7): 522-7, 2010 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-20539312

RESUMEN

Argonaute (AGO) proteins are core components of RNA-induced silencing complexes and have essential roles in RNA-mediated gene silencing. They are characterized by a bilobal architecture, consisting of one lobe containing the amino-terminal and PAZ domains and another containing the MID and PIWI domains. Except for the PAZ domain, structural information on eukaryotic AGO domains is not yet available. In this study, we report the crystal structure of the MID domain of the eukaryotic AGO protein QDE-2 from Neurospora crassa. This domain adopts a Rossmann-like fold and recognizes the 5'-terminal nucleotide of a guide RNA in a manner similar to its prokaryotic counterparts. The 5'-nucleotide-binding site shares common residues with a second, adjacent ligand-binding site, suggesting a mechanism for the cooperative binding of ligands to the MID domain of eukaryotic AGOs.


Asunto(s)
Proteínas Fúngicas/química , Neurospora crassa/química , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Alineación de Secuencia
12.
Nat Commun ; 13(1): 4107, 2022 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-35840613

RESUMEN

Unrestrained ketogenesis leads to life-threatening ketoacidosis whose incidence is high in patients with diabetes. While insulin therapy reduces ketogenesis this approach is sub-optimal. Here, we report an insulin-independent pathway able to normalize diabetic ketogenesis. By generating insulin deficient male mice lacking or re-expressing Toll-Like Receptor 4 (TLR4) only in liver or hepatocytes, we demonstrate that hepatic TLR4 in non-parenchymal cells mediates the ketogenesis-suppressing action of S100A9. Mechanistically, S100A9 acts extracellularly to activate the mechanistic target of rapamycin complex 1 (mTORC1) in a TLR4-dependent manner. Accordingly, hepatic-restricted but not hepatocyte-restricted loss of Tuberous Sclerosis Complex 1 (TSC1, an mTORC1 inhibitor) corrects insulin-deficiency-induced hyperketonemia. Therapeutically, recombinant S100A9 administration restrains ketogenesis and improves hyperglycemia without causing hypoglycemia in diabetic mice. Also, circulating S100A9 in patients with ketoacidosis is only marginally increased hence unveiling a window of opportunity to pharmacologically augment S100A9 for preventing unrestrained ketogenesis. In summary, our findings reveal the hepatic S100A9-TLR4-mTORC1 axis in non-parenchymal cells as a promising therapeutic target for restraining diabetic ketogenesis.


Asunto(s)
Diabetes Mellitus Experimental , Cetosis , Animales , Calgranulina B/metabolismo , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/metabolismo , Insulina/metabolismo , Cuerpos Cetónicos/metabolismo , Hígado/metabolismo , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo
13.
Mol Cell Oncol ; 8(4): 1975473, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34616878

RESUMEN

Accurate chromosome segregation depends on tight regulation of the protease separase, which cleaves the ring-shaped cohesin complex that entraps the two sister chromatids. We recently reported structures of human separase bound to its inhibitors securin or the cyclin-dependent kinase 1 (CDK1)-cyclin B1 (CCNB1)-cyclin-dependent kinases regulatory subunit 1 (CKS1) complex and discovered an array of molecular mechanisms that block cohesin-cleavage.

14.
Elife ; 102021 09 14.
Artículo en Inglés | MEDLINE | ID: mdl-34519269

RESUMEN

The mTORC1 kinase complex regulates cell growth, proliferation, and survival. Because mis-regulation of DEPTOR, an endogenous mTORC1 inhibitor, is associated with some cancers, we reconstituted mTORC1 with DEPTOR to understand its function. We find that DEPTOR is a unique partial mTORC1 inhibitor that may have evolved to preserve feedback inhibition of PI3K. Counterintuitively, mTORC1 activated by RHEB or oncogenic mutation is much more potently inhibited by DEPTOR. Although DEPTOR partially inhibits mTORC1, mTORC1 prevents this inhibition by phosphorylating DEPTOR, a mutual antagonism that requires no exogenous factors. Structural analyses of the mTORC1/DEPTOR complex showed DEPTOR's PDZ domain interacting with the mTOR FAT region, and the unstructured linker preceding the PDZ binding to the mTOR FRB domain. The linker and PDZ form the minimal inhibitory unit, but the N-terminal tandem DEP domains also significantly contribute to inhibition.


Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Sitios de Unión , Microscopía por Crioelectrón , Escherichia coli , Regulación de la Expresión Génica , Humanos , Procesamiento de Imagen Asistido por Computador , Péptidos y Proteínas de Señalización Intracelular/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Modelos Moleculares , Dominios PDZ , Unión Proteica , Conformación Proteica , Proteínas Recombinantes , Serina-Treonina Quinasas TOR/genética
15.
Nat Commun ; 11(1): 3464, 2020 07 10.
Artículo en Inglés | MEDLINE | ID: mdl-32651375

RESUMEN

DOCK (dedicator of cytokinesis) proteins are multidomain guanine nucleotide exchange factors (GEFs) for RHO GTPases that regulate intracellular actin dynamics. DOCK proteins share catalytic (DOCKDHR2) and membrane-associated (DOCKDHR1) domains. The structurally-related DOCK1 and DOCK2 GEFs are specific for RAC, and require ELMO (engulfment and cell motility) proteins for function. The N-terminal RAS-binding domain (RBD) of ELMO (ELMORBD) interacts with RHOG to modulate DOCK1/2 activity. Here, we determine the cryo-EM structures of DOCK2-ELMO1 alone, and as a ternary complex with RAC1, together with the crystal structure of a RHOG-ELMO2RBD complex. The binary DOCK2-ELMO1 complex adopts a closed, auto-inhibited conformation. Relief of auto-inhibition to an active, open state, due to a conformational change of the ELMO1 subunit, exposes binding sites for RAC1 on DOCK2DHR2, and RHOG and BAI GPCRs on ELMO1. Our structure explains how up-stream effectors, including DOCK2 and ELMO1 phosphorylation, destabilise the auto-inhibited state to promote an active GEF.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Calorimetría , Proteínas Activadoras de GTPasa/genética , Factores de Intercambio de Guanina Nucleótido/genética , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Cinética , Microscopía Electrónica , Fosforilación , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismo , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismo
16.
Nat Commun ; 10(1): 4502, 2019 10 03.
Artículo en Inglés | MEDLINE | ID: mdl-31582740

RESUMEN

The cytosolic antibody receptor TRIM21 possesses unique ubiquitination activity that drives broad-spectrum anti-pathogen targeting and underpins the protein depletion technology Trim-Away. This activity is dependent on formation of self-anchored, K63-linked ubiquitin chains by the heterodimeric E2 enzyme Ube2N/Ube2V2. Here we reveal how TRIM21 facilitates ubiquitin transfer and differentiates this E2 from other closely related enzymes. A tri-ionic motif provides optimally distributed anchor points that allow TRIM21 to wrap an Ube2N~Ub complex around its RING domain, locking the closed conformation and promoting ubiquitin discharge. Mutation of these anchor points inhibits ubiquitination with Ube2N/Ube2V2, viral neutralization and immune signalling. We show that the same mechanism is employed by the anti-HIV restriction factor TRIM5 and identify spatially conserved ionic anchor points in other Ube2N-recruiting RING E3s. The tri-ionic motif is exclusively required for Ube2N but not Ube2D1 activity and provides a generic E2-specific catalysis mechanism for RING E3s.


Asunto(s)
Lisina/metabolismo , Ribonucleoproteínas/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitinación/fisiología , Secuencias de Aminoácidos/genética , Factores de Restricción Antivirales , Biocatálisis , Cristalografía por Rayos X , Células HEK293 , Células HeLa , Humanos , Modelos Moleculares , Mutación , Resonancia Magnética Nuclear Biomolecular , Unión Proteica/genética , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/química , Ubiquitina-Proteína Ligasas/metabolismo
17.
Elife ; 72018 11 26.
Artículo en Inglés | MEDLINE | ID: mdl-30460895

RESUMEN

The biogenesis of 60S ribosomal subunits is initiated in the nucleus where rRNAs and proteins form pre-60S particles. These pre-60S particles mature by transiently interacting with various assembly factors. The ~5000 amino-acid AAA+ ATPase Rea1 (or Midasin) generates force to mechanically remove assembly factors from pre-60S particles, which promotes their export to the cytosol. Here we present three Rea1 cryoEM structures. We visualise the Rea1 engine, a hexameric ring of AAA+ domains, and identify an α-helical bundle of AAA2 as a major ATPase activity regulator. The α-helical bundle interferes with nucleotide-induced conformational changes that create a docking site for the substrate binding MIDAS domain on the AAA +ring. Furthermore, we reveal the architecture of the Rea1 linker, which is involved in force generation and extends from the AAA+ ring. The data presented here provide insights into the mechanism of one of the most complex ribosome maturation factors.


Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/química , Adenosina Trifosfato/química , ARN Ribosómico/química , Proteínas Ribosómicas/química , Subunidades Ribosómicas Grandes de Eucariotas/genética , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , ATPasas Asociadas con Actividades Celulares Diversas/genética , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Adenosina Difosfato/química , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Sitios de Unión , Fenómenos Biomecánicos , Clonación Molecular , Microscopía por Crioelectrón , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Cinética , Modelos Moleculares , Biogénesis de Organelos , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , ARN de Hongos/química , ARN de Hongos/metabolismo , ARN Ribosómico/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/enzimología , Subunidades Ribosómicas Grandes de Eucariotas/ultraestructura , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidad por Sustrato
18.
Essays Biochem ; 61(5): 543-560, 2017 11 08.
Artículo en Inglés | MEDLINE | ID: mdl-29118099

RESUMEN

Structure-based drug design plays a central role in therapeutic development. Until recently, protein crystallography and NMR have dominated experimental approaches to obtain structural information of biological molecules. However, in recent years rapid technical developments in single particle cryo-electron microscopy (cryo-EM) have enabled the determination to near-atomic resolution of macromolecules ranging from large multi-subunit molecular machines to proteins as small as 64 kDa. These advances have revolutionized structural biology by hugely expanding both the range of macromolecules whose structures can be determined, and by providing a description of macromolecular dynamics. Cryo-EM is now poised to similarly transform the discipline of structure-based drug discovery. This article reviews the potential of cryo-EM for drug discovery with reference to protein ligand complex structures determined using this technique.


Asunto(s)
Microscopía por Crioelectrón/métodos , Diseño de Fármacos , Complejos Multiproteicos/química , Proteínas/química , Bibliotecas de Moléculas Pequeñas/química , Animales , Microscopía por Crioelectrón/instrumentación , Cristalografía por Rayos X , Descubrimiento de Drogas , Escherichia coli/química , Humanos , Ligandos , Modelos Moleculares , Complejos Multiproteicos/agonistas , Complejos Multiproteicos/antagonistas & inhibidores , Proteínas/agonistas , Proteínas/antagonistas & inhibidores , Saccharomyces cerevisiae/química , Bibliotecas de Moléculas Pequeñas/síntesis química , Relación Estructura-Actividad
19.
Nat Struct Mol Biol ; 24(4): 414-418, 2017 04.
Artículo en Inglés | MEDLINE | ID: mdl-28263324

RESUMEN

Separase is a caspase-family protease that initiates chromatid segregation by cleaving the kleisin subunits (Scc1 and Rec8) of cohesin, and regulates centrosome duplication and mitotic spindle function through cleavage of kendrin and Slk19. To understand the mechanisms of securin regulation of separase, we used single-particle cryo-electron microscopy (cryo-EM) to determine a near-atomic-resolution structure of the Caenorhabditis elegans separase-securin complex. Separase adopts a triangular-shaped bilobal architecture comprising an N-terminal tetratricopeptide repeat (TPR)-like α-solenoid domain docked onto the conserved C-terminal protease domain. Securin engages separase in an extended antiparallel conformation, interacting with both lobes. It inhibits separase by interacting with the catalytic site through a pseudosubstrate mechanism, thus revealing that in the inhibited separase-securin complex, the catalytic site adopts a conformation compatible with substrate binding. Securin is protected from cleavage because an aliphatic side chain at the P1 position represses protease activity by disrupting the organization of catalytic site residues.


Asunto(s)
Microscopía por Crioelectrón , Securina/ultraestructura , Separasa/ultraestructura , Secuencias de Aminoácidos , Animales , Caenorhabditis elegans , Humanos , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Estabilidad Proteica , Estructura Secundaria de Proteína , Securina/química , Separasa/química , Especificidad por Sustrato
20.
Nat Struct Mol Biol ; 20(11): 1289-97, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-24121232

RESUMEN

The CCR4-NOT deadenylase complex is a master regulator of translation and mRNA stability. Its NOT module orchestrates recruitment of the catalytic subunits to target mRNAs. We report the crystal structure of the human NOT module formed by the CNOT1, CNOT2 and CNOT3 C-terminal (-C) regions. CNOT1-C provides a rigid scaffold consisting of two perpendicular stacks of HEAT-like repeats. CNOT2-C and CNOT3-C heterodimerize through their SH3-like NOT-box domains. The heterodimer is stabilized and tightly anchored to the surface of CNOT1 through an unexpected intertwined arrangement of peptide regions lacking defined secondary structure. These assembly peptides mold onto their respective binding surfaces and form extensive interfaces. Mutagenesis of individual interfaces and perturbation of endogenous protein ratios cause defects in complex assembly and mRNA decay. Our studies provide a structural framework for understanding the recruitment of the CCR4-NOT complex to mRNA targets.


Asunto(s)
Proteínas Represoras/química , Proteínas Represoras/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Cristalografía por Rayos X , Análisis Mutacional de ADN , Humanos , Modelos Biológicos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Multimerización de Proteína , ARN Mensajero/metabolismo , Receptores CCR4/metabolismo , Proteínas Represoras/genética , Factores de Transcripción/genética
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