Threonyl-tRNA synthetase (PDB ID: 1QF6) from Escherichia coli functions as both an catalytic and regulatory protein (5). As an enzyme, threonyl-tRNA synthetase (ThrRS) maintains translational fidelity by improving aminoacylation efficiency and selectivity (1, 4, 5). As a regulatory protein, ThrRS adapts its own expression to the cellular demands by repressing the translation of its own mRNA (1, 4, 5). ThrRS is a member of the aminoacyl-tRNA synthetase (aaRS) family (1, 2). aaRSs catalyze the synthesis of aminoacyl-tRNAs by charging a specific tRNA molecule with its cognate amino acid in the cytoplasm and/or mitochondria (2). ThrRS is a cytoplasmic aaRS that esterifies threonine to its cognate 76 nucleotide-long tRNA (Figure A) via the ThrRS-tRNA^Thr complex (2). According to ExPASY, E. coli ThrRS has an isoelectric point (pI) of 5.80 and molecular weight of 74014.30 Da (8). 

Aminoacylation of tRNA^Thr is a two-step reaction crucial to protein synthesis (2). In the first step, ThrRS binds the cognate amino acid, i.e. threonine, and an ATP molecule to form a threonyl-adenylate intermediate (Thr-AMP), and a pyrophosphate molecule (PPi) is released (2, 10). In the second step, threonine is transferred to the 3’ ribose hydroxyl moiety of the terminal adenosine of the cognate tRNA, i.e. tRNA^Thr (2, 10). The AMP molecule is released, followed by the charged tRNA (2). Thus, aminoacylation prepares the charged tRNA by covalently linking threonine to the tRNA with the appropriate triplet anticodon. A ribosome can then transfer threonine from the charged tRNA to the growing polypeptide, according to the genetic code. For this reason, aminoacyl-tRNA synthetases must be able to discriminate against structurally similar, noncognate amino acids. Key structural features responsible for the specificity and function of threonyl-tRNA synthetase are (i) its accommodation of AMP, Zn2+ and threonine in the active site and (ii) its cross-subunit interactions with the anticodon loop and acceptor stem of tRNA^Thr (1, 5). 

ThrRS is an α2-dimeric enzyme with each monomer containing 642 residues (1). The secondary structure composition of each monomer is 38% α-helical (27 helices; 245 residues) and 26% β-sheet (41 strands; 172 residues) (13). It belongs to the class II aaRSs that approach the tRNA acceptor stem from the major groove side of the tRNA and aminoacylate the 3’-OH of the terminal adenosine of their cognate tRNAs (1). Class II aaRSs are also characterized by three signature motifs (9). Motif 1 […Pro…] consists of a α-helix linked to a β strand (9). Motif 2 [PheArgxGlu] is comprised of two antiparallel β-strands connected by a long loop (9). Motif 3 [Gly/x3GluArg] consists of a β-strand followed by an α-helix (9). Each monomer can be distinguished into four domains – two N-terminal domains (N1 and N2) joined via a linker helix to the core formed by the catalytic and C-terminal domains (1). Residues 281-298 form motif 1 in the catalytic domain of both monomers, which participates in the homodimer interface (1). 

The C-terminal domain (residues 535-642) is a mixed α/β domain made up of four antiparallel and one parallel β strand surrounded by three α helices (1). The class IIa-conserved C-terminal domain approaches the anticodon loop from the major groove side (1). The anticodon for E. coli threonine is BGU, B being C, G, or U (1). G35 and U36 are the identity determinants of E. coli tRNA^Thr (1). These two bases form a hydrogen bond between N2 of G35 and O4 of U36 (1). Additionally, N1 of G35 interacts with the highly conserved Glu600, and O2 of U36 interacts with the strictly conserved R609 (1). 

The catalytic domain (residues 243-534) contains the three class II signature motifs and is constructed from six antiparallel β-strands surrounded by three α helices (1). This module contains important residues that bind threonine as well as zinc- and AMP-binding sites that facilitate the aminoacylation of cognate tRNA with L-threonine (1, 10). The binding of these substrates induces conformational changes within the mobile regions of the catalytic domain. While the small substrates can bind in any order, they must be in place before productive tRNA binding can occur (10). 

The residues in the active site that confer specificity for threonine are Arg363, Gln381, Asp383, Gln484, and Tyr462 (10). Arg363 (motif 2 loop) and Tyr462 (threonine loop) both undergo movements with their respective loops that reshape the active site cavity upon threonine binding (10). The conformational change of the threonine loop leads to 14° rotation that brings this loop closer to the ordering loop (residues 301-317) in the catalytic domain (10). In the motif 2 loop, the displacement of the main chain allows the guanidinium group of Arg363 to interact with the carboxylate moiety of threonine (10).

AMP Binding Pocket.  Residues evolutionarily conserved in aaRS class II motifs 2 and 3 facilitate ATP recognition and catalysis (1). The motif 2-arginine (Arg363) interacts with the α phosphate (1). The motif 2-aromatic residue (Phe379) and motif 3-arginine (Arg520) stack on both sides of the adenine (1). 

The linker helix (residues 225-242) connects the catalytic core to the N-terminal region (1). The N-terminal region (residues 1-224) is composed of two distinct domains, the N1 module and N2 module (1). The N1 module (residues 1-62) has an α+β topology of five-stranded mixed sheet system and two helices (1). The N2 module (residues 63-224) is comprised of a pair of perpendicularly oriented antiparallel β sheets of four and three strands, respectively, that surround a central α helix that forms the core of the domain (1).

Together, the catalytic and N2 domains recognize the acceptor stem (1). The catalytic domain approaches the major groove side of the acceptor stem, contacting only the CCA end (1). The CCA end enters a deep cleft with the terminal adenosine (A76) intercalated between the class II-invariant Arg363 and Tyr313 residues (1).  The oxygen atoms of A76 participate in hydrogen bonds: O2’ with His309 and Tyr462, O3’ with Gln484, and O4’ with Tyr313 (1). This network of hydrogen bonds situates the terminal ribose favorably for esterification by threonine. That is, O3’ is in a position to accept the amino acid from the threonyl-AMP intermediate formed in the first step of aminoacylation (1). 

The N2 domain has a novel fold that wraps around the minor groove side of the acceptor stem, clamping the stem between the catalytic and N2 domains (1). This fold is maintained by weak forces between the N2 domain and the first two base pairs of the acceptor stem on the minor groove side (1). The interactions involve both main chain (Gly203-CO…N2-G71) and side chain moieties (Tyr205-OH…N2-G1) (1). 

Besides operating as a catalyst for tRNA aminoacylation, ThrRS also behaves as a regulatory protein that represses the translation of its own mRNA (3, 4). Its catalytic and C-terminal domains bind to two anticodon arm-like structures of the operator upstream of initiation codon, while its N-terminal domain competitively inhibits ribosome binding (5). The mimicry between tRNA and mRNA operator suggests structural analogies between the two recognition modes (1, 3, 5). 

PSI-BLAST compares the 1° structure of proteins with a lower E-value indicative of greater sequence similarity. Dickeya dadantii threonyl-tRNA synthetase has an E-value of 0.0 when compared to E. coli threonyl-tRNA synthetase (6). DALI compares proteins with E. coli ThrRS according to their 3° structures with a higher Z-score indicative of greater structural similarity. Compared to E. coli ThrRS, Homo sapiens cytoplasmic threonyl-tRNA ligase (PDB ID = 4HWT) has a Z-score of 51.9, and Saccharomyces cervisiae mitochondrial threonyl-tRNA synthetase (PDB IB = 3UH0) has a Z-score of 48.2 (7). 

While threonyl-tRNA synthetase classically regulates protein synthesis, it is also related to the immune response (11). “Autoimmune diseases are a group of clinically diverse conditions, characterized by the production of abnormal antibody and/or cellular immune responses that adversely effect host tissues” (12). Anti-synthetase syndromes are one class of autoimmune diseases resulting in the chronic inflammation of muscles, known as myositis (12). In myositis, threonyl-tRNA synthetase acts as an autoantigen that is targeted by the PL-7 autoantibody, inhibiting the aminoacylation activity of ThrRS (11, 12). While the etiology of how aminoacyl-tRNA synthetases, like ThrRS, become autoantigens is incomplete, identifying epitopes for these autoantibodies may reveal insight into their mechanisms of action and provide an avenue for therapeutic interventions.