1 INTRODUCTION
Protein-protein or protein-nucleic acid interactions drive a plethora of biological processes. For the interaction to be biologically fruitful, a protein must be capable of not only choosing its cognate partner from the cellular soup but also be able to discriminate against non-cognate partners present in its environment. A classic example is the case of aminoacyl-tRNA synthetases (aaRSs), whose function is to aminoacylate or charge its cognate tRNA and discriminate against all other non-cognate tRNA (1). It is now established that each tRNA type, corresponding to a particular amino acid, possesses unique nucleotides called identity determinants that allow its recognition by cognate aaRS and anti-determinants that discriminate against noncognate aaRSs (2). An added layer of recognition/discrimination is encountered in glutamyl tRNA-synthetases (GluRS) in some bacteria whose genomes lack glutaminyl-tRNA synthetase (GlnRS) (3,4).
GluRS is a class-I aminoacyl-tRNA synthetase that catalyzes the glutamylation of tRNAGlu (3). In absence of GlnRS, GluRS in these bacteria are non-discriminatory (ND) and glutamylates both tRNAGlu and tRNAGln. Glutamylation of tRNAGln produces the mismatched product Glu-tRNAGln (5,6). The misacylated Glu-tRNAGln is then further edited to the correct product Gln-tRNAGln by the enzyme glutamyl-tRNAGln amidotransferase (gatCAB) through a transamidation pathway (7). All ancient versions of bacterial GluRS were tRNAGln-non-discriminatory GluRS (ND-GluRS). During the course of evolution, bacteria acquired GlnRS of an eukaryotic origin through horizontal gene transfer (4,8,9). The newly acquired GlnRS was evolutionarily selected in some bacteria, where the native ND-GluRS that charged both tRNAGlu and tRNAGlnevolved into a tRNAGln-discriminatory (D-GluRS) that charged only tRNAGlu. In addition to ND-GluRS and D-GluRS, a large number of proteobacterial species (for example Helicobacter pylori (10), Acidithiobacillus ferrooxidans (11)) possess two copies of GluRS (GluRS1 and GluRS2) with distinct tRNAGlx-specificities (GluRS1: mostly tRNAGlu-specific; GluRS2: tRNAGln-specific), suggesting a gene duplication of the primordial version of GluRS.
The structural features of Thermus thermophilus  GluRS (Tth -GluRS) have been extensively studied (12,13). GluRS is composed of two structural domains. The N-terminal domain, also known as the catalytic domain, contains the L-glu and ATP binding sites, along with the class-I specific signature motifs HIGH and KMSKS in the ATP binding site along with. This domain also encompasses a sparse binding interface with the acceptor stem and the D-helix nucleotides of tRNAGlu. The other domain (C-terminal) interacts with the anticodon nucleotides of tRNAGlu and is aptly known as the anticodon-binding domain.
Although the most important structural insights for bacterial GluRSs came from Tth -GluRS, a non-proteobacterial GluRS, majority of biochemical studies for the mechanistic understanding of GluRS:tRNA interactions have been performed on GluRS/tRNAGlu in the proteobacterium Escherichia coli (14–17). Mutational studies performed on the E. coli GluRS (Eco -D-GluRS) and tRNAGlu identified several ‘hot-spots’ (important amino acids and nucleotides) for efficient glutamylation reaction (16). For example, it has been shown that tRNAGlu-specificity of GluRS in E. coli and some other proteo-bacterial GluRSs arise due to subtle conformational differences between tRNAGlu and tRNAGln, originating at the D-helix (Figure 1A, Figure S1) — augmented (presence of base-triple interaction 13:22:46 and the absence of nucleotide 47) in tRNAGlu versus non-augmented (absence of base-triple interaction 13:22:46 and the presence of nucleotide 47) in tRNAGln (8,18). Interestingly, this is not true in case of non-proteobacterium T. thermophilus , which, despite possessing a D-GluRS, displays augmented D-helix in both tRNAGlu and in tRNAGln. A zinc ion present in the catalytic domain of Eco -GluRS was shown to play a critical role glutamylation reaction (19), although many bacterial GluRSs do not contain a bound Zn2+, including Tth -GluRS, implying the irregular occurrence of the zinc atom (20). In another study, when an arginine residue (R266) in the tRNA-binding interface of Eco -GluRS was mutated to leucine, glutamylation efficiency of the protein was drastically reduced (more than 2500 fold) (16). Interestingly, sequence analysis of bacterial GluRSs revealed that this arginine residue is exclusively present only in proteobacterial GluRS. In other words, hot-spot signatures for GluRS-tRNAGlxinteraction are not homogeneously conserved in all bacterial GluRSs (4), indicating that factors responsible for the interactions are phylum-specific and not universal. Therefore, to completely understand sequence and structural signatures that drive specificity of tRNAGlx-glutamylation reaction in bacteria, the sequence and structural signatures must be analyzed in a phylum-specific manner and the structural insights obtained from Tth -GluRS may not be enough to understand the results of the functional studies performed on Eco -GluRS (4).
From the perspective of GluRS evolution, the proteobacterial domain in bacteria had experienced multiple sets of events (horizontal gene transfer, gene duplication and perhaps domain fusion) while adapting a tRNAGlu-specific aminoacylation pathway (4). In order to achieve tRNAGlu-specificity, the evolving GluRS must have undergone major adaptations, especially in residues lining its tRNA-binding interface. To understand the rationale behind such adaptations in proteobacterial GluRS, structural insights from crystal structures of proteobacterial GluRS are needed. Further, since a number of mutational studies related to tRNAGlx-specificity have been performed on Eco -GluRS, it is important to analyzeEco -GluRS structure to elucidate clues behind tRNAGlx-specificity. Here, we report the crystal structure of Eco -GluRS. From structural and sequence analysis of a large number of bacterial GluRS, both proteo- and non-proteobacterial, we identify structural features present in proteobacterial GluRSs that are required for tRNAGlu-specificity.
Our results highlight that the specific structural feature responsible for the tRNAGlx-specificity of bacterial GluRSs is the presence or the absence of an unique “towards tRNA” or “away from tRNA” conformation adopted by a short loop connecting Helix 8 and Helix 9. The “towards tRNA” conformation is compatible with the augmented D-helix but incompatible with the non-augmented D-helix, while the “away from tRNA” conformation is compatible with both.