Crystal structures that suggest late development of genetic code components for differentiating aromatic side chains

Abstract

Early forms of the genetic code likely generated "statistical" proteins, with similar side chains occupying the same sequence positions at different ratios. In this scenario, groups of related side chains were treated by aminoacyl-tRNA synthetases as a single molecular species until a discrimination mechanism developed that could separate them. The aromatic amino acids tryptophan, tyrosine, and phenylalanine likely constituted one of these groups. A crystal structure of human tryptophanyl-tRNA synthetase was solved at 2.1 A with a tryptophanyl-adenylate bound at the active site. A cocrystal structure of an active fragment of human tyrosyl-tRNA synthetase with its cognate amino acid analog was also solved at 1.6 A. The two structures enabled active site identifications and provided the information for structure-based sequence alignments of approximately 45 orthologs of each enzyme. Two critical positions shared by all tyrosyl-tRNA synthetases and tryptophanyl-tRNA synthetases for amino acid discrimination were identified. The variations at these two positions and phylogenetic analyses based on the structural information suggest that, in contrast to many other amino acids, discrimination of tyrosine from tryptophan occurred late in the development of the genetic code.

Fig. 1.

Classification of aminoacyl-tRNA synthetases adapted from ref. . The 20 synthetases are divided into 2 classes of 10 enzymes each. The exceptional class I LysRS is shown in gray. Highlighted with a yellow box, TyrRS and TrpRS from class Ic are paired with PheRS from class IIc.

Fig. 2.

Structure of the dimeric human TrpRS with one monomer shown in color. The circled CP1 insertion of the Rossmann fold domain forms the dimerization interface. All three domains [N-terminal appended domain (blue), Rossmann fold catalytic domain (yellow), and anticodon recognition domain (green)] were resolved in one monomer of the dimer with a disordered linker of 21 residues connecting the N-domain and the Rossmann fold domain. However, in the other monomer, the first 96 residues, which include the N-terminal domain, the linker region, and part of the Rossmann fold catalytic domain, were completely disordered. A bound Trp-AMP was found only in the monomer with the resolved N-domain.

Fig. 3.

(A) Structure-based alignment of human TyrRS, B. stearothermophilus TyrRS, Thermus thermophilus TyrRS, human TrpRS, and B. stearothermophilus TrpRS. Variable regions, which usually correspond to terminal or loop regions, were removed to give a total of 173 aa in the alignment of all sequences. The secondary structure elements of human TyrRS were superimposed on top of the alignment (α,α-helices; η,3₁₀ helices; β,β-strands). “HIGH” and “KMSKS” signature sequences were colored in blue, and the two positions of amino acid recognition residues (inα8 andβ2) were colored in orange. (B) Active site of human TyrRS for the recognition of the tyrosinol side chain, which represent the active site of all eukaryotic, archaeal, and one group of bacterial TyrRSs including B. stearothermophilus TyrRS. The tyrosinol hydroxyl group is a hydrogen bond donor to the carboxylate oxygen of Asp in α8 and an acceptor for the hydroxyl of a Tyr in β2. (C) Active site of Thermus thermophilus TyrRS, which represent the other group of bacterial TyrRS. Here, the hydroxyl of the Tyr is replaced by the ε-amino of a Lys at the same position in β2 to donate a hydrogen bond to the tyrosinol hydroxyl group. (D) Active site of human TrpRS for the recognition of the tryptophan side chain, which represents the active site of all of the eukaryotic and one archaeal TrpRS from P. abyssi. The indole nitrogen of the tryptophan side chain accepts just one hydrogen bond from the hydroxyl of a Tyr in β2. (E) Active site of B. stearothermophilus TrpRS, which represent the active site of all of the bacterial and, except P. abyssi, all other archaeal TrpRSs. The indole nitrogen of the tryptophan side chain here accepts the one hydrogen bond from the carboxylate group of Asp in α8.

Fig. 4.

Simplified phylogenetic tree calculated from the alignment of 93 sequences of TyrRSs and TrpRSs by using maximum parsimony, neighbor joining, and maximum likelihood methods. The numbers in the branch nodes correspond to bootstrap frequencies after using maximum parsimony (1,000 cycles) and maximum likelihood (100 cycles) analyses. The numbers in parentheses of each group indicate the total number of sequences associated with that group. The two key residues for amino acid recognition of each group are listed on the tree in orange. The ancestral sequence estimated by maximum likelihood method for the central node of the tree has α8-D/β2-Y.