RESUMEN
Modified nucleotides in non-coding RNAs, such as tRNAs and snRNAs, represent an important layer of gene expression regulation through their ability to fine-tune mRNA maturation and translation. Dysregulation of such modifications and the enzymes installing them have been linked to various human pathologies including neurodevelopmental disorders and cancers. Several methyltransferases (MTases) are regulated allosterically by human TRMT112 (Trm112 in Saccharomyces cerevisiae), but the interactome of this regulator and targets of its interacting MTases remain incompletely characterized. Here, we have investigated the interaction network of human TRMT112 in intact cells and identify three poorly characterized putative MTases (TRMT11, THUMPD3 and THUMPD2) as direct partners. We demonstrate that these three proteins are active N2-methylguanosine (m2G) MTases and that TRMT11 and THUMPD3 methylate positions 10 and 6 of tRNAs, respectively. For THUMPD2, we discovered that it directly associates with the U6 snRNA, a core component of the catalytic spliceosome, and is required for the formation of m2G, the last 'orphan' modification in U6 snRNA. Furthermore, our data reveal the combined importance of TRMT11 and THUMPD3 for optimal protein synthesis and cell proliferation as well as a role for THUMPD2 in fine-tuning pre-mRNA splicing.
Asunto(s)
Precursores del ARN , Proteínas de Saccharomyces cerevisiae , Humanos , Precursores del ARN/genética , Precursores del ARN/metabolismo , Empalme del ARN , Empalmosomas/metabolismo , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proliferación Celular/genética , Biosíntesis de Proteínas , Metiltransferasas/genética , ARNt Metiltransferasas/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Most eukaryotic mRNAs harbor a characteristic 5' m7GpppN cap that promotes pre-mRNA splicing, mRNA nucleocytoplasmic transport and translation while also protecting mRNAs from exonucleolytic attacks. mRNA caps are eliminated by Dcp2 during mRNA decay, allowing 5'-3' exonucleases to degrade mRNA bodies. However, the Dcp2 decapping enzyme is poorly active on its own and requires binding to stable or transient protein partners to sever the cap of target mRNAs. Here, we analyse the role of one of these partners, the yeast Pby1 factor, which is known to co-localize into P-bodies together with decapping factors. We report that Pby1 uses its C-terminal domain to directly bind to the decapping enzyme. We solved the structure of this Pby1 domain alone and bound to the Dcp1-Dcp2-Edc3 decapping complex. Structure-based mutant analyses reveal that Pby1 binding to the decapping enzyme is required for its recruitment into P-bodies. Moreover, Pby1 binding to the decapping enzyme stimulates growth in conditions in which decapping activation is compromised. Our results point towards a direct connection of Pby1 with decapping and P-body formation, both stemming from its interaction with the Dcp1-Dcp2 holoenzyme.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Endorribonucleasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Adenosina Trifosfato/metabolismo , Dominio Catalítico , Proteínas de Unión al ADN/química , Endopeptidasas/química , Endopeptidasas/metabolismo , Endorribonucleasas/química , Holoenzimas/química , Holoenzimas/metabolismo , Ligasas/metabolismo , Modelos Moleculares , Orgánulos/enzimología , Orgánulos/metabolismo , Unión Proteica , Dominios Proteicos , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/química , Factores de Transcripción/químicaRESUMEN
N6-methyladenosine (m6A) has recently been found abundantly on messenger RNA and shown to regulate most steps of mRNA metabolism. Several important m6A methyltransferases have been described functionally and structurally, but the enzymes responsible for installing one m6A residue on each subunit of human ribosomes at functionally important sites have eluded identification for over 30 years. Here, we identify METTL5 as the enzyme responsible for 18S rRNA m6A modification and confirm ZCCHC4 as the 28S rRNA modification enzyme. We show that METTL5 must form a heterodimeric complex with TRMT112, a known methyltransferase activator, to gain metabolic stability in cells. We provide the first atomic resolution structure of METTL5-TRMT112, supporting that its RNA-binding mode differs distinctly from that of other m6A RNA methyltransferases. On the basis of similarities with a DNA methyltransferase, we propose that METTL5-TRMT112 acts by extruding the adenosine to be modified from a double-stranded nucleic acid.
Asunto(s)
Adenosina/química , Regulación Neoplásica de la Expresión Génica , Metiltransferasas/química , ARN Mensajero/química , ARN Ribosómico 18S/química , Adenosina/genética , Adenosina/metabolismo , Secuencia de Bases , Sitios de Unión , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas , Línea Celular Tumoral , Cristalografía por Rayos X , Eliminación de Gen , Células HCT116 , Humanos , Metiltransferasas/genética , Metiltransferasas/metabolismo , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Ribosómico 18S/genética , ARN Ribosómico 18S/metabolismo , Transducción de Señal , Especificidad por SustratoRESUMEN
DEAD-box helicases play central roles in the metabolism of many RNAs and ribonucleoproteins by assisting their synthesis, folding, function and even their degradation or disassembly. They have been implicated in various phenomena, and it is often difficult to rationalize their molecular roles from in vivo studies. Once purified in vitro, most of them only exhibit a marginal activity and poor specificity. The current model is that they gain specificity and activity through interaction of their intrinsically disordered domains with specific RNA or proteins. DDX3 is a DEAD-box cellular helicase that has been involved in several steps of the HIV viral cycle, including transcription, RNA export to the cytoplasm and translation. In this study, we investigated DDX3 biochemical properties in the context of a biological substrate. DDX3 was overexpressed, purified and its enzymatic activities as well as its RNA binding properties were characterized using both model substrates and a biological substrate, HIV-1 gRNA. Biochemical characterization of DDX3 in the context of a biological substrate identifies HIV-1 gRNA as a rare example of specific substrate and unravels the extent of DDX3 ATPase activity. Analysis of DDX3 binding capacity indicates an unexpected dissociation between its binding capacity and its biochemical activity. We further demonstrate that interaction of DDX3 with HIV-1 gRNA relies both on specific RNA determinants and on the disordered N- and C-terminal regions of the protein. These findings shed a new light regarding the potentiality of DDX3 biochemical activity supporting its multiple cellular functions.
Asunto(s)
ARN Helicasas DEAD-box , Infecciones por VIH/virología , VIH-1/genética , ARN Guía de Kinetoplastida/metabolismo , ARN Helicasas DEAD-box/química , ARN Helicasas DEAD-box/aislamiento & purificación , ARN Helicasas DEAD-box/fisiología , Humanos , Cinética , Unión Proteica , Especificidad por SustratoRESUMEN
Protein synthesis is a complex and highly coordinated process requiring many different protein factors as well as various types of nucleic acids. All translation machinery components require multiple maturation events to be functional. These include post-transcriptional and post-translational modification steps and methylations are the most frequent among these events. In eukaryotes, Trm112, a small protein (COG2835) conserved in all three domains of life, interacts and activates four methyltransferases (Bud23, Trm9, Trm11 and Mtq2) that target different components of the translation machinery (rRNA, tRNAs, release factors). To clarify the function of Trm112 in archaea, we have characterized functionally and structurally its interaction network using Haloferax volcanii as model system. This led us to unravel that methyltransferases are also privileged Trm112 partners in archaea and that this Trm112 network is much more complex than anticipated from eukaryotic studies. Interestingly, among the identified enzymes, some are functionally orthologous to eukaryotic Trm112 partners, emphasizing again the similarity between eukaryotic and archaeal translation machineries. Other partners display some similarities with bacterial methyltransferases, suggesting that Trm112 is a general partner for methyltransferases in all living organisms.
Asunto(s)
Proteínas Arqueales/fisiología , Proteínas Bacterianas/fisiología , Haloferax volcanii/enzimología , Procesamiento Postranscripcional del ARN , ARNt Metiltransferasas/fisiología , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Conjuntos de Datos como Asunto , Activación Enzimática , Células Eucariotas/enzimología , Evolución Molecular , Holoenzimas/fisiología , Inmunoprecipitación , Espectrometría de Masas , Metilación , Modelos Moleculares , Unión Proteica , Conformación Proteica , Mapeo de Interacción de Proteínas , Proteómica , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Especificidad de la Especie , ARNt Metiltransferasas/deficiencia , ARNt Metiltransferasas/genéticaRESUMEN
Viral internal ribosomes entry site (IRES) elements coordinate the recruitment of the host translation machinery to direct the initiation of viral protein synthesis. Within hepatitis C virus (HCV)-like IRES elements, the sub-domain IIId(1) is crucial for recruiting the 40S ribosomal subunit. However, some HCV-like IRES elements possess an additional sub-domain, termed IIId2, whose function remains unclear. Herein, we show that IIId2 sub-domains from divergent viruses have different functions. The IIId2 sub-domain present in Seneca valley virus (SVV), a picornavirus, is dispensable for IRES activity, while the IIId2 sub-domains of two pestiviruses, classical swine fever virus (CSFV) and border disease virus (BDV), are required for 80S ribosomes assembly and IRES activity. Unlike in SVV, the deletion of IIId2 from the CSFV and BDV IRES elements impairs initiation of translation by inhibiting the assembly of 80S ribosomes. Consequently, this negatively affects the replication of CSFV and BDV. Finally, we show that the SVV IIId2 sub-domain is required for efficient viral RNA synthesis and growth of SVV, but not for IRES function. This study sheds light on the molecular evolution of viruses by clearly demonstrating that conserved RNA structures, within distantly related RNA viruses, have acquired different roles in the virus life cycles.
Asunto(s)
Sitios Internos de Entrada al Ribosoma/genética , Pestivirus/genética , Picornaviridae/genética , ARN Viral/genética , Animales , Secuencia de Bases , Sitios de Unión/genética , Virus de la Enfermedad de la Frontera/genética , Virus de la Enfermedad de la Frontera/fisiología , Línea Celular , Virus de la Fiebre Porcina Clásica/genética , Virus de la Fiebre Porcina Clásica/fisiología , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Conformación de Ácido Nucleico , Pestivirus/fisiología , Picornaviridae/fisiología , ARN Viral/química , ARN Viral/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , PorcinosRESUMEN
In the late phase of the HIV virus cycle, the unspliced genomic RNA is exported to the cytoplasm for the necessary translation of the Gag and Gag-pol polyproteins. Three distinct translation initiation mechanisms ensuring Gag production have been described with little rationale for their multiplicity. The Gag-IRES has the singularity to be located within Gag ORF and to directly interact with ribosomal 40S. Aiming at elucidating the specificity and the relevance of this interaction, we probed HIV-1 Gag-IRES structure and developed an innovative integrative modelling strategy to take into account all the gathered information. We propose a novel Gag-IRES secondary structure strongly supported by all experimental data. We further demonstrate the presence of two regions within Gag-IRES that independently and directly interact with the ribosome. Importantly, these binding sites are functionally relevant to Gag translation both in vitro and ex vivo. This work provides insight into the Gag-IRES molecular mechanism and gives compelling evidence for its physiological importance. It allows us to propose original hypotheses about the IRES physiological role and conservation among primate lentiviruses.
Asunto(s)
VIH-1/genética , Sitios Internos de Entrada al Ribosoma , Iniciación de la Cadena Peptídica Traduccional , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Genes Reporteros , VIH-1/metabolismo , Humanos , Células Jurkat , Cinética , Luciferasas/genética , Luciferasas/metabolismo , Modelos Moleculares , Conformación de Ácido Nucleico , Sistemas de Lectura Abierta , Subunidades Ribosómicas Pequeñas de Eucariotas/ultraestructura , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
As obligatory intracellular parasites, viruses rely on cellular machines to complete their life cycle, and most importantly they recruit the host ribosomes to translate their mRNA. The Hepatitis C viral mRNA initiates translation by directly binding the 40S ribosomal subunit in such a way that the initiation codon is correctly positioned in the P site of the ribosome. Such a property is likely to be central for many viruses, therefore the description of host-pathogen interaction at the molecular level is instrumental to provide new therapeutic targets. In this study, we monitored the 40S ribosomal subunit and the viral RNA structural rearrangement induced upon the formation of the binary complex. We further took advantage of an IRES viral mutant mRNA deficient for translation to identify the interactions necessary to promote translation. Using a combination of structure probing in solution and molecular modeling we establish a whole atom model which appears to be very similar to the one obtained recently by cryoEM. Our model brings new information on the complex, and most importantly reveals some structural rearrangement within the ribosome. This study suggests that the formation of a 'kissing complex' between the viral RNA and the 18S ribosomal RNA locks the 40S ribosomal subunit in a conformation proficient for translation.
Asunto(s)
Hepacivirus/genética , Sitios Internos de Entrada al Ribosoma/genética , ARN Viral/genética , Subunidades Ribosómicas Pequeñas de Eucariotas/genética , Animales , Secuencia de Bases , Sitios de Unión/genética , Sistema Libre de Células , Codón Iniciador/genética , Microscopía por Crioelectrón , Células HeLa , Hepacivirus/metabolismo , Hepacivirus/fisiología , Interacciones Huésped-Patógeno , Humanos , Sustancias Macromoleculares/metabolismo , Sustancias Macromoleculares/ultraestructura , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , Iniciación de la Cadena Peptídica Traduccional/genética , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Ribosómico 18S/genética , ARN Ribosómico 18S/metabolismo , ARN Viral/química , ARN Viral/metabolismo , Conejos , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismoRESUMEN
Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus, the etiological agent of Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). One of the key viral proteins that contributes to tumorigenesis is vFLIP, a viral homolog of the FLICE inhibitory protein. This KSHV protein interacts with the NFκB pathway to trigger the expression of antiapoptotic and proinflammatory genes and ultimately leads to tumor formation. The expression of vFLIP is regulated at the translational level by an internal ribosomal entry site (IRES) element. However, the precise mechanism by which ribosomes are recruited internally and the exact location of the IRES has remained elusive. Here we show that a 252-nt fragment directly upstream of vFLIP, within a coding region, directs translation. We have established its RNA structure and demonstrate that IRES activity requires the presence of eIF4A and an intact eIF4G. Furthermore, and unusually for an IRES, eIF4E is part of the complex assembled onto the vFLIP IRES to direct translation. These molecular interactions define a new paradigm for IRES-mediated translation.
Asunto(s)
Herpesvirus Humano 8/genética , ARN Viral/química , Proteínas Virales/genética , Proteínas Virales/metabolismo , Sitios de Unión , Línea Celular Tumoral , Regulación Viral de la Expresión Génica , Células HEK293 , Humanos , Modelos Moleculares , Conformación de Ácido Nucleico , ARN Viral/genética , Ribosomas/metabolismo , Transcripción GenéticaRESUMEN
Initiation of translation on Type II IRESs, such as those of EMCV and FMDV viruses, has been well documented in the recent years. For EMCV, the current model argues for a mechanism in which the key interaction necessary for the pre-initiation complex recruitment is eIF4G binding to the central J-K domains of EMCV-IRES. Here we demonstrate that, in contrast with the current model, the molecular mechanism of EMCV-IRES involves direct recruitment of the 40S subunit. Importantly, we identified a specific structural element that prevents the correct positioning of the initiation codon in the close vicinity of the ribosomal P site. This work clarifies how this interaction could not be anticipated by earlier studies and allows us to propose a new model for initiation complex assembly on EMCV-IRES. The role attributed to eIF4G/4A can thus be refined as stabilizing/promoting the conformational changes that are necessary for IRES function, thus resembling the role conventionally assigned to ITAFs. This raises the interesting possibility that IRESs are primarily ribosome binders, some of which having partly lost the ability to fold into the active structure without the help of proteins.
Asunto(s)
Regiones no Traducidas 5' , Virus de la Encefalomiocarditis/genética , Modelos Genéticos , Iniciación de la Cadena Peptídica Traduccional , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Factor 4A Eucariótico de Iniciación/metabolismo , Factor 4G Eucariótico de Iniciación/metabolismo , Conformación de Ácido Nucleico , Sistemas de Lectura Abierta , ARN Viral/química , ARN Viral/metabolismoRESUMEN
The multiprotein exon junction complex (EJC), deposited by the splicing machinery, is an important constituent of messenger ribonucleoprotein particles because it participates to numerous steps of the mRNA lifecycle from splicing to surveillance via nonsense-mediated mRNA decay pathway. By an unknown mechanism, the EJC also stimulates translation efficiency of newly synthesized mRNAs. Here, we show that among the four EJC core components, the RNA-binding protein metastatic lymph node 51 (MLN51) is a translation enhancer. Overexpression of MLN51 preferentially increased the translation of intron-containing reporters via the EJC, whereas silencing MLN51 decreased translation. In addition, modulation of the MLN51 level in cell-free translational extracts confirmed its direct role in protein synthesis. Immunoprecipitations indicated that MLN51 associates with translation-initiating factors and ribosomal subunits, and in vitro binding assays revealed that MLN51, alone or as part of the EJC, interacts directly with the pivotal eukaryotic translation initiation factor eIF3. Taken together, our data define MLN51 as a translation activator linking the EJC and the translation machinery.
Asunto(s)
Factor 3 de Iniciación Eucariótica/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Biosíntesis de Proteínas , Transporte Biológico , Células HEK293 , Células HeLa , Humanos , Inmunoprecipitación , Intrones , Estructura Terciaria de Proteína , Empalme del ARN , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/metabolismoRESUMEN
Multidrug resistance has become a serious concern in the treatment of bacterial infections. A prominent role is ascribed to the active efflux of xenobiotics out of the bacteria by a tripartite protein machinery. The mechanism of drug extrusion is rather well understood, thanks to the X-ray structures obtained for the Escherichia coli TolC/AcrA/AcrB model system and the related Pseudomonas aeruginosa OprM/MexA/MexB. However, many questions remain unresolved, in particular the stoichiometry of the efflux pump assembly. On the basis of blue native polyacrylamide gel electrophoresis (BN-PAGE) (Wittig et al., Nat. Protoc. 2006, 1, 418-428), we analyzed the binding stoichiometry of both palmitylated and non-palmitylated MexA with the cognate partner OprM trimer at different ratios and detergent conditions. We found that ß-octyl glucopyranoside (ß-OG) detergent was not suitable for this technique. Then we proved that MexA has to be palmitylated in order to stabilized the complex formation with OprM. Finally, we provided evidence for a two by two (2, 4, 6, or upper) binding of palmitylated MexA per trimer of OprM.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Electroforesis en Gel de Poliacrilamida/métodos , Proteínas de Transporte de Membrana/química , Complejos Multiproteicos/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Resistencia a Múltiples Medicamentos , Proteínas de Transporte de Membrana/metabolismo , Complejos Multiproteicos/metabolismo , Ácidos Palmíticos/química , Ácidos Palmíticos/metabolismo , Unión ProteicaRESUMEN
Flavin adenine dinucleotide (FAD) synthetase is an essential enzyme responsible for the synthesis of FAD by adenylation of riboflavin monophosphate (FMN). We have solved the 1.9 A resolution structure of Fad1, the yeast FAD synthetase, in complex with the FAD product in the active site. The structure of Fad1 shows it to be a member of the PP-ATPase superfamily. Important conformational differences in the two motifs involved in binding the phosphate moieties of FAD compared to the Candida glabrata FMNT ortholog suggests that this loop is dynamic and undergoes substantial conformational changes during its catalytic cycle.
Asunto(s)
Flavina-Adenina Dinucleótido/química , Nucleotidiltransferasas/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimología , Candida glabrata/química , Candida glabrata/enzimología , Dominio Catalítico , Cristalografía por Rayos X , Flavina-Adenina Dinucleótido/metabolismo , Modelos Moleculares , Nucleotidiltransferasas/metabolismo , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Genomic RNA of primate lentiviruses serves both as an mRNA that encodes Gag and Gag-Pol polyproteins and as a propagated genome. Translation of this RNA is initiated by standard cap dependant mechanism or by internal entry of the ribosome. Two regions of the genomic RNA are able to attract initiation complexes, the 5' untranslated region and the gag coding region itself. Relying on probing data and a phylogenetic study, we have modelled the secondary structure of HIV-1, HIV-2 and SIV(Mac) coding region. This approach brings to light conserved secondary-structure elements that were shown by mutations to be required for internal entry of the ribosome. No structural homologies with other described viral or cellular IRES can be identified and lentiviral IRESes show many peculiar properties. Most notably, the IRES present in HIV-2 gag coding region is endowed with the unique ability to recruit up to three initiation complexes on a single RNA molecule. The structural and functional properties of gag coding sequence define a new type of IRES. Although its precise role is unknown, the conservation of the IRES among fast evolving lentiviruses suggests an important physiological role.
Asunto(s)
VIH-2/genética , Iniciación de la Cadena Peptídica Traduccional , ARN Viral/química , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Ectima Contagioso , Factores Eucarióticos de Iniciación/metabolismo , Genoma Viral , VIH-1/genética , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , ARN Viral/metabolismo , Virus de la Inmunodeficiencia de los Simios/genéticaRESUMEN
Loss of N7-methylguanosine (m7G) modification is involved in the recently discovered rapid tRNA degradation pathway. In yeast, this modification is catalyzed by the heterodimeric complex composed of a catalytic subunit Trm8 and a noncatalytic subunit Trm82. We have solved the crystal structure of Trm8 alone and in complex with Trm82. Trm8 undergoes subtle conformational changes upon Trm82 binding which explains the requirement of Trm82 for activity. Cocrystallization with the S-adenosyl-methionine methyl donor defines the putative catalytic site and a guanine binding pocket. Small-angle X-ray scattering in solution of the Trm8-Trm82 heterodimer in complex with tRNA(Phe) has enabled us to propose a low-resolution structure of the ternary complex which defines the tRNA binding mode of Trm8-Trm82 and the structural elements contributing to specificity.
Asunto(s)
ARN de Hongos/química , ARN de Transferencia de Fenilalanina/química , Saccharomyces cerevisiae/química , Sitios de Unión , Cristalografía por Rayos X , Guanosina/análogos & derivados , Modelos Moleculares , Conformación de Ácido Nucleico , ARN de Hongos/genética , ARN de Hongos/aislamiento & purificación , ARN de Transferencia de Fenilalanina/genética , ARN de Transferencia de Fenilalanina/aislamiento & purificación , Saccharomyces cerevisiae/genética , Difracción de Rayos XRESUMEN
Protein release factor eRF1 in Saccharomyces cerevisiae, in complex with eRF3 and GTP, is methylated on a functionally crucial Gln residue by the S-adenosylmethionine-dependent methyltransferase Ydr140w. Here we show that eRF1 methylation, in addition to these previously characterized components, requires a 15-kDa zinc-binding protein, Ynr046w. Co-expression in Escherichia coli of Ynr046w and Ydr140w allows the latter to be recovered in soluble form rather than as inclusion bodies, and the two proteins co-purify on nickel-nitrilotriacetic acid chromatography when Ydr140w alone carries a His tag. The crystal structure of Ynr046w has been determined to 1.7 A resolution. It comprises a zinc-binding domain built from both the N- and C-terminal sequences and an inserted domain, absent from bacterial and archaeal orthologs of the protein, composed of three alpha-helices. The active methyltransferase is the heterodimer Ydr140w.Ynr046w, but when alone, both in solution and in crystals, Ynr046w appears to be a homodimer. The Ynr046w eRF1 methyltransferase subunit is shared by the tRNA methyltransferase Trm11p and probably by two other enzymes containing a Rossman fold.
Asunto(s)
Metiltransferasas/fisiología , Factores de Terminación de Péptidos/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Secuencia de Aminoácidos , Animales , Escherichia coli/metabolismo , Glutamina/química , Humanos , Metiltransferasas/metabolismo , Datos de Secuencia Molecular , Níquel/química , Factores de Terminación de Péptidos/química , Unión Proteica , Estructura Terciaria de Proteína , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , Zinc/química , Dedos de Zinc , ARNt MetiltransferasasRESUMEN
Human multidrug resistance protein 1 (MRP1) is a membrane protein that belongs to the ATP-binding cassette (ABC) superfamily of transport proteins. MRP1 contributes to chemotherapy failure by exporting a wide range of anti-cancer drugs when over expressed in the plasma membrane of cells. Here, we report the first high-resolution crystal structure of human MRP1-NBD1. Drug efflux requires energy resulting from hydrolysis of ATP by nucleotide binding domains (NBDs). Contrary to the prokaryotic NBDs, the extremely low intrinsic ATPase activity of isolated MRP1-NBDs allowed us to obtain the structure of wild-type NBD1 in complex with Mg2+/ATP. The structure shows that MRP1-NBD1 adopts a canonical fold, but reveals an unexpected non-productive conformation of the catalytic site, providing an explanation for the low intrinsic ATPase activity of NBD1 and new hypotheses on the cooperativity of ATPase activity between NBD1 and NBD2 upon heterodimer formation.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/química , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/metabolismo , Magnesio/metabolismo , Nucleótidos/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Secuencia de Aminoácidos , Ácido Aspártico/metabolismo , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Dimerización , Histidina/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Homología Estructural de ProteínaRESUMEN
We have recently applied in vitro evolution methods to create in Neocarzinostatin a new binding site for a target molecule unrelated to its natural ligand. The main objective of this work was to solve the structure of some of the selected binders in complex with the target molecule: testosterone. Three proteins (1a.15, 3.24 and 4.1) were chosen as representative members of sequence families that came out of the selection process within different randomization schemes. In order to evaluate ligand-induced conformational adaptation, we also determined the structure of one of the proteins (3.24) in the free and complexed forms. Surprisingly, all these mutants bind not one but two molecules of testosterone in two very different ways. The 3.24 structure revealed that the protein spontaneously evolved in the system to bind two ligand molecules in one single binding crevice. These two binding sites are formed by substituted as well as by non-variable side-chains. The comparison with the free structure shows that only limited structural changes are observed upon ligand binding. The X-ray structures of the complex formed by 1a.15 and 4.1 Neocarzinostatin mutants revealed that the two variants form very similar dimers. These dimers were observed neither for the uncomplexed variants nor for wild-type Neocarzinostatin but were shown here to be induced by ligand binding. Comparison of the three complexed forms clearly suggests that these unanticipated structural responses resulted from the molecular arrangement used for the selection experiments.
Asunto(s)
Evolución Molecular , Testosterona/metabolismo , Cinostatina/química , Cinostatina/metabolismo , Sitios de Unión/genética , Cristalografía por Rayos X , Técnicas In Vitro , Ligandos , Modelos Moleculares , Unión Proteica , Conformación ProteicaRESUMEN
Class I release factors bind to ribosomes in response to stop codons and trigger peptidyl-tRNA hydrolysis at the P site. Prokaryotic and eukaryotic RFs share one motif: a GGQ tripeptide positioned in a loop at the end of a stem region that interacts with the ribosomal peptidyl transferase center. The glutamine side chain of this motif is specifically methylated in both prokaryotes and eukaryotes. Methylation in E. coli is due to PrmC and results in strong stimulation of peptide chain release. We have solved the crystal structure of the complex between E. coli RF1 and PrmC bound to the methyl donor product AdoHCy. Both the GGQ domain (domain 3) and the central region (domains 2 and 4) of RF1 interact with PrmC. Structural and mutagenic data indicate a compact conformation of RF1 that is unlike its conformation when it is bound to the ribosome but is similar to the crystal structure of the protein alone.