PMID:9715912
| Citation |
Arnez, JG, Flanagan, K, Moras, D and Simonson, T (1998) Engineering an Mg2+ site to replace a structurally conserved arginine in the catalytic center of histidyl-tRNA synthetase by computer experiments. Proteins 32:362-80 |
|---|---|
| Abstract |
Histidyl-tRNA synthetase (HisRS) differs from other class II aminoacyl-tRNA synthetases (aaRS) in that it harbors an arginine at a position where the others bind a catalytic Mg2+ ion. In computer experiments, four mutants of HisRS from Escherichia coli were engineered by removing the arginine and introducing a Mg2+ ion and residues from seryl-tRNA synthetase (SerRS) that are involved in Mg2+ binding. The mutants recreate an active site carboxylate pair conserved in other class II aaRSs, in two possible orders: Glu-Asp or Asp-Glu, replacing Glu-Thr in native HisRS. The mutants were simulated by molecular dynamics in complex with histidyl-adenylate. As controls, the native HisRS was simulated in complexes with histidine, histidyl-adenylate, and histidinol. The native structures sampled were in good agreement with experimental structures and biochemical data. The two mutants with the Glu-Asp sequence showed significant differences in active site structure and Mg2+ coordination from SerRS. The others were more similar to SerRS, and one of them was analyzed further through simulations in complex with histidine, and His+ATP. The latter complex sampled two Mg2+ positions, depending on the conformation of a loop anchoring the second carboxylate. The lowest energy conformation led to an active site geometry very similar to SerRS, with the principal Mg2+ bridging the alpha- and beta-phosphates, the first carboxylate (Asp) coordinating the ion through a water molecule, and the second (Glu) coordinating it directly. This mutant is expected to be catalytically active and suggests a basis for the previously unexplained conservation of the active site Asp-Glu pair in class II aaRSs other than HisRS. |
| Links | |
| Keywords |
Adenosine Triphosphate/metabolism; Arginine/chemistry; Arginine/metabolism; Binding Sites; Catalysis; Computer Simulation; Conserved Sequence; Histidine-tRNA Ligase/chemistry; Histidine-tRNA Ligase/genetics; Histidine-tRNA Ligase/metabolism; Magnesium/chemistry; Magnesium/metabolism; Models, Molecular; Mutation; Protein Engineering; Thermus thermophilus/enzymology |
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