CRYSTAL-STRUCTURE ANALYSIS OF THE ACTIVATION OF HISTIDINE BY THERMUS-THERMOPHILUS HISTIDYL-TRANSFER-RNA SYNTHETASE

The crystal structure at 2.7 Angstrom resolution of histidyl-tRNA synt hetase (HisRS) from Thermus thermophilus in complex with its amino aci d substrate histidine has been determined. In the crystal asymmetric u nit there are two homodimers, each subunit containing 421 amino acid r esidues. Each monomer of the enzyme consists of three domains: (1) an N-terminal catalytic domain containing a six-stranded antiparallel bet a-sheet and the three motifs common to all class II aminoacyl-tRNA syn thetases, (2) a 90-residue C-terminal alpha/beta domain which is commo n to most class IIa synthetases and is probably involved in recognizin g the anticodon stem-loop of tRNA(His), and (3) a HisRS-specific alpha -helical domain inserted into the catalytic domain, between motifs II and III. The position of the insertion domain above the catalytic site suggests that it could clamp onto the acceptor stem of the tRNA durin g aminoacylation. Two HisRS-specific peptides, 259-RGLDYY and 285-GGRY DG, are intimately involved in forming the binding site for the histid ine, a molecule of which is found in the active site of each monomer. The structure of HisRS in complex with histidyl adenylate, produced en zymatically in the crystal, has been determined at 3.2 Angstrom resolu tion. This structure shows that the HisRS-specific Arg-259 interacts d irectly with the a-phosphate of the adenylate on the opposite side to the usual conserved motif 2 arginine. Arg-259 thus substitutes for the divalent cation observed in seryl-tRNA synthetase and plays a crucial catalytic role in the mechanism of histidine activation.