RESUMEN
The three-dimensional structure of the seryl-tRNA synthetase from Thermus thermophilus has been determined and refined at 2.5 A resolution. The final model consists of a dimer of 421 residues each and 190 water molecules. The R-factor is 18.4% for all the data between 10 and 2.5 A resolution. The structure is very similar to that of the homologous enzyme from Escherichia coli, with an r.m.s. difference of 1.5 A for the 357 alpha-carbon atoms considered equivalent. The comparison of the two structures indicates increased hydrophobicity, reduced conformational entropy and reduced torsional strain as possible mechanisms by which thermostability is obtained in the enzyme from the thermophile.
Asunto(s)
Serina-ARNt Ligasa/ultraestructura , Thermus thermophilus/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/ultraestructura , Cristalografía por Rayos X , Escherichia coli/enzimología , Proteínas Fúngicas/ultraestructura , Calor , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Desnaturalización Proteica , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/enzimología , Alineación de Secuencia , Homología de Secuencia , Propiedades de SuperficieRESUMEN
The secondary structures of metazoan mitochondrial (mt) tRNAs(Ser) deviate markedly from the paradigm of the canonical cloverleaf structure; particularly, tRNA(Ser)(GCU) corresponding to the AGY codon (Y=U and C) is highly truncated and intrinsically missing the entire dihydrouridine arm. None of the mt serine isoacceptors possesses the elongated variable arm, which is the universal landmark for recognition by seryl-tRNA synthetase (SerRS). Here, we report the crystal structure of mammalian mt SerRS from Bos taurus in complex with seryl adenylate at an atomic resolution of 1.65 A. Coupling structural information with a tRNA-docking model and the mutagenesis studies, we have unraveled the key elements that establish tRNA binding specificity, differ from all other known bacterial and eukaryotic systems, are the characteristic extensions in both extremities, as well as a few basic residues residing in the amino-terminal helical arm of mt SerRS. Our data further uncover an unprecedented mechanism of a dual-mode recognition employed to discriminate two distinct 'bizarre' mt tRNAs(Ser) by alternative combination of interaction sites.
Asunto(s)
Bovinos/genética , Mitocondrias/genética , Modelos Moleculares , ARN de Transferencia de Serina/genética , Serina-ARNt Ligasa/metabolismo , Serina-ARNt Ligasa/ultraestructura , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/metabolismo , Secuencia de Aminoácidos , Animales , Emparejamiento Base/genética , Codón/genética , Cristalografía , Análisis Mutacional de ADN , Datos de Secuencia Molecular , Alineación de Secuencia , Serina/metabolismo , Serina-ARNt Ligasa/genéticaRESUMEN
The low temperature crystal structure of the ternary complex of Thermus thermophilus seryl-tRNA synthetase with tRNA(Ser) (GGA) and a non-hydrolysable seryl-adenylate analogue has been refined at 2.7 angstrom resolution. The analogue is found in both active sites of the synthetase dimer but there is only one tRNA bound across the two subunits. The motif 2 loop of the active site into which the single tRNA enters interacts within the major groove of the acceptor stem. In particular, a novel ring-ring interaction between Phe262 on the extremity of this loop and the edges of bases U68 and C69 explains the conservation of pyrimidine bases at these positions in serine isoaccepting tRNAs. This active site takes on a significantly different ordered conformation from that observed in the other subunit, which lacks tRNA. Upon tRNA binding, a number of active site residues previously found interacting with the ATP or adenylate now switch to participate in tRNA recognition. These results shed further light on the structural dynamics of the overall aminoacylation reaction in class II synthetases by revealing a mechanism which may promote an ordered passage through the activation and transfer steps.
Asunto(s)
ARN de Transferencia de Serina/ultraestructura , Serina-ARNt Ligasa/ultraestructura , Thermus thermophilus/enzimología , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Escherichia coli/enzimología , Sustancias Macromoleculares , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Conformación Proteica , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Aminoacylation rate determinations for a series of variant RNA minihelix substrates revealed that Escherichia coli seryl-tRNA synthetase (SerRS) recognizes the 1--72 through 5--68 base pairs of the E.coli tRNA(Ser) acceptor stem with the major recognition elements clustered between positions 2--71 and 4--69. The rank order of effects of canonical base pair substitutions at each position on kcat/Km was used to assess the involvement of major groove functional groups in recognition. Conclusions based on the biochemical data are largely consistent with the interactions revealed by the refined structure of the homologous Thermus thermophilus tRNA(Ser)-SerRS complex that Cusack and colleagues report in the accompanying paper. Disruption of an end-on hydrophobic interaction between the major groove C5(H) of pyrimidine 69 and an aromatic side chain of SerRS is shown to significantly decrease kcat/Km of a minihelix substrate. This type of interaction provides a means by which proteins can recognize the binary information of 'degenerate' sequences, such as the purine-pyrimidine base pairs of tRNA(Ser). The 3--70 base pair is shown to contribute to recognition by SerRS even though it is not contacted specifically by the protein. The latter effect derives from the organization of the specific contacts that SerRS makes with the neighboring 2--71 and 4--69 acceptor stem base pairs.