Universal pathway for posttransfer editing reactions: Insights from the crystal structure of TtPheRS with puromycin

At the amino acid binding and recognition step, phenylalanyl-tRNA synthetase (PheRS) faces the challenge of discrimination between cognate phenylalanine and closely similar noncognate tyrosine. Resampling of Tyr-tRNA^Phe to PheRS increasing the number of correctly charged tRNA molecules has recently been revealed. Thus, the very same editing site of PheRS promotes hydrolysis of misacylated tRNA species, associated both with cis- and trans-editing pathways. Here we report the crystal structure of Thermus thermophilus PheRS (TtPheRS) at 2.6 Å resolution, in complex with phenylalanine and antibiotic puromycin mimicking the A76 of tRNA acylated with tyrosine. Starting from the complex structure and using a hybrid quantum mechanics/molecular mechanics approach, we investigate the pathways of editing reaction catalyzed by TtPheRS. We show that both 2′ and 3′ isomeric esters undergo mutual transformation via the cyclic intermediate orthoester, and the editing site can readily accommodate a model of Tyr-tRNA^Phe where deacylation occurs from either the 2′- or 3′-OH. The suggested pathway of the hydrolytic reaction at the editing site of PheRS is of sufficient generality to warrant comparison with other class I and class II aminoacyl-tRNA synthetases.A key role in genetic code translation play aminoacyl-tRNA synthetases (aaRSs), providing linkage of amino acids to tRNAs. Before activation, at the amino acid recognition step, some aaRSs face a challenge of discrimination among amino acids with closely similar chemical structure. The rate of erroneous aminoacylation products generated in vivo is no more than one error per 10⁴−10⁵ correct reactions (1). To ensure such an extent of accuracy, aaRSs developed a multisieve mechanism of proofreading (2, 3). The existence of a proofreading activity has been demonstrated for both class I and class II aaRSs. Among aaRSs on record, about half of them are capable of selecting between amino acids resembling each other (4).The class aminoacyl-tRNA synthetases (aaRSs), namely IleRS, ValRS, and LeuRS, are characterized by a conserved connective polypeptide 1 (CP1) editing domain forming insertion into the catalytic core, except in cases of bacterial and mitochondrial LeuRSs, where the CP1 occurs at a different point of insertion (5). MetRS also falls into class I, but its CP1 lacks editing activity (6). The editing domains of class II aaRSs (ThrRS, ProRS, AlaRS, PheRS) are more diverse in amino acid sequence and in the distinguishing features of their folds. Kinetic experiments carried out for SerRS revealed the presence of a tRNA-independent pretransfer editing pathway (7).Detailed analyses of posttransfer editing were performed for class I LeuRS and class II ThrRS (8, 9). The structures of the LeuRS posttransfer complex imply the existence of water molecules that are specifically coordinated, to play the role of attacking nucleophiles. The alanine-scanning mutagenesis of the editing site has failed to identify key residues directly involved in catalysis (8). Thus, it was proposed that the CP1 domain simply binds the substrates in a configuration that favors attack by a water molecule, which itself is appropriately positioned by the set of key residues. The crystal structure of the editing domain from ThrRS complexed with Ser-A76 reveals two water molecules located on either side of the hydrolyzed bond (9). This study underlines the crucial role played by tRNA in substrate-assisted catalysis, in positioning the catalytic water molecules along with the protein side chains (9, 10).The 3D structures of Thermus thermophilus phenylalanyl-tRNA synthetase (TtPheRS) and its complexes with functional substrates (11–14) revealed that the catalytic α subunit exerts control over aminoacylation reaction whereas the major role of the β subunit lies in the recognition and binding of cognate tRNA^Phe and hydrolysis of misacylated tRNA (Fig. 1A). The early fast kinetic study demonstrated that tyrosine is indeed transferred to tRNA^Phe, and the misacylated tRNA is rapidly hydrolyzed (15). Later, it was established that editing activity of the bacterial and archaeal/eukaryotic PheRSs is associated with the active site located at the interface region between B3 and B4 domains in the β subunit (16–18).Open in a separate windowFig. 1.(A) Structure of the TtPheRS complex with puromycin and phenylalanine. The protein is shown in cartoon representation; the ligands puromycin (red) and phenylalanine (dark blue) are shown in space-filling representation. The domain architecture of one αβ-heterodimer is shown with the N-terminal coiled coil of the α-subunit colored cyan, catalytic domains A1 and A2 colored red, and structural domains of the β-subunit domains from B1 to B8 colored differently. The symmetry-related heterodimer is denoted with asterisks. (B) The editing site cavity of TtPheRS with bound puromycin. The electron density map (colored in red), calculated as described in SI Materials and Methods with coefficients (F_obs − F_calc) contoured at 2.5σ. Crystal structure of the TtPheRS complex in space-filling representation (colored gray) rendered to show protein surface interacting with puromycin.Here we present the crystal structure of TtPheRS, in complex with phenylalanine at the “synthetic” (aminoacylation) site and puromycin (mimicking the A76 of tRNA misacylated with Tyr) at the editing site. The natural substrate’s ester moiety represents an isoelectronic analog of the puromycin amide group, wherein the NH group is replaced with an ester oxygen atom. The appearance of puromycin at the editing site is accompanied by changes in the positions of some bound water molecules or even by their loss, compared with TtPheRS complex with Tyr (17). Loss of the water molecules, supposedly underlying the nucleophilic attack on the carbonyl carbon of the ester bond, gives grounds for revisiting the hydrolytic mechanism at work in bacterial PheRSs (17, 19). The suggested pathway of hydrolytic reaction is of sufficient generality to warrant comparison with those of other class I and class II aaRSs.