Prolyl-tRNA Synthetase
Created by Amanda Wang
Prolyl-tRNA synthetase (PDB ID = 1H4T) from Thermus thermophilus (ProRSTT) is a class IIa aminoacyl-tRNA synthetase that catalyzes the esterification of proline to the 3’-hydroxyl group of ribose on the 3’ end of its cognate tRNAPro isoacceptors. This tRNA charging is achieved through two steps. First, ProRSTT activates proline, using ATP to form a prolyl-adenylate (Pro-AMP) intermediate complex. The proline is transferred to the 3’ terminal end of tRNAPro (1). ProRSTT is part of the second genetic code that ensures fidelity during information transfer from nucleic acids to peptides and accurate protein synthesis (2). In order to do so, ProRSTT must be able to distinguish between chemically similar amino acids by a factor of at least 104 (3). ProRSTT is divided into two major classes: eukaryote/archaeon-like and prokaryote-like. Conformation differences between species-specific ProRS could be the basis of pathogen specific antibiotics (4).
ProRSTT exists physiologically as an asymmetric homodimer with a molecular weight of 108 kDa (1) and an isoelectric point of 5.95 (5). Each monomer consists of 477 residues and three domains. The N-terminal catalytic domain formed by residues 1-273 consists of three motifs in a six-stranded antiparallel β-sheet flanked by α-helices. It is involved in the catalysis of tRNA charging (4). The C-terminal anticodon-binding domain spans residues 290-377, forming an α/β fold made of a five-stranded mixed β-sheet surrounded by three α-helices. The β-sheet contacts the tRNA stem-loop from the major groove side in this domain, recognizing two nucleotide bases: G-35 and G-36 (6). The zinc-binding domain follows through residues 402-477. This compact domain is comprised of a four-stranded mixed β-sheet with two flanking α-helices has four cysteine residues that form a tetrahedral complex with a zinc ion. The zinc ion is thought to play a non-essential role in stabilizing the C-terminal domain (4). The protein is 35% α-helix and 27% β-sheet (7).
Both reactions catalyzed by ProRSTT are preformed at the same active site. This is achieved through conformational changes of the enzyme which bring the site of anticodon-binding and the site of catalysis, which are normally 70 Å apart, close enough to allow charging (8). ProRSTT ensures specific recognition of proline through an induced fit mechanism, where movements of loops and side chains create a buried pocket perfectly complementary to the polarity of proline. Non-polar Trp-158, Glu-160, and Trp-259 residues in the catalytic domain complement the hydrophobicity of proline. Polar Arg-142 and His-230 also in the catalytic domain hydrogen bond to the proline carboxyl group. Phe-205 is found on the mobile proline-binding loop, which swings shut upon proline binding to bury the proline ring in the pocket (3).
The enzyme stabilizes proline activation in two ways. Positively charged His-230 interacts with the oxygen that bridges ATP to proline (3). ATP is stabilized by cation binding sites on either side of the β- and γ-phosphates, and the pyrophosphate produced is stabilized by a conserved C-terminal tyrosine, whose negatively charged carboxyl group points directly into the active site (4). This causes a conformational change of the N-terminal catalytic domain due to hydrogen bonding between Thr-153 on motif two and the adenosine of ATP (8). The α-helix H4 ‘ordering loop’ is also moved in a lever arm effect due to hydrogen bonding between His-83 and Ala-206 on the proline binding loop and between Glu-90 and Trp-143 on motif two. Alpha-helix H4 in turn also maintains the two loops in a closed, ordered conformation (3). It has been suggested that a fully ordered functional active site necessary for tRNA 3’-acceptor stem binding and proline transfer requires the prior formation of prolyl-adenylate due to the subsequent conformational changes (4). This is a demonstration of how the function of the enzyme changes as the structure changes. The induced fit causes ProRSTT to have a higher affinity for Pro-AMP, allowing it to remain tightly bound and avoid hydrolysis. When the reaction is complete, the reduced affinity allows the products to be released more easily (3).
The structure of the anticodon-binding domain is responsible for the enzyme’s specificity for proline. The recognition of two nucleotide bases G-35 and G-36 on tRNAPro through hydrogen bonding is necessary and sufficient for the identification of all tRNAPro isoacceptors (9) because the proline occupies a full codon group. This is true for almost all species, as G-35 and G-36 are highly conserved in tRNAPro, and it agrees with the requirement of the genetic code, where the first nucleotide is the wobble position. This is reflected in the positioning of the nucleotides in the binding site. Residues Ile-295, Pro-332, and Phe-336 form a hydrophobic path for G-35 and G-36 (6). In all class IIa synthetases, the position occupied by Phe-336 is a highly conserved aromatic or large hydrophobic residue, which is necessary for forming an edge when interacting with G-36 (6). These residues all form very tight hydrogen bonds with the tRNA acceptor stem, resulting in enzymatic specificity. Nucleotide G-34, however, does not form extensive hydrogen bonds with ProRSTT. The loop of protein near G-34 is the most mobile in the anticodon-binding domain, and G-34 only forms one hydrogen bond with Asp-298 (6).
The primary structures of prokaryote-like and eukaryote/archaeon-like ProRS are significantly different. Prokaryote-like ProRS has around 570 residues per subunit, a characteristic insertion domain of 180 residues in the N-terminal catalytic domain, and no C-terminal zinc-binding domain. Eukaryote/arhcaeon-like ProRS, on the other hand, has a characteristic C-terminal extension of around eighty residues after the C-terminal anticodon-binding domain and no insertion domain. The bacterial insertion domain is hypothesized to have proofreading ability. This extra editing domain (INS) has an active site that is capable of hydrolyzing mischarged alanine-tRNAPro, allowing the mischarged amino acid to be released from the enzyme (10). Eukaryote/archaeon-like ProRS, on the other hand, can achieve the required specificity through the induced fit mechanism, and therefore does not require proofreading ability (3). This shows how different necessities of function correlates to different structures. This difference in structure also presents an opportunity for the targeting of pathogen specific prolyl-tRNA synthetases (and other structurally similar aminoacyl-tRNA synthetases) using antibiotics (4).
Threonyl-tRNA synthetase (ThrRS) is a related class IIa aminoacyl-tRNA synthetase. Its core regions are very similar, resulting in similar catalytic mechanisms (4). The similarities between ThrRS and ProRS make ThrRS a good model for the active form of ProRSTT, which has yet to be crystallized (4). Basic Local Alignment Search Tool (BLAST) was used to compare the protein sequence of ProRSTT to database sequences to find proteins with regions of similar primary structure (11). BLAST produces an E-value for each subject. Differences in sequence homology result in an increase in E-value. An E-value that is less than 0.5 is considered significant in this case. The Dali server was used to find proteins with similar tertiary structure. Dali compares the 3D intramolecular distances between a pair of proteins and assigns a Z-score. A Z-score of above two means the subject has significantly similar folds (12). Threonyl-tRNA synthetase (PDB ID =1EVK) from Escherichia coli produced an E-value of 3e-11 and a Z-score of 27.3. Thr-tRNA synthetase has 159 matching residues and twenty three identical residues in the catalytic domain with ProRSTT (4).
Threonyl-tRNA synthetase also requires only two nucleotide bases for recognition of tRNA: G-35 and U-36 (6). The tRNA core regions for both differ only in the conformation of nucleotide thirty eight (4). The mechanism for specificity, however, is very different. ThrRS uses its zinc ligand to form a pentacoordinate intermediate with the amino group and side chain hydroxyl to ensure only amino acids with a β-position hydroxyl group are activated. Here, zinc is neither structural nor catalytic, but is used as a cofactor (13). tRNAs from similar classes are able to maintain fidelity in the genetic code through specificity for one amino acid through similar small differences in protein structure.
Histidyl-tRNA synthetase from Thermus thermophilus (HisRSTT) is another closely related class IIa aminoacyl-tRNA synthetase that was not found in the database queries due to significant structural differences. HisRSTT does, however, bind histidine in a very similar mechanism to that of ProRSTT. The well-crystallized structures of HisRSTT in different stages of its enzymatic reaction make it a good model for ProRSTT. HisRSTT has a histidine binding loop that is analogous to the proline binding loop. HisRSTT too is ordered by the binding of its amino acid, and moves a distance of up to 8 Å. As in ProRSTT, the histidine binding loop is only in the ordered conformation if the correct amino acid is bound. HisRSTT also has a topologically equivalent ‘ordering loop’. Unlike in ProRSTT, the ordering loop in HisRSTT does not require the formation of the amino-adenylate intermediate in order to be stabilized. HisRSTT forms two hydrogen bonds with histidine, which provide more stabilization, as compared to the one formed by ProRSTT (3).