The Crystal Structures Of The Editing Domain Of E.coli Leucyl-tRNA Synthetase And Its Complexes With Methionine And Isoleucine

Aminoacyl-tRNA synthetases (aaRSs) are responsible for the covalent link ofamino acids to their cognate tRNAs via aminoacylation reaction and play a vital rolein maintaining the fidelity of protein synthesis. Leucyl-tRNA synthetase (LeuRS)can link not only the cognate leucine but also the nearly cognate isoleucine andmethionine to tRNA^Leu. The editing domain of LeuRS deacylates the mischargedIle-tRNALeu and Met-tRNA^Leu. We detemined the crystal structures of the editingdomain of E. coli LeuRS (ecLeuRS-ED) in both apo form and complexes withmethionine and isoleucine, respectively. These structures provide new insight intothe molecular basis of the editing function of ecLeuRS-ED. Analyses of thesestructures revealed the precise binding and recognition mode of Met and Ile at theediting active site. The editing active site is consisted of a number of conservedamino acids which are involved in precise recognition and binding of the noncognateamino acids. The substrate-binding pocket has a rigid structure which has anoptimal fit in stereochemistry for isoleucine and methionine, but has steric hindrancefor leucine. Based on our structural and previously available biochemical data, wepropose that ecLeuRS-ED uses a lock-and-key mechanism to recognize anddiscriminate the amino acids. Hydrolysis of the mischarged amino acids in theediting reaction will rely on the proper binding and correct recognition of both mainchain and side chain of the substrate. In general, the editing active site ofLeuRS-ED has a very rigid structure (a lock) with a defined stereochemistry and usesa number of conserved residues to precisely bind and recognize the misactivatedamino acids and discriminate and reject the cognate amino acid, specifically usingMet336, Asp345, and possibly Thr247 for main-chain recognition and Thr252,Met336, and Val338 for side-chain recognition. An amino acid substrate (a key)that fits the substrate-binding pocket in both geometry (size and length) and chemicalproperty can bind into the pocket and be positioned properly for hydrolysis. Aminoacids that cannot fit into the editing site would be rejected because they are either toosmall to bind tightly at the active site (such as Ala) or have steric hindrance with thesurrounding residues (such as Leu). Structural comparison also reveals that allsubclass Ia aaRSs share a conserved structure core of the editing domain andconserved residues at the editing active site, suggesting that these enzymes might usea common mechanism for the editing function.