RESUMEN
The Mitochondrial Complex I Assembly (MCIA) complex is essential for the biogenesis of respiratory Complex I (CI), the first enzyme in the respiratory chain, which has been linked to Alzheimer's disease (AD) pathogenesis. However, how MCIA facilitates CI assembly, and how it is linked with AD pathogenesis, is poorly understood. Here we report the structural basis of the complex formation between the MCIA subunits ECSIT and ACAD9. ECSIT binding induces a major conformational change in the FAD-binding loop of ACAD9, releasing the FAD cofactor and converting ACAD9 from a fatty acid ß-oxidation (FAO) enzyme to a CI assembly factor. We provide evidence that ECSIT phosphorylation downregulates its association with ACAD9 and is reduced in neuronal cells upon exposure to amyloid-ß (Aß) oligomers. These findings advance our understanding of the MCIA complex assembly and suggest a possible role for ECSIT in the reprogramming of bioenergetic pathways linked to Aß toxicity, a hallmark of AD.
Asunto(s)
Enfermedad de Alzheimer , Complejo I de Transporte de Electrón , Humanos , Oxidación-Reducción , Complejo I de Transporte de Electrón/metabolismo , Metabolismo Energético , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismoRESUMEN
To eliminate specific or aberrant transcripts, eukaryotes use nuclear RNA-targeting complexes that deliver them to the exosome for degradation. S. pombe MTREC, and its human counterpart PAXT, are key players in this mechanism but inner workings of these complexes are not understood in sufficient detail. Here, we present an NMR structure of an MTREC scaffold protein Red1 helix-turn-helix domain bound to the Iss10 N-terminus and show this interaction is required for proper cellular growth and meiotic mRNA degradation. We also report a crystal structure of a Red1-Ars2 complex explaining mutually exclusive interactions of hARS2 with various ED/EGEI/L motif-possessing RNA regulators, including hZFC3H1 of PAXT, hFLASH or hNCBP3. Finally, we show that both Red1 and hZFC3H1 homo-dimerize via their coiled-coil regions indicating that MTREC and PAXT likely function as dimers. Our results, combining structures of three Red1 interfaces with in vivo studies, provide mechanistic insights into conserved features of MTREC/PAXT architecture.
Asunto(s)
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Proteínas Portadoras/metabolismo , Humanos , Meiosis , ARN/metabolismo , Estabilidad del ARN/genética , ARN Mensajero/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMEN
Fatty acid ß-oxidation (FAO) and oxidative phosphorylation (OXPHOS) are mitochondrial redox processes that generate ATP. The biogenesis of the respiratory Complex I, a 1â MDa multiprotein complex that is responsible for initiating OXPHOS, is mediated by assembly factors including the mitochondrial complex I assembly (MCIA) complex. However, the organisation and the role of the MCIA complex are still unclear. Here we show that ECSIT functions as the bridging node of the MCIA core complex. Furthermore, cryo-electron microscopy together with biochemical and biophysical experiments reveal that the C-terminal domain of ECSIT directly binds to the vestigial dehydrogenase domain of the FAO enzyme ACAD9 and induces its deflavination, switching ACAD9 from its role in FAO to an MCIA factor. These findings provide the structural basis for the MCIA complex architecture and suggest a unique molecular mechanism for coordinating the regulation of the FAO and OXPHOS pathways to ensure an efficient energy production.
Asunto(s)
Complejo I de Transporte de Electrón/química , Flavina-Adenina Dinucleótido/metabolismo , Mitocondrias/metabolismo , Acil-CoA Deshidrogenasas/genética , Acil-CoA Deshidrogenasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Microscopía por Crioelectrón , Complejo I de Transporte de Electrón/metabolismo , Metabolismo Energético , Flavina-Adenina Dinucleótido/química , Humanos , Fosforilación Oxidativa , Dominios y Motivos de Interacción de Proteínas , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Neurons are extremely energy demanding cells and highly dependent on the mitochondrial oxidative phosphorylation (OXPHOS) system. Mitochondria generate the energetic potential via the respiratory complexes I to IV, which constitute the electron transport chain (ETC), together with complex V. These redox reactions release energy in the form of ATP and also generate reactive oxygen species (ROS) that are involved in cell signaling but can eventually lead to oxidative stress. Complex I (CI or NADH:ubiquinone oxidoreductase) is the largest ETC enzyme, containing 44 subunits and the main contributor to ROS production. In recent years, the structure of the CI has become available and has provided new insights into CI assembly. A number of chaperones have been identified in the assembly and stability of the mature holo-CI, although they are not part of its final structure. Interestingly, CI dysfunction is the most common OXPHOS disorder in humans and defects in the CI assembly process are often observed. However, the dynamics of the events leading to CI biogenesis remain elusive, which precludes our understanding of how ETC malfunctioning affects neuronal integrity. Here, we review the current knowledge of the structural features of CI and its assembly factors and the potential role of CI misassembly in human disorders such as Complex I Deficiencies or Alzheimer's and Parkinson's diseases.
RESUMEN
In plants, MADS domain transcription factors act as central regulators of diverse developmental pathways. In Arabidopsis thaliana, one of the most central members of this family is SEPALLATA3 (SEP3), which is involved in many aspects of plant reproduction, including floral meristem and floral organ development. SEP3 has been shown to form homo and heterooligomeric complexes with other MADS domain transcription factors through its intervening (I) and keratin-like (K) domains. SEP3 function depends on its ability to form specific protein-protein complexes; however, the atomic level determinants of oligomerization are poorly understood. Here, we report the 2.5-Å crystal structure of a small portion of the intervening and the complete keratin-like domain of SEP3. The domains form two amphipathic alpha helices separated by a rigid kink, which prevents intramolecular association and presents separate dimerization and tetramerization interfaces comprising predominantly hydrophobic patches. Mutations to the tetramerization interface demonstrate the importance of highly conserved hydrophobic residues for tetramer stability. Atomic force microscopy was used to show SEP3-DNA interactions and the role of oligomerization in DNA binding and conformation. Based on these data, the oligomerization patterns of the larger family of MADS domain transcription factors can be predicted and manipulated based on the primary sequence.
Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Homeodominio/química , Proteínas de Homeodominio/metabolismo , Proteínas de Dominio MADS/química , Proteínas de Dominio MADS/metabolismo , Multimerización de Proteína , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Cromatografía en Gel , Cristalografía por Rayos X , ADN de Plantas/metabolismo , Microscopía de Fuerza Atómica , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Mutantes/química , Regiones Promotoras Genéticas/genética , Unión Proteica , Estructura Terciaria de Proteína , Relación Estructura-ActividadRESUMEN
DNA helicases are responsible for unwinding the duplex DNA, a key step in many biological processes. UvrD is a DNA helicase involved in several DNA repair pathways. We report here crystal structures of Deinococcus radiodurans UvrD (drUvrD) in complex with DNA in different nucleotide-free and bound states. These structures provide us with three distinct snapshots of drUvrD in action and for the first time trap a DNA helicase undergoing a large-scale spiral movement around duplexed DNA. Our structural data also improve our understanding of the molecular mechanisms that regulate DNA unwinding by Superfamily 1A (SF1A) helicases. Our biochemical data reveal that drUvrD is a DNA-stimulated ATPase, can translocate along ssDNA in the 3'-5' direction and shows ATP-dependent 3'-5', and surprisingly also, 5'-3' helicase activity. Interestingly, we find that these translocase and helicase activities of drUvrD are modulated by the ssDNA binding protein. Analysis of drUvrD mutants indicate that the conserved ß-hairpin structure of drUvrD that functions as a separation pin is critical for both drUvrD's 3'-5' and 5'-3' helicase activities, whereas the GIG motif of drUvrD involved in binding to the DNA duplex is essential for the 5'-3' helicase activity only. These special features of drUvrD may reflect its involvement in a wide range of DNA repair processes in vivo.
Asunto(s)
ADN Helicasas/metabolismo , ADN/química , ADN/metabolismo , Deinococcus/enzimología , Conformación de Ácido Nucleico , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Cristalografía por Rayos X , ADN Helicasas/química , ADN Helicasas/genética , ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , Modelos Moleculares , Mutagénesis , Conformación ProteicaRESUMEN
In higher plants, the MADS-box genes encode a large family of transcription factors (TFs) involved in key developmental processes, most notably plant reproduction, flowering and floral organ development. SEPALLATA 3 (SEP3) is a member of the MADS TF family and plays a role in the development of the floral organs through the formation of multiprotein complexes with other MADS-family TFs. SEP3 is divided into four domains: the M (MADS) domain, involved in DNA binding and dimerization, the I (intervening) domain, a short domain involved in dimerization, the K (keratin-like) domain important for multimeric MADS complex formation and the C (C-terminal) domain, a largely unstructured region putatively important for higher-order complex formation. The entire K domain along with a portion of the I and C domains of SEP3 was crystallized using high-throughput robotic screening followed by optimization. The crystals belonged to space group P2(1)2(1)2, with unit-cell parameters a = 123.44, b = 143.07, c = 49.83â Å, and a complete data set was collected to 2.53â Å resolution.
Asunto(s)
Proteínas de Arabidopsis/química , Arabidopsis/química , Proteínas de Homeodominio/química , Queratinas/química , Factores de Transcripción/química , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cristalografía por Rayos X , Escherichia coli/genética , Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Queratinas/genética , Queratinas/metabolismo , Datos de Secuencia Molecular , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Chlorogenic acids (CGAs) are a group of phenolic secondary metabolites produced by certain plant species and an important component of coffee (Coffea spp.). The CGAs have been implicated in biotic and abiotic stress responses, while the related shikimate esters are key intermediates for lignin biosynthesis. Here, two hydroxycinnamoyl-coenzyme A shikimate/quinate hydroxycinnamoyl transferases (HCT/HQT) from coffee were biochemically characterized. We show, to our knowledge for the first time, that in vitro, HCT is capable of synthesizing the 3,5-O-dicaffeoylquinic acid diester, a major constituent of the immature coffee grain. In order to further understand the substrate specificity and catalytic mechanism of the HCT/HQT, we performed structural and mutagenesis studies of HCT. The three-dimensional structure of a native HCT and a proteolytically stable lysine mutant enabled the identification of important residues involved in substrate specificity and catalysis. Site-directed mutagenesis confirmed the role of residues leucine-400 and phenylalanine-402 in substrate specificity and of histidine-153 and the valine-31 to proline-37 loop in catalysis. In addition, the histidine-154-asparagine mutant was observed to produce 4-fold more dichlorogenic acids compared with the native protein. These data provide, to our knowledge, the first structural characterization of a HCT and, in conjunction with the biochemical and mutagenesis studies presented here, delineate the underlying molecular-level determinants for substrate specificity and catalysis. This work has potential applications in fine-tuning the levels of shikimate and quinate esters (CGAs including dichlorogenic acids) in different plant species in order to generate reduced or elevated levels of the desired target compounds.
Asunto(s)
Ácido Clorogénico/química , Café/química , Ácido Quínico/análogos & derivados , Aciltransferasas/química , Aciltransferasas/genética , Secuencia de Aminoácidos , Aminoácidos/química , Dominio Catalítico , Cromatografía Líquida de Alta Presión , Coffea/química , Coffea/genética , Activación Enzimática , Escherichia coli/química , Escherichia coli/genética , Ésteres/química , Isomerismo , Conformación Molecular , Mutagénesis Sitio-Dirigida , Proteínas de Plantas/química , Proteínas de Plantas/genética , Biosíntesis de Proteínas , Ácido Quínico/química , Semillas/química , Semillas/genética , Alineación de Secuencia , Especificidad por SustratoRESUMEN
UvrA proteins are key actors in DNA damage repair and play an essential role in prokaryotic nucleotide excision repair (NER), a pathway that is unique in its ability to remove a broad spectrum of DNA lesions. Understanding the DNA binding and damage recognition activities of the UvrA family is a critical component for establishing the molecular basis of this process. Here we report the structure of the class II UvrA2 from Deinococcus radiodurans in two crystal forms. These structures, coupled with mutational analyses and comparison with the crystal structure of class I UvrA from Bacillus stearothermophilus, suggest a previously unsuspected role for the identified insertion domains of UvrAs in both DNA binding and damage recognition. Taken together, the available information suggests a model for how UvrA interacts with DNA and thus sheds new light on the molecular mechanisms underlying the role of UvrA in the early steps of NER.
Asunto(s)
Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Daño del ADN , ADN Bacteriano/metabolismo , Deinococcus/enzimología , Adenosina Trifosfatasas/clasificación , Adenosina Trifosfatasas/aislamiento & purificación , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión/genética , Cristalización , Análisis Mutacional de ADN , Reparación del ADN , ADN Bacteriano/química , ADN Bacteriano/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Dimerización , Geobacillus stearothermophilus/enzimología , Hidrólisis , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína/genética , Homología de Secuencia de Aminoácido , Temperatura , Factores de TiempoRESUMEN
Highly pathogenic strains of Helicobacter pylori use a type IV secretion system to inject the CagA protein into human gastric cells. There, CagA associates with the inner side of the membrane and is tyrosine-phosphorylated at EPIYA motifs by host kinases. The phosphorylation triggers a series of interactions between CagA and human proteins that result in a dramatic change of cellular morphology. Structural and functional analyses of the protein have proved difficult, due to the proteolytically sensitive nature of the recombinant protein. To circumvent these difficulties, we applied ESPRIT, a library-based construct screening method, to generate a comprehensive set of 5'-randomly deleted gene fragments. Screening of 18 432 constructs for soluble expression resulted in a panel of 40 clones, which were further investigated by large-scale purification. Two constructs of approximately 25 and 33 kDa were particularly soluble and were purified to near homogeneity. CagA fragments larger than 40 kDa were prone to heavy proteolysis at the C-terminus, with a favoured cleavage site near the first EPIYA motif. Thus, these well-expressed recombinant constructs isolated are likely to be similar to those observed following natural proteolysis in human cells, and open the way for structural and functional studies requiring large amounts of purified material.