Active-site assembly in glutaminyl-tRNA synthetase by tRNA-mediated induced fit.

Aminoacyl tRNA-synthetases comprise an ancient class of enzymes that are crucial to maintaining fidelity in the genetic code. They catalyze specific aminoacyl-tRNA synthesis in a two-step reaction. First, energy from ATP hydrolysis is utilized to generate an activated aminoacyl adenylate intermediate possessing a mixed anhydride linkage (1). In a second step, the 2′ or 3′ ribose oxygen of the 3′-terminal A76 tRNA nucleotide attacks the carbonyl carbon atom of this linkage, forming aminoacyl-tRNA with release of AMP. The tRNA synthetases are divided into two classes possessing topologically distinct active-site domains (2). Other domains in the structure are primarily involved in discriminating against noncognate tRNAs, and diverge significantly among the enzymes. Crystal structures of all twenty canonical tRNA synthetases, many determined in multiple liganded states including as complexes with tRNA, reveal the common presence of induced-fit conformational rearrangements (3). Interestingly, despite the common active-site Rossmann fold (class I) or mixed α/β domain (class II), the conformational changes upon ligand binding are not conserved. Instead, distinct rearrangements, often including both global repositioning of domains and local movements of surface loops and side-chains, appear to be idiosyncratic to each enzyme (4). The ubiquity of induced fit in tRNA synthetases reflects the common presence of this phenomenon in RNA-protein interactions generally (5). Catalytic roles of induced fit may include (i) exploiting intrinsic flexibility to lower the free-energy barrier for complex formation, and/or (ii) fostering specificity via incomplete triggering of rearrangements in noncognate complexes, leading to the improper juxtaposition of reactive moieties on bound substrates (6, 7). E. coli glutaminyl-tRNA synthetase (GlnRS) is a monomeric class I enzyme that has served as a model system to address the detailed stereochemical pathway of catalysis and the molecular origins of specificity for the amino acid and tRNA (8). GlnRS is one of four class I tRNA synthetases that require binding of cognate tRNA to catalyze amino acid activation, suggesting that structural transitions associated with tRNA complex formation are crucial to formation of the correct active site conformation (9). Substantial other evidence also points to the importance of induced fit in mediating GlnRS substrate specificity. First, fluorescence experiments have yielded direct physical evidence for enzyme rearrangements upon tRNA binding (10, 11). Second, mutation of tRNAGln anticodon nucleotide U35 sharply reduces both the rate of the chemical steps for aminoacylation as well as the affinity for glutamine, demonstrating the existence of a structural pathway for long-range communication from the C-terminal β-barrel domains to the active site some 40 A distant (12). Third, comparison of the crystal structures of unliganded GlnRS and the GlnRS-tRNAGln complex shows in detail how tRNA binding generates structural changes throughout the entire enzyme structure, particularly with respect to the positioning of key active site peptides that bind glutamine and ATP (4, 13). When tRNA binds to GlnRS, the 110 amino acid acceptor-binding domain (ABD; residues 100-209), which is inserted between the two halves of the dinucleotide fold (DNF), makes a rigid body rotation of about 10° towards the active site (Figure 1). In addition to properly positioning a complementary surface for binding the hairpinned single-stranded tRNA 3′-acceptor end, rotation of the ABD also results in the reorganization of a DNF surface loop spanning amino acids 64-76 within the N-terminal half of the fold (depicted in purple in Figure 1). This surface loop is only loosely ordered in the unliganded enzyme. Its reorientation has several important consequences: (i) the side chain of Asp66 is repositioned to point into the substrate binding pocket, where it makes a direct salt bridge with the α-NH3+ group of the amino acid; (ii) the side-chain carboxylate of Glu73 makes a new contact with the carboxylate of Glu34. The new Glu34-Glu73 interaction, which presumably also involves uptake of a proton for charge neutralization between the carboxylates, apparently assists in reorganization of the active-site peptide spanning Pro32-Pro33-Glu34-Pro35, and facilitates formation of a new Asp66-Pro33 main-chain hydrogen-bond. Together, these active site motions also allow binding of the tRNA-A76 ribose sugar directly adjacent to the ATP α-phosphate, as required for catalysis (14) (Figures ​(Figures2,2, ​,33). Figure 1 Superimposition of unliganded (gray) and tRNA-bound (blue) GlnRS structures based on protein backbone atoms within the dinucleotide fold, as defined previously (4). Peptide segments implicated in the induced-fit rearrangement studied here are colored ... Figure 2 Interactions in and adjacent to the GlnRS active site. The QSI inhibitor is shown in red and tRNA is depicted in green. Hydrogen bonds are shown as dotted gold lines. Peptides in GlnRS are colored as in Figure 1. Figure 3 Stereoview of interactions made by Glu34 in the GlnRS active site. Atoms are colored as follows: carbon, green; oxygen, red; nitrogen, blue; phosphoris, orange. Waters are shown as red spheres, and hydrogen bonds are shown by dashed black lines. This examination of crystal structures strongly suggests that the unusual intramolecular Glu34-Glu73 contact formed upon tRNA binding is central to mediating active site assembly by induced fit. Moreover, another role for Glu34 has been proposed based on examination of the crystal structure of GlnRS bound to tRNAGln and the aminoacyl adenylate analog 5′-O-[N-(L-glutaminyl)sulphamoyl] adenosine (QSI) (15). In that study, it was suggested that the Glu34 carboxylate functions as a general base in the second step of the aminoacylation reaction, by increasing the nucleophilicity of the 2′-OH of tRNA A76 via a water-mediated interaction. An earlier alternative proposal, however, is that a nonbridging oxygen of the glutaminyl adenylate α-phosphate group functions as a general base for this reaction (14). The latter mechanism has been suggested to operate in the class II aspartyl (AspRS) and histidyl-tRNA synthetases (HisRS) as well (16, 17). Indeed, since this mechanism is substrate-mediated, it could function generally in the tRNA synthetase enzyme family. Here we investigate the roles of Glu34 and Glu73 in catalysis by E. coli GlnRS by mutational analysis coupled to steady-state and pre-steady state kinetic measurements of glutaminyl-tRNA synthesis. By single-turnover kinetics, we find that the rate of the chemical steps for Gln-tRNAGln synthesis by the E34Q mutant is nearly identical to that of the wild-type enzyme, indicating that the carboxylate group of Glu34 does not have an obligate role in facilitating the second-step aminoacyl transfer. Surprisingly, rates of aminoacyl-tRNA synthesis in mutants of the more distal Glu73 residue are slower than rates measured for mutants at Glu34, suggesting a unique role for the former side-chain in promoting proper juxtaposition of the A76 ribose in the active site. Mutations at both positions weaken the binding affinity for glutamine at the opposite side of the active site, highlighting the interdependent nature of substrate binding and the extraordinary precision of architectural construction that is required to properly juxtapose all three reactive moieties for the overall two-step reaction.