Ligation of tRNAs with their cognate amino acids, by aminoacyl-tRNA synthetases, establishes the genetic code. Throughout evolution, tRNAAla selection by alanyl-tRNA synthetase (AlaRS) has depended predominantly on a single wobble base pair in the acceptor stem, G3•U70, mainly on the kcat level. Here we report the crystal structures of an archaeal AlaRS in complex with tRNAAla with G3•U70 and its A3•U70 variant. AlaRS interacts with both the minor- and major-groove sides of G3•U70, widening the major groove. The geometry difference between G3•U70 and A3•U70 is transmitted along the acceptor stem to the 3′-CCA region. Thus, the 3′-CCA region of tRNAAla with G3•U70 is oriented to the reactive route that reaches the active site, whereas that of the A3•U70 variant is folded back into the “non-reactive route”. This novel mechanism enables the single wobble pair to dominantly determine the specificity of tRNA selection, by an approximate 100-fold difference in kcat.

Mistranslation from confusion of serine for alanine by alanyl-tRNA synthetases (AlaRSs) has profound functional consequences1-3. Throughout evolution, two editing-checkpoints prevent disease-causing mistranslation from confusing glycine or serine for alanine at the active site of AlaRS. In both bacteria and mice, Ser poses a bigger challenge than Gly1,2. One checkpoint is the AlaRS editing center, while the other is from widely distributed AlaXps—free-standing, genome-encoded editing proteins that clear Ser-tRNAAla. The paradox of misincorporating both a smaller (glycine) and a larger amino acid (serine) suggests a deep conflict for nature-designed AlaRS. To understand the chemical basis for this conflict, kinetic and mutational analysis, together with nine crystal structures, provided snapshots of adenylate formation for each amino acid. An inherent dilemma is posed by constraints of a structural design that pins down the α–amino group of the bound amino acid using an acidic residue. This design, of more than 3 billion years, creates a serendipitous interaction with the serine OH that is difficult to avoid. Apparently not able to find better architecture for recognition of alanine, the serine misactivation problem was solved through free-standing AlaXps, which appeared contemporaneously with early AlaRSs. The results reveal unconventional problems and solutions arising from the historical design of the protein synthesis machinery.

Protein synthesis involves the accurate attachment of amino acids to their matching tRNA molecules. Mistranslating the amino acids serine or glycine for alanine is prevented by the function of independent but collaborative aminoacylation and editing domains of alanyl-tRNA synthetases (AlaRSs). Here we show that the C-Ala domain plays a key role in AlaRS function. The C-Ala domain is universally tethered to the editing domain both in AlaRS and in many homologous free-standing, editing proteins. Crystal structure and functional analyses showed that C-Ala forms an ancient single-stranded nucleic acid binding motif that promotes cooperative binding of both aminoacylation and editing domains to tRNAAla. In addition, C-Ala may have played an essential role in the evolution of AlaRSs by coupling aminoacylation to editing to prevent mistranslation. The algorithm of the genetic code is established in the first reaction of protein synthesis. In this reaction, aminoacyl-tRNA synthetases (AARSs) catalyze the attachment of amino acids to their cognate transfer RNAs (tRNAs) that bear the triplet anticodons of the genetic code. When a tRNA is acylated with the wrong amino acid, mistranslation occurs if the misacylated tRNA is released from the synthetase, captured by elongation factor, and used at the ribosome for peptide synthesis. To prevent mistranslation, some AARSs have separate editing activities that hydrolyze the misacylated amino acid from the tRNA (1–3). Because an editing-defective tRNA synthetase is toxic to bacterial and mammalian cells (4, 5), and is causally linked to disease in animals (6), strong selective pressure retains these editing activities throughout evolution.

The question of how dispersed mutations in one protein engender the same gain-of-function phenotype is of great interest. Here we focus on mutations in glycyl-tRNA synthetase (GlyRS) that cause an axonal form of Charcot-Marie-Tooth (CMT) diseases, the most common hereditary peripheral neuropathies. Because the disease phenotype is dominant, and not correlated with defects in the role of GlyRS in protein synthesis, the mutant proteins are considered to be neomorphs that gain new functions from altered protein structure. Given that previous crystal structures showed little conformational difference between dimeric wild-type and CMT-causing mutant GlyRSs, the mutant proteins were investigated in solution by hydrogen-deuterium exchange (monitored by mass spectrometry) and small-angle X-ray scattering to uncover structural changes that could be suppressed by crystal packing interactions. Significantly, each of five spatially dispersed mutations induced the same conformational opening of a consensus area that is mostly buried in the wild-type protein. The identified neomorphic surface is thus a candidate for making CMT-associated pathological interactions, and a target for disease correction. Additional result showed that a helix-turn-helix WHEP domain that was appended to GlyRS in metazoans can regulate the neomorphic structural change, and that the gain of function of the CMT mutants might be due to the loss of function of the WHEP domain as a regulator.Overall, the results demonstrate how spatially dispersed and seemingly unrelated mutations can perpetrate the same localized effect on a protein.aminoacyl tRNA synthetase | hereditary motor and sensory neuropathy | dimer formation

AlaXp is a widely distributed (from bacteria to humans) genome-encoded homolog of the editing domain of alanyltRNA synthetases. Editing repairs the confusion of serine and glycine for alanine through clearance of mischarged (with Ser or Gly) tRNA Ala . Because genome-encoded fragments of editing domains of other synthetases are scarce, the AlaXp redundancy of the editing domain of alanyl-tRNA synthetase is thought to reflect an unusual sensitivity of cells to mistranslation at codons for Ala. Indeed, a small defect in the editing activity of alanyltRNA synthetase is causally linked to neurodegeneration in the mouse. Although limited earlier studies demonstrated that AlaXp deacylated mischarged tRNA Ala in vitro, the significance of this activity in vivo has not been clear. Here we describe a bacterial system specifically designed to investigate activity of AlaXp in vivo. Serine toxicity, experienced by a strain harboring an editing-defective alanyl-tRNA synthetase, was rescued by an AlaXp-encoding transgene. Rescue was dependent on amino acid residues in AlaXp that are needed for its in vitro catalytic activity. Thus, the editing activity per se of AlaXp was essential for suppressing mistranslation. The results support the idea that the unique widespread distribution of AlaXp arises from the singular difficulties, for translation, poised by alanine.The editing activities of tRNA synthetases provide a major safeguard against mistranslation, the insertion of the wrong amino acid at a specific codon (1-4). This insertion arises from small amounts of tRNA mischarging, a phenomenon that is intrinsic to the active sites of many of the synthetases. This mischarging results from the inherent inability of the enzyme binding pockets to discriminate rigorously between closely similar amino acid side chains, especially to achieve the level of discrimination needed for the high accuracy of the genetic code. Examples include the synthesis of Val-tRNAIle by isoleucyl-tRNA synthetase (3, 5), Thr-tRNAVal by valyl-tRNA synthetase (6, 7), Val-tRNA Leu by leucyl-tRNA synthetase (8 -10), Ala-tRNA Pro by prolyl-tRNA synthetase (11, 12), Ser-tRNA Thr by threonyl-tRNA synthetase (13), Ile-tRNA Phe by phenylalanyl-tRNA synthetase (4), and Ser-or Gly-tRNA Ala by alanyltRNA synthetase (AlaRS) 2 (14, 15). Normally, the mischarged tRNAs are cleared by a distinct editing activity, specific to each synthetase. This activity is able to distinguish the correct from the incorrect amino acid fused to its cognate tRNA. In addition to the editing activity that is part of the synthetase and encoded as a separate domain (16 -18), there are a few instances of freestanding editing domain homologs encoded by various genomes (12, 19 -22). The most widespread (through all three kingdoms of life) of these is AlaXp, a small protein that is a homolog of the editing domain of AlaRS and has been shown to edit Ser-tRNA Ala and Gly-tRNA Ala in vitro (21-24). Not clear, however, is whether AlaXp plays a role in vivo in guarding against mistranslation.The importan...