Histidyl-tRNA synthetase
Created by Tanaporn Wangsanut
Histidyl-tRNA synthetase (PDB = 1H4V) from Thermus thermophilus plays an important role in protein synthesis by covalently linking histidine to tRNA that contains corresponding triplet anticodon. Once tRNA binds to its corresponding amino acid, a ribosome can transfer the amino acid from tRNA onto a growing polypeptide, according to the genetic code. Hence, aminoacyl-tRNA synthetase must be able to discriminate against chemically similar, non-cognate amino acids, leading to specificity of each aminoacyl-tRNA synthetase. As a result, most aminoacyl-tRNA synthetase enzyme binds tightly with its target amino acid. The product of this enzymatic activity, however, is difficult to release as it binds strongly to the enzyme. To allow for the release of the product from the enzyme, an “induced fit mechanism” can be employed. Histidyl-tRNA Synthetase uses induced conformational change to ensure the amino acid specificity. The molecular weight of histidyl-tRNA synthetase is 47222.46 Da, and its isoelctric point (pI) is 5.93. The ligand of histidyl-tRNA-synthetase is sulfate ion.
The structure of Histidyl-tRNA synthetase directly correlates to the function of the enzyme. Histidyle-tRNA synthetase is a homo-dimeric enzyme, containing 421 amino acid residues. These amino acid residues can be categorized by polarity into four groups: hydrophobic, polar, positively charged, or negatively charged amino acids. The secondary structure of histadyl-tRNA synthetase is 41% alpha helix (19 helices; 175 residues) and 20% beta sheets (16 strands; 87 residues). The dimeric interface is made up from three main structural components: a long alpha and beta strand segment, a beta strand region rich in aromatic residues, and a beta ribbon that reaches over the top of the interface (Arnaz, 1). There are three domains in each monomeric subunit of HisRS : the N-terminal catalytic domain which contains three motifs (motif1-3), a 90 to 100-residue C-terminal alpha/beta domain which is probably involved in recognizing the anticodon stem-loop of tRNA(His), and a HisRS-specific alpha helical domain inserted between motifs 2 and 3 of the catalytic domain. The catalytic domain consists of six strands of antiparallel beta sheets, shown in green flanked by three alpha helix motifs, shown in red. The histidine side chain-binding pocket is found on the catalytic domain. The C-terminal domain contains four parallel/one antiparallel beta strands, and three alpha helix strands (Francklyn, 268).
Aminoacyl-tRNA synthetase first binds with ATP molecules, and forms adenylate – aminoacyl tRNA synthetase (aaRS-AMP), which then binds to the corresponding amino acid. The interaction between aaRS with ATP is crucial for its function, and therefore the ATP binding site on the enzyme is highly conserved. For histidyl-tRNA synthetase, residues on the beta strand and loop portion of motif 2 are responsible for the ATP binding site. Residue Phe-125 is responsible for specific recognition of the adenine ring of ATP. Residue Thr-281 accounts for specific recognition of the ribose portion of ATP, and the N6 amino group of ATP is recognized by residue Tyr-122 (Francklyn, 269). Furthermore, residues Arg-113 and Glu-115 on the motif 2- loop interact with the phosphate group of ATP. ATP binds to histidyl tRNA synthetase through the induced-fit mechanism.
Histidine, a substrate of histidyl-tRNA synthetase, binds to active sites on catalytic domain of each monomer. The active site is formed by two conserved peptides, 259-RGLDYY and 285-GGRYDG. Some of the critical residues for histidine recognition and activation are Arg-112, Arg-259, and Tyr-264. Unlike the other class of aminoacylt-tRNA synthetase that tends to use divalent cations such as Mn2+ to catalyze the amino acid activation, histidyle-tRNA synthetase uses a positively charged Arg-259 to substitute for histidine activation. Hence, Arg-259 plays a crucial role in the mechanism of histidine activation.
During histidine binding, induced fit mechanism occurs by two important conformational changes. First, conformational change occurs at the Histidine-1 loop (consisting of 259- RGLDYY), in which Tyr-263 and Tyr-264 residues construct the histidine side-chain binding pocket. Catalytic Arg-259 forms hydrogen bonds to Tyr-264, Glu-270 and the histidine carboxyl group, allowing for the correct positioning of the complex structure that can interact with the α-phosphate group. Second, histidine binding induces conformational change at the “ordering loop”, by which residues 57 to 62 undergo the disordered/ordered transition of the ordering loop. Two hydrogen bonds stabilize the closed structure of the histidine binding loop and ordering loop. These two hydrogen bonds between Val-54 and Thr-58 on the ordering loop with Asp-262 and Gly-260 on the histidine-1 loop may be responsible for the co-operative ordering of the two loops during histidine binding.
The induced conformational mechanism seen in the binding of histidine to histidyl-tRNA synthetase corresponds to global conformational change found in other proteins. These changes are coupled to global inter-domain and inter-subunit conformational changes, which have an essential functional effect on tRNA binding and the aminoacylation step of the reaction. The C- terminal domain orients in a different direction compared to its catalytic domain, and therefore the C-terminal will interact with only the catalytic domain of the other subunit. This restricted interaction dictates the inter-domain and inter-subunit conformational changes. Namely, during substrate binding, there is a relative movement of the two subunits (3.8° rotation) and the anti-codon binding domain of one subunit moves essentially as a rigid body with the catalytic domain of the other subunit. These movements are a result of the co-operative conformational change in the active site, which is induced by histidine binding, and also involved the histidine 1 loop and the ordering loop (Yaremchuk, 995).
Histidyl-tRNA synthetase from Thermus thermophilus is compared with other proteins by using bioinformatics searches on PSI-BLAST, Dali and Expasy. PSI-BLAST is a program used to find proteins with a similar primary structure to a protein query. An E value below 0.5 demonstrates a significant similarity between two proteins. The Dali Server is a method for finding proteins with tertiary structure similarities to a query. Proteins with similar folds will have a Z- score above 2. Histidyl-tRNA synthetase (PDB=1QE0), shown in grey, from Staphylococcus aureus, is the same enzyme found in Thermus thermophilus, and therefore these two enzymes contain both the same primary structure and folding pattern. Histidyl-tRNA synthetase from S. aureus contains two chain subunits, A and B. The results of DALI, (PDB=1QE0-A, Z=44.4, rmsd= 0.4) and protein Blast (E=0.00) searches show that HisRT from S. aureus has both primary and tertiary similarities to HisRS from Thermus thermophilus. However, HisRS from these two organisms are different in several parts. Unlike histidyl-tRNA synthestase from Thermus thermophilus that contains 421 amino acid residues, S. aureus consists of 420 amino acid residues (Qiu, 1). Moreover, there are several different important loops in HisRS from S. aureus compared to Thermus thermophilus. These differences include the histidine A motif (Arg-257- Tyr-262) that is essential for substrate recognition, a loop (Gly-52 to Lys-62) that seems to control the communication between the histidine and ATP binding sites, the motif 2 loop (Glu-114 to Arg-120) that binds ATP, and the insertion domain that likely binds tRNA.
Histidyl-tRNA synthetase is associated with autoimmune diseases, such as rheumatic arthritis or myositis disease because HisRS can act as an antigen by inducing leukocyte migration. Indeed, HisRS induces CD4+ and CD8+ lymphocytes, interleukin (IL)-2–activated monocytes, and immature dendritic cells (iDCs) to migrate. Thus, autoantigenic aminoacyl– tRNA synthetases, perhaps liberated from damaged muscle cells, may perpetuate the development of myositis by recruiting mononuclear cells that induce innate and adaptive immune responses (Howard, 1). Hopefully, identifying epitopes responsible for these autoantibodies may reveal the complex mechanisms involved in these pathologies, and ultimately lead to novel therapeutic interventions for patients.