Role for a Conserved Structural Motif in Assembly of a Class I Aminoacyl-tRNA Synthetase Active Site

Abstract

The catalytic domains of class I aminoacyl-tRNA synthetases are built around a conserved Rossmann nucleotide binding fold, with additional polypeptide domains responsible for tRNA binding or hydrolytic editing of misacylated substrates. Structural comparisons identified a conserved motif bridging the catalytic and anticodon binding domains of class Ia and Ib enzymes. This stem contact fold (SCF) has been proposed to globally orient each enzyme's cognate tRNA by interacting with the inner corner of the L-shaped tRNA. Despite the structural similarity of the SCF among class Ia/Ib enzymes, the sequence conservation is low. We replaced amino acids of the MetRS SCF with portions of the structurally similar glutaminyl-tRNA synthetase (GlnRS) motif or with alanine residues. Chimeric variants retained significant tRNA methionylation activity, indicating that structural integrity of the helix-turn-strand-helix motif contributes more to tRNA aminoacylation than does amino acid identity. In contrast, chimeras were significantly reduced in methionyl adenylate synthesis, suggesting a role for the SCF in formation of a structured active site domain. A highly conserved aspartic acid within the MetRS SCF is proposed to make an electrostatic interaction with an active site lysine; these residues were replaced with alanines or conservative substitutions. Both methionyl adenylate formation and methionine transfer were impaired, and activity was not significantly recovered by making the compensatory double substitution.

Misacylation of specific nonmethionyl tRNAs by a bacterial methionyl-tRNA synthetase

“…Similarly, replacement of EcMRS residues 363 LSSRID 368 , which form the loop of the stem contact fold motif, with the structurally equivalent 313 IGVTKQ 318 residues of E. coli glutaminyl-tRNA synthetase showed little effect on mismethionylation. This replacement also had minimal effect on cognate tRNA aminoacylation efficiency, consistent with the hypothesis that the class Ia/Ib stem contact fold orients the tRNA core on the enzyme without making base-specific contacts (23).…”

“…Transcripts were generated by in vitro transcription of overlapping oligonucleotides and purified as previously described (23,37). The concentration of active tRNA molecules was determined by aminoacylation plateaus (Fig.…”

“…4A). Substitutions included those at highly conserved residues previously shown to have severe impacts on methionyl-adenylate formation (D369) or anticodon recognition (W461) and subsequent reduced levels of cognate tRNA Met CAU methionylation (22,23). Other substitutions were made at residues not directly involved in either anticodon binding or catalysis, but hypothesized to participate in functional coupling of these domains (24,25).…”

“…Substitutions at W221, F277, and D369 result in a decrease of mismethionylation. Asp-369 is highly conserved among MRSs; the carboxylate side chain is in close contact to Lys-295 in the enzyme active site, and the D369A variant is defective in methionyl-adenylate formation (23). Similarly, F277A exhibits decreased amino acid activation.…”

Modulation of Aminoacylation and Editing Properties of Leucyl-tRNA Synthetase by a Conserved Structural Module

“…1A). Both the KMSKS loop and this structural mod-ule constitute the stem contact (SC)-fold (13)(14)(15)(16)(17)(18). The last ␣-helix of the SC-fold is almost coaxial to the first ␣-helix of the helix bundle domain but is separated by a short linker segment in most class I synthetases (13).…”