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Isoleucyl-tRNA synthetase (PDB ID: 1FFY) is a type of aminoacyl-tRNA synthetase found in Staphylococcus aureus (1). Aminoacyl-tRNA synthetases (aaRSs) catalyze the aminoacylation reaction that binds the 3’ ribose of tRNA to its associated amino acid and each of the 20 amino acids has its own associated aaRS (2). aaRSs can produce correctly bound tRNA with an accuracy of at least 10,000:1 (3). Specifically, isoleucyl-tRNA synthetase (ileRS) produces less than one error in 3,000 instances (1). For IleRS to operate at these high levels of specificity, it must contain a way to edit incorrectly bound tRNAIle molecules.

IleRS has a molecular weight of 104,884.55 Da and an isoelectric point of 5.3 (4). It exists as a monomer and does not form a complex with other proteins. IleRS contains eight domains that can be organized into three main regions, each of which has its own function. The first region, which consists of three domains at the C-terminus and one at the N-terminus, recognizes unusual anticodon loop conformations. The second region, which consists of the Rossman fold domain, constitutes the core that activates and transfers the isoleucine molecule to the tRNA, termed the synthetic active site. The third region, which consists of the main editing domain, removes any incorrectly bonded amino acid, termed the editing active site (1). IleRS forms a complex with tRNA and isoleucine (or mupirocin, as discussed later). In order for the protein to function, it must first bind with Ile. In the ligand free structure of the protein, the editing domain overlaps the active site, where the tRNA is meant to bind. Once Ile is bound, it interacts with the editing domain, allowing the protein to bind with tRNA and form the completed complex (1). IleRS operates in a two-step, double sieve mechanism. The first sieve, the active site, excludes larger amino acids from being acylated. However, smaller and similar amino acids are allowed to proceed through the binding process. In the case of IleRS, the most common mistake is to aminoacylate Val, since it differs from Ile by only one methyl group. The editing site, therefore, must act as the second sieve for higher specificity (5). The synthetic and editing active sites are 34 Å apart and are located at the bottom of two deep clefts in the protein. An antiparallel pair of α-helices along the long axis of the aaRS binds with a small groove in the tRNA acceptor stem (1). The 3’ terminus of tRNAIle must bind with the synthetic active site to bind with Ile. However, due to the positions of Ade-73 and Cyt-74, it is impossible for tRNAIle to bind to the active site. It therefore must adopt a hairpin-like conformation at the 3’ terminus, which allows Cyt-74 to fit in a small crevice in the protein and the phosphate backbone of the tRNAIle to rest alongside a string of basic residues, reducing the steric strain and adding stability (1). The editing domain is characterized by the residues Tyr-392 and His-394, which are responsible for the specificity of the editing activity (1). Various mutations of these two residues have been shown to alter the degree of specificity, proving their critical nature (6).

The 20 aaRSs can be divided into two classes based on the organization of the editing site. IleRS is a Class 1 aaRS, which indicates the presence of a Rossmann domain that contains the critical amino acid motifs HMGH and KMSKS (7). Class 1 aaRSs can also be divided into three subclasses (Ia, Ib, and Ic) depending on the peptide sequence and structure of the active and editing sites. IleRS is in Class 1a along with methionyl-, valyl-, leucyl-, cysteinyl-, and arginyl-tRNA synthetases and as such shares similar sequences and structures with these proteins (8). The Rossman fold of ileRS contains two specific connective peptide chains (CP1 and CP2) that contain similar sequences across ileRS from different organisms. CP1 has been shown to be 276 residues long (specifically, residues 157-433) with residues 378-444 being critical in binding ileRS to tRNA (6). The Rossman fold has a hydrophobic pocket framed by residues Val-588, Met-65, Leu-69, Met-596, Trp-621, Val-631, and Ile-633, which houses the Ile in preparation for aminoacylation to tRNAIle (7).

S. aureus is a common cause for noscomical infections in humans and is usually treated with mupirocin, as the more common antibiotic methicillin is not effective (9). The 14-methyl terminus on mupirocin mimics Ile and therefore bonds to the ileRS active synthetic site instead of the intended Ile, interrupting the aminoacylation and therefore protein synthesis in the bacteria (10). Lys-595 in the characteristic KMSKS sequence binds to the carboxylate tail of mupirocin and adds stability to the complex (1). The mupirocin is able to bond in this fashion because it can fit in the hydrophobic pocket of the Rossman fold. However, certain samples of S. aureus ileRS have shown low levels of resistance to the antibiotic due to mutations in the protein. For example, mutations V588F and V631F have been shown to be the most direct causes to low-level mupirocin resistance (10,7). Val-588 is a part of the important hydrophobic pocket in the Rossman fold. Since it is a rather small amino acid, it allows room for the mupirocin molecule to take the place of the Ile. Val-588 also forms hydrogen bonds with mupirocin, thus adding stability to the complex (7). Replacing this residue with Phe would alter the structure of the hydrophobic pocket since it is a much more bulky residue than Val. To accommodate this switch, the fold would have to undergo a torsional rotation around the phenylalanyl chi-1 bond, which would not leave adequate room for the mupirocin (7). Since Val-631 also is a part of the hydrophobic pocket, the mutation V631F would have a similar effect.

Leucyl-tRNA synthetase, or leuRS (PDB ID: 4arc) from Escherichia coli is another Class 1a synthetase and thus is similar in function and structure to ileRS in that the editing site is characterized by a Rossman fold. LeuRS has a molecular weight of 99,397.08 Da and an isoelectric point of 5.38, meaning this protein is slightly smaller than ileRS and can function in slightly more basic environments (4). The sequence of leuRS was compared to that of ileRS by the Position-Specific-Iterated Basic Local Alignment Search Tool (PSI-BLAST). PSI-BLAST compares the primary structures of proteins and assigns an E value based on the sequence homology and any gaps in the structure. E values below 0.5 indicate significant similarities for proteins and are considered significant. LeuRS showed an E value of 3 x10-168 (11). The tertiary structure of leuRS was compared to that of ileRS using the Dali Server, which use a sum-of-pairs method to compare the intramolecular distances of the protein in question to those stored in the database and it generates a Z score. For proteins, a Z score above 2 indicates significant similarities in the tertiary strucure. The Dali server generated a Z score of 27.6 for leuRS (12). As a class 1a aaRS, leuRS contains the critical KMSKS peptide sequence in the synthetic active site (13). The structure of the active synthetic site, and the position of the class 1a conserved Tyr-43 also requires the tRNA to adapt a hairpin conformation in order to form the complex. As it is in ileRS, the synthetic active site is roughly 34-35 Å away from the editing active site, meaning the activated tRNA must move from the synthetic site to the editing site for proofreading (13).