ENGINEERING AN MG2-TRANSFER-RNA SYNTHETASE BY COMPUTER EXPERIMENTS( SITE TO REPLACE A STRUCTURALLY CONSERVED ARGININE IN THE CATALYTIC CENTER OF HISTIDYL)

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, f our 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 recr eate an active site carboxylate pair conserved in other class II aaRSs , in two possible orders: Glu-Asp or Asp-Glu, replacing Glu-Thr in nat ive 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 n ative structures sampled were in good agreement with experimental stru ctures and biochemical data. The two mutants with the Glu-Asp sequence showed significant differences in active site structure and Mg2+ coor dination from SerRS. The others were more similar to SerRS, and one of them was analyzed further through simulations in complex with histidi ne, and His+ATP. The latter complex sampled two Mg2+ positions, depend ing on the conformation of a loop anchoring the second carboxylate. Th e lowest energy conformation led to an active site geometry very simil ar to SerRS, with the principal Mg2+ bridging the alpha- and beta-phos phates, the first carboxylate (Asp) coordinating the ion through a wat er molecule, and the second (Glu) coordinating it directly. This mutan t 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. Proteins 32:362-380, 1998. (C) 1998 Wiley-Liss, Inc.