The active site of yeast aspartyl-tRNA synthetase: structural and functional aspects of the aminoacylation reaction.

Abstract

The crystal structures of the various complexes formed by yeast aspartyl‐tRNA synthetase (AspRS) and its substrates provide snapshots of the active site corresponding to different steps of the aminoacylation reaction. Native crystals of the binary complex tRNA‐AspRS were soaked in solutions containing the two other substrates, ATP (or its analog AMPPcP) and aspartic acid. When all substrates are present in the crystal, this leads to the formation of the aspartyl‐adenylate and/or the aspartyl‐tRNA. A class II‐specific pathway for the aminoacylation reaction is proposed which explains the known functional differences between the two classes while preserving a common framework. Extended signature sequences characteristic of class II aaRS (motifs 2 and 3) constitute the basic functional unit. The ATP molecule adopts a bent conformation, stabilized by the invariant Arg531 of motif 3 and a magnesium ion coordinated to the pyrophosphate group and to two class‐invariant acidic residues. The aspartic acid substrate is positioned by a class II invariant acidic residue, Asp342, interacting with the amino group and by amino acids conserved in the aspartyl synthetase family. The amino acids in contact with the substrates have been probed by site‐directed mutagenesis for their functional implication.

The Adenylation Domain of Tyrocidine Synthetase 1

“…It has been proposed that the (S/T)GT(T/S)GXPKG consensus sequence may be related to the phosphate-binding loop found in several ATPbinding proteins of known structure (Fry et al, 1986;Knighton et al, 1891 ;Tanaka et a]., 1992;De Bondt et al, 1993;Iwamoto et al, 1993;Cavarelli et al, 1994;Scheffzek et al, 1995). Mutational analysis has revealed that residue Lys186 from the (SIT)GT(TIS)GXPKG motif of TY 1 is the most critical residue for its function; however, the available data do not distinguish clearly between an essential role in substrate recognition and binding, and a function related to the cleavage of pyrophosphate and stabilization of the reaction products (Gocht and Marahiel, 1994).…”

Unique protein architecture of alanyl-tRNA synthetase for aminoacylation, editing, and dimerization

“…Discussion tRNA Interactions. In the crystal structures of tRNA-bound class-II aaRSs, including ThrRS, seryl-tRNA synthetase, and aspartyl-tRNA synthetase, the amino acid acceptor arm of the tRNA binds to a common site on the class II aminoacylation domain (20,36,37). The binding site corresponds to the groove formed between Mid1 and Mid2 in the tRNA-recognition domain of A. fulgidus AlaRS.…”

Toward understanding phosphoseryl-tRNA Cys formation: The crystal structure of Methanococcus maripaludis phosphoseryl-tRNA synthetase

“…Structural superposition of a catalytic domain of SepRS with a complex of S. cerevisiae AspRS bound to tRNA and ATP (33) indicates that the tungstate is a marker for the position of a phosphate moiety in phosphoserine rather than one of the phosphates of an ATP substrate. ATP in the active site of class II synthetases adopts a bent conformation (9,33) and consequently, the distance between the tungsten and the ␣, ␤, and ␥ phosphorous atoms of the ATP from the superposed complex and the tungsten are 6, 9, and 11 Å, respectively. The superposition of the two synthetases aligns R324 in M. maripaludis SepRS with R531 in S. cerevisiae AspRS, a residue that interacts with the ␥ phosphate of ATP.…”