Yeast arginyl-tRNA synthetase (PBD ID: 1BS2) from Saccharomyces cerevisiae catalyzes the esterification of arginine to its complementary tRNA (1). The esterification reaction forms functional tRNAs that help form proteins that require the amino acid arginine. Without arginyl-tRNA synthetase (argRS), the yeast would not be able to form new proteins requiring the amino acid arginine. Low levels of argRS would slow down translation of proteins with arginine residues possibly leading to ubiquitination and subsequent degradation of a nascent protein entering the endoplasmic reticulum (2). The aminoacylation reaction occurs in a two step process. In the first step, the amino acid, arginine, is bound to ATP in the presence of Mg2+ to form an aminoacyl adenylate. In the second step, the aminoacyl group, still bound to the catalyst, is transferred to the cognate tRNA(3). In the formation of most aminoacyl-tRNAs, the enzymatic synthetase does not require the presence of tRNA to form the aminoacyl adenylate. However, argRS is one of three aminoacyl-tRNA synthetases (aaRS) that require the presence of tRNA to undergo adenylation of the amino acid (4). This is likely due to ATP being unable to completely enter the binding pocket until the induced fit of the tRNA opens up the active site (3).
The synthetase binds to three ligands, two of which are the arginine and tRNA that are esterified together. The third ligand is ATP, whose hydrolysis forms an intermediate arginine-ADP complex before the arginine is esterified to tRNA (1). The argRS also contains the associated metal ion Mg2+ which stabilizes the formation of the aminoacyl adenylate intermediate (3).
Yeast arginyl-tRNA synthetase was analyzed using several protein databanks to characterize the protein and compare it to similar proteins. An Expasy search reveals that arginyl-tRNA synthetase has a molecular weight of 69.525kDa and an isoelectric point at a pH of 6.33 (5). This indicates that the protein is negatively charged at neutral pH. As the protein operates in the cytosol, the protein has a net negative charge in its native environment (1). Analysis of the yeast protein utilizing a protein BLAST search indicated a high similarity of the yeast synthetase to arginyl-tRNA synthetase from Homo sapiens (PBD ID: 4Q2T) and Escherichia coli (PBD ID: 4OBY) (6). The similarity of yeast arginyl-tRNA synthetase to Homo sapiens arginyl-tRNA synthetase has an E-value of 0 (6). The E-value compares amino acids between proteins and assigns a score based on the similarity between the sequences. A low E-value corresponds to high similarity. Therefore, the E-value of 0 indicates an extremely high similarity between the protein sequences of the two separate species. The same holds true when comparing yeast arginyl-tRNA synthetase to E. coli arginyl-tRNA synthetase, which also has a BLAST E-value of 0 (6). Even though the E-value is so small, there exist many differences in protein primary structure. In fact, only 28% of amino acids are directly matched to one another, and only 49% are “positives” or amino acids that can be substituted for one another with minimal change in function. Notable amino acids are Ser-151, Gln-375, Tyr-34 and His-162 which are all important amino acids in the active site that are conserved between proteins. Another notable amino acid is Glu-148, which in the human protein is positively substituted with aspartate and is responsible for a salt bridge interaction (1) (6). While, there are many differences between the amino acid sequences of the protein, the amino acids important to the function of the protein seem to be conserved between the species.
A Dali search of the protein yielded Z-scores for structural similarity of the yeast protein to the protein of Homo sapiens and E. coli. The Z-score value is based on the similarity of the 3-D structures of the proteins. A Z-score over 2 is considered significantly similar. The Z-score of the comparison of Homo sapiens arginyl-tRNA synthetase to yeast arginyl-tRNA synthetase is 44.0 indicating a high similarity in tertiary structure (7). The Z-score of the comparison of yeast arginyl-tRNA synthetase to E. coli arginyl-tRNA synthetase is 38.2, also indicating a high similarity in tertiary structure. The similar tertiary structure is to be expected because the proteins perform homologous actions in separate organisms. The Z scores arise from high similarity in structure, but still contain fundamental differences. Most notably, the yeast protein has a large number of alpha helices in relation to both E. coli and Homo sapiens (3).
The secondary structure of the protein contains 30 alpha helices, 20 beta sheets, and 58 random coils(1). The structure contains a higher fraction of alpha helices than the related protein in Homo sapiens (3). The secondary structure gives rise to a complex tertiary structure that includes an active site, a Rossman fold, and a tRNA recognition site (1). The active site consists of multiple amino acids responsible for stabilizing the arginine ligand, with several focusing on the carboxy group (1). A Rossman fold is a structure that binds nucleotides like NADPH, FAD, and ATP (8). In arginyl-tRNA synthetase, the Rossman fold is responsible for binding ATP until the arginine-ADP intermediate can be formed before esterification to tRNA (3). The presence of the Rossman fold classifies argRS as a class 1 aminoacyl-tRNA synthetase (8). Arginyl-tRNA synthetase can further be distinguished from other aminoacyl-tRNAs by its inability to bind ATP without the induced fit of the tRNA opening up the binding pocket for a sugar on the ATP (3). This makes the Rossman fold important to the proper functioning of the protein, as the ATP cannot be bound by the arginine until the proper tRNA complexes with the enzymatic system. The tRNA recognition site is responsible for binding the correct tRNA. Multiple tRNAs exist, and the one containing an anti-codon that corresponds to arginine must be selected for.
The protein consists of a single subunit with 5 domains, additional domain 1 (Add-1), additional domain 2 (Add-2), insertion domain 1 (Ins-1), insertion domain 2 (Ins-2), and the Rossman fold. Add-1 is the N-terminal domain and contains 4 beta sheets and 5 alpha helices. The Add-1 domain is responsible for tRNA recognition. Insertion domain 1 closes off one side of the active site and contains helix H11, which is part of the nucleotide binding fold. Insertion domain 2 corresponds to a connecting peptide 1 (CP1) which connects the two halves of the Rossman fold (1). The Rossman fold is nucleotide binding complex that helps bind ATP in the formation of the intermediate during the esterification of arginine to tRNA (3). The CP1 in yeast arginyl-tRNA synthetase is composed of the helix H12, beta strand S10, and the antiparallel beta sheet formed from strands S8 and S9. The C-terminal Add-2 domain contains 10 alpha helices and only one beta strand, S13. The Add-2 domain works with the Add-1 domain in recognition of the tRNA. The Add-2 domain is heavily implicated in its work as the anti-codon binding domain due to its similarity in spatial structure to other amino acid tRNA synthetases (1).
Important functional groups in the active site of arginyl-tRNA synthetase are involved in stabilizing arginine. Most of the characterized amino acid groups occur in the arginine binding pocket. The amino group on Asn-153 performs stable hydrogen bonding interactions with the backbone carboxylate on the arginine ligand, along with His-162. Ser-151 also stabilizes the complex through hydrogen bonding interactions . Gln-375 also forms hydrogen bonding interactions with the arginine carboxylate, but also acts as a hydrogen acceptor for Tyr-347. Tyr-347 first accepts the hydrogen from from the arginine substrate before passing it off to Gln-375. Glu-148 and Asp-351 form salt bridge interactions with the carboxylate group on the arginine substrate. His-159 forms water-mediated interactions with the substrate. Outside of the binding pocket for ATP, there is a peptide composed of six amino acids, Ser-480, Phe-481, Glu-482, Gly-483, Asp-484, and Thr-485, in the Add-2 domain that form an omega loop at the surface of the protein. This omega loop is the anti-codon recognition site that serves as a switch to activate the protein. This way, the protein is only activated when the tRNA specific for arginine binds to the surface of argRS. Gly-483 is specifically important to the functioning of the omega loop, as mutations that code for Ser-483 instead of Gly-483 are found to be lethal. Gly-483 provides needed structural support to the protein at the tRNA recognition site (1).