Cysteinyl-tRNA synthetase (PDB ID: 1U0B) is an amino acyl-tRNA synthetase of Escherichia coli that forms a complex with tRNA^Cys (1). Amino acyl-tRNA synthetases (aaRSs) perform specific aminoacylation of tRNA with their equivalent amino acids (2). With a molecular weight of 52202.09 Da, cysteinyl-tRNA synthetase (CysRS) is the smallest monomeric class I tRNA synthetase, and thus is of importance in defining the limiting principles of tRNA recognition and aminoacylation among this subgroup of enzymes (1, 3). There are two classes of aaRSs that vary based on their nucleotide binding domains. Class I aaRSs have catalytic domains containing the classical nucleotide binding Rossmann fold which differentiate them from class II aaRSs that have an antiparallel beta sheet flanked by alpha helices (2). The Rossmann fold is one of five structural components of CysRS. Succeeding the Rossmann fold are the ‘Connective Polypeptide’ (CP) domain, the Stem-Contact fold (SC), the helical bundle domain and the C-terminal anticodon binding (AB) domain (1). Class Ia aaRSs, encompassing leucyl-tRNA, arginyl-tRNA, isoleucyl-tRNA, methionyl-tRNA, valyl-tRNA and cysteinyl-tRNA synthetases, have a class specific helix bundle domain (9).

CysRS has two general pathways for recognizing the globular tertiary structure and anticodon stem loop domains of tRNA^Cys (1). These mechanisms allow for the discrimination of nucleic acid sequences. Regulatory proteins, such as CysRS, recognize specific nucleic acid sequences through direct readout and indirect readout. Direct readout involves base-amino acid contact between the tRNA^Cys anticodon stem loop domain and CysRS, which determines the stability of the CysRS-tRNA^Cys complex. Indirect readout is performed through tRNA conformation and flexibility. Indirect readout determines sequence specificity by the change in intramolecular conformational energy of the tRNA upon complex formation. Intrinsic conformation of the tRNA and the deformation of the nucleic acid upon the formation of the complex can both contribute to the globular tertiary structure recognition (5). The structure of CysRS shows that it has diversified from other class Ia enzymes by developing separate domains for direct and indirect recognition (1).

The AB domain, adjacent to the anticodon loop, is made up of 60 amino acids forming mixed α/β folds. This domain provides the direct readout through hydrogen bonding with all three anticodon nucleotides (G-34, C-35, and A-36). The α-helical bundle, responsible for direct readout in other class Ia synthetases, has no influence on the direct readout mechanism of CysRS. GCA anticodon nucleotides are stacked on top of each other inside of the complementary cavity of the AB domain. The helix-loop-helix motif (residues 413-443) interacts with G-34 and C-35 nucleotides. The exocyclic N₂ and exocyclic O₆ of G-34 form hydrogen bonds with Asp-436 and Arg-427. G-34 is also stabilized by stacking with the Trp-432 indole ring. The guanidinium of Arg-439 forms a hydrogen bond with the O₂ of G-35. Asp-451, found on the two-stranded antiparallel β-sheets (residues 446-461), interacts with both C-35 and A-36 nucleotides. The carboxylate of Asp-451 forms a hydrogen bond with the N₄ of C-35 while the main chain amide forms a single bond with the exocyclic amine of A-36. Arg-423 provides stability to the direct readout by forming an ion pair with Asp-451 and orienting the residue to interact with C-35 and A-36.  A high level of conservation of residues in the AB domain exists, while residues involved in indirect readout are less conserved among class I variants. The AB domain of CysRS is specialized for anticodon loop selection while for other aaRSs, large N-terminal and C-terminal domains reach around their appropriate tRNAs to bind the core regions (1).

A large fraction of binding interactions involved in indirect readout occur between the alpha helical bundle domain of CysRS and the tRNA D and anticodon stems (the vertical arm of the L shaped tRNA). The bend in the tRNA^Cys molecule is stabilized by the hydrogen bonds and electrostatic interactions between the sugar phosphate backbone and CysRS. The most unusual feature of the conformation of tRNA^Cys core is the G-15•G-48 Levitt pair. This Levitt pair involves the exocyclic O6 of G15 hydrogen bonded with N₁ and N₂ groups of G-48. The unique tertiary fold of the core of tRNACys is a result of the interactions of this G-15•G-48 Levitt pair and surrounding nucleotides. The G-15•G-48 pair is insufficient to form the unique globular core structure of tRNACys and needs to be supported by other interactions. Asn-351 to G-15 phosphate contact mediates tRNA recognition by indirect readout. The tRNACys structure is stabilized by a salt bridge created by water-mediated hydrogen bonds linking G-15 phosphate with Asp-348 and Lys-12 (1).

The Rossmann fold, first identified in 1974, is one of the most studied dinucleotide-binding folds. It is a single β-α-β-α-β motif that binds a mononucleotide. Two β-α-β-α-β motifs form a six stranded parallel beta-sheet flanked by alpha helices (4). The Rossmann fold active site domain of CysRS acylates the tRNA^Cys at the 2’OH of A-76 (1). A zinc ion is bound at the base of the active site cleft where it interacts with the side chains Cys-28, Cys-209, His-234, and Glu-238. The zinc ion has a key role in amino acid discrimination by forming a fifth ligand interaction with the cysteine thiolate substrate. A tetranucleotide (UCCA-76) forms a single stranded hairpin structure that curves into the active site. The hairpin is stabilized by a large surface loop (residues 159-174) of the ‘CP’ domain. Increased stabilization of the hairpin occurs though interactions with the G-1 – C-71 base pair (1).

Class I complexes form complete active sites that are closed and ready for catalytic activity only in the presence of ATP or of an aminoacyl adenylate. The structure of CysRS indicates that Trp-205 may act as a gatekeeper, only allowing aminoacylation to occur when both cysteine and ATP are bound to the active site. Generally, class I tRNA synthetases bind ATP at Gly-39 (1).

Methionyl-tRNA synthetase (MetRS) from Thermus thermophiles HB8 (PDB ID: 1A8H) is a class I amino acyl-tRNA synthetases that forms a complex with tRNA^Met. To provide better comparison it is important to note that the T.thermophiles MetRS structure is similar to the E.coli MetRS structure as determined by electron density map analysis (6). CysRS has an isoelectric point (pI) of 5.33, a more acidic pI than MetRS at a value of 6.14. MetRS is slightly larger than CysRS with a molecular weight of 57938.55 Da as determined by the protein identification and analysis tools on the ExPASy server (3). MetRS, with a primary structure made up of 500 amino acids, is longer than the 461 amino acid sequence of CysRS (10). The primary structure of CysRS was compared to methionyl-tRNA synthetase (MetRS) using a Position-Specific-Iterated Basic Local Alignment Search Tool (PSI-BLAST) and gave an E value of 10-74(7). Increased sequence homology decreases the E value, and an E value of less than 0.05 is considered significant for proteins. The tertiary structure of CysRS was compared to MetRS using the Dali Server and gave a Z score of 24.5 (8). The Dali Server uses a sum-of-pairs method, the similarity between intramolecular distances is compared, and the Z score is evaluated. A Z score of above 2 indicates similar protein folds between proteins. The ‘CP’ domain, found in all class Ia aaRSs, contains an extra antiparallel β-strand in MetRS that does not exist in CysRS and other class Ia aaRSs (6). The Rossmann fold and AB domain are highly conserved between CysRS and MetRS, as well as all other class I aaRSs (6, 1). Similar to CysRS, a zinc ion is essential for the aminoacylation activity of the MetRS. The structural differences between these proteins allow for specific aminoacylation reactions to occur with the appropriate amino acids. 

Ultimately, CysRS is distinct from other class I aaRSs because of its separation of domains to perform direct and indirect recognition. The selectivity of CysRS is dependent on these two mechanisms which are driven by intermolecular interactions throughout its five domains. MetRS, while being specific to methionine as opposed to cysteine, has similar primary and tertiary structures to CysRS. This demonstrates that minor variations in structure, such as the addition of a beta sheet, can lead to significant variations in function.