Crystal structure of Pyrococcus horikoshii tryptophanyl-tRNA synthetase and structure-based phylogenetic analysis suggest an archaeal origin of tryptophanyl-tRNA synthetase

The ancient and ubiquitous aminoacyl-tRNA synthetases constitute a valuable model system for studying early evolutionary events. So far, the evolutionary relationship of tryptophanyl- and tyrosyl-tRNA synthetase (TrpRS and TyrRS) remains controversial. As TrpRS and TyrRS share low sequence homology but high structural similarity, a structure-based method would be advantageous for phylogenetic analysis of the enzymes. Here, we present the first crystal structure of an archaeal TrpRS, the structure of Pyrococcus horikoshii TrpRS (pTrpRS) in complex with tryptophanyl-5′ AMP (TrpAMP) at 3.0 Å resolution which demonstrates more similarities to its eukaryotic counterparts. With the pTrpRS structure, we perform a more complete structure-based phylogenetic study of TrpRS and TyrRS, which for the first time includes representatives from all three domains of life. Individually, each enzyme shows a similar evolutionary profile as observed in the sequence-based phylogenetic studies. However, TyrRSs from Archaea/Eucarya cluster with TrpRSs rather than their bacterial counterparts, and the root of TrpRS locates in the archaeal branch of TyrRS, indicating the archaeal origin of TrpRS. Moreover, the short distance between TrpRS and archaeal TyrRS and that between bacterial and archaeal TrpRS, together with the wide distribution of TrpRS, suggest that the emergence of TrpRS and subsequent acquisition by Bacteria occurred at early stages of evolution.     

A short peptide insertion crucial for angiostatic activity of human tryptophanyl-tRNA synthetase

Human tryptophanyl-tRNA synthetase (TrpRS) is secreted into the extracellular region of vascular endothelial cells. The splice variant form (mini TrpRS) functions in vascular endothelial cell apoptosis as an angiostatic cytokine. In contrast, the closely related human tyrosyl-tRNA synthetase (TyrRS) functions as an angiogenic cytokine in its truncated form (mini TyrRS). Here, we determined the crystal structure of human mini TrpRS at a resolution of 2.3 A and compared the structure with those of prokaryotic TrpRS and human mini TyrRS. Deletion of the tRNA anticodon-binding (TAB) domain insertion, consisting of eight residues in the human TrpRS, abolished the enzyme's apoptotic activity for endothelial cells, whereas its translational catalysis and cell-binding activities remained unchanged. Thus, we have identified the inserted peptide motif that activates the angiostatic signaling.     

Evidence for the early divergence of tryptophanyl- and tyrosyl-tRNA synthetases

Each amino acid is attached to its cognate tRNA by a distinct aminoacyl-tRNA synthetase (aaRS). The conventional evolutionary view is that the modern complement of synthetases existed prior to the divergence of eubacteria and eukaryotes. Thus comparisons of prokaryotic and eukaryotic aminoacyl-tRNA synthetases of the same type (charging specificity) should show greater sequence similarities than comparisons between synthetases of different types—and this is almost always so. However, a recent study [Ribas de Pouplana L, Furgier M, Quinn CL, Schimmel P (1996) Proc Natl Acad Sci USA 93:166–170] suggested that tryptophanyl- (TrpRS) and tyrosyl-tRNA (TyrRS) synthetases of the Eucarya (eukaryotes) are more similar to each other than either is to counterparts in the Bacteria (eubacteria). Here, we reexamine the evolutionary relationships of TyrRS and TrpRS using a broader range of taxa, including new sequence data from the Archaea (archaebacteria) as well as species of Eucarya and Bacteria. Our results differ from those of Ribas de Pouplana et al.: All phylogenetic methods support the separate monophyly of TrpRS and TyrRS. We attribute this result to the inclusion of the archaeal data which might serve to reduce long branch effects possibly associated with eukaryotic TrpRS and TyrRS sequences. Furthermore, reciprocally rooted phylogenies of TrpRS and TyrRS sequences confirm the closer evolutionary relationship of Archaea to eukaryotes by placing the root of the universal tree in the Bacteria. Received: 7 December 1996 / Accepted: 11 February 1997     

Ancient adaptation of the active site of tryptophanyl-tRNA synthetase for tryptophan binding

The amino acid binding domains of the tryptophanyl (TrpRS)- and tyrosyl-tRNA synthetases (TyrRS) of Bacillus stearothermophilus are highly homologous. These similarities suggest that conserved residues in TrpRS may be responsible for both determining tryptophan recognition and discrimination against tyrosine. This was investigated by the systematic mutation of TrpRS residues based upon the identity of homologous positions in TyrRS. Of the four residues which interact directly with the aromatic side chain of tryptophan (Phe5, Met129, Asp132, and Val141) replacements of Asp132 led to significant changes in the catalytic efficiency of Trp aminoacylation (200-1250-fold reduction in k(cat)/K(M)) and substitution of Val141 by the larger Glu side chain reduced k(cat)/K(M) by 300-fold. Mutation of Pro127, which determines the position of active-site residues, did not significantly effect Trp binding. Of the mutants tested, D132N TrpRS also showed a significant reduction in discrimination against Tyr, with Tyr acting as a competitive inhibitor but not a substrate. The analogous residue in B. stearothermophilusTyrRS (Asp176) has also been implicated as a determinant of amino acid specificity in earlier studies [de Prat Gay, G., Duckworth, H. W., and Fersht, A. R. (1993) FEBS Lett. 318, 167-171]. This striking similarity in the function of a highly conserved residue found in both TrpRS and TyrRS provides mechanistic support for a common origin of the two enzymes.     

Inhibition of tumor angiogenesis by a natural fragment of a tRNA synthetase

Human tyrosyl- and tryptophanyl-tRNA synthetases (TyrRS and TrpRS, respectively) link protein synthesis to signal-transduction pathways, including angiogenesis. Fragments of TyrRS stimulate angiogenesis, whereas those of TrpRS (T2-TrpRS) inhibit angiogenesis. Thus, these two synthetases acquired opposing activities during evolution, possibly as a coordinated mechanism for regulating angiogenesis. The recent identification of the cellular target of T2-TrpRS sheds light into the mechanism of angiogenesis inhibition. This mechanism provides a molecular basis for the lack of effect of T2-TrpRS on the normal vasculature. With these features, we suggest that this fragment of a tRNA synthetase might safely be used to arrest neovascularization of tumors. In particular, an anti-angiogenesis agent that stops the growth of tumor vessels without affecting normal vessels might serve as an adjunct to cytotoxic therapy.     

Highly differentiated motifs responsible for two cytokine activities of a split human tRNA synthetase.

While native human tyrosyl-tRNA synthetase (TyrRS) is inactive as a cell-signaling molecule, it can be split into two distinct cytokines. The enzyme is secreted under apoptotic conditions in culture where it is cleaved into an N-terminal fragment that harbors the catalytic site and into a C-domain fragment found only in the mammalian enzymes. The N-terminal fragment is an interleukin-8 (IL-8)-like cytokine, whereas the released C-domain is an endothelial-monocyte-activating polypeptide II (EMAP II)-like cytokine. Although the IL-8-like activity of the N-fragment depends on an ELR motif found in alpha-chemokines and conserved among mammalian TyrRSs, here we show that a similar (NYR) motif in the context of a lower eukaryote TyrRS does not confer the IL8-like activity. We also show that a heptapeptide from the C-domain has EMAP II-like chemotaxis activity for mononuclear phagocytes and polymorphonuclear leukocytes. Eukaryote proteins other than human TyrRS that have EMAP II-like domains have variants of the heptapeptide motif. Peptides based on these sequences are inactive as cytokines. Thus, the cytokine activities of split human TyrRS depend on highly differentiated motifs that are idiosyncratic to the mammalian system.     

Induction of angiogenesis by a fragment of human tyrosyl-tRNA synthetase

The first step of protein synthesis is catalyzed by aminoacyl-tRNA synthetases. In addition, certain mammalian tRNA synthetases link protein synthesis to cytokine signaling pathways. In particular, human tyrosyl-tRNA synthetase (TyrRS) can be split by proteolysis into two fragments having distinct cytokine activities. One of the TyrRS fragments (mini TyrRS) contains features identical to those in CXC chemokines (like interleukin-8) that also act as angiogenic factors. Here mini TyrRS (but not full-length TyrRS) is shown to stimulate chemotaxis of endothelial cells in vitro and stimulate angiogenesis in each of two in vivo animal models. The angiogenic activity of mini TyrRS can be opposed by anti-angiogenic chemokines like IP-10. Thus, a biological fragment of human tyrosyl-tRNA synthetase links protein synthesis to regulation of angiogenesis.     

Mutational switching of a yeast tRNA synthetase into a mammalian-like synthetase cytokine

Aminoacyl-tRNA synthetases catalyze the attachment of amino acids to their cognate tRNAs. A link was recently established between protein biosynthesis and cytokine signal transduction. Human tyrosyl-tRNA synthetase can be split into two fragments, each of which has a distinct cytokine function. This activity is specific to the human enzyme. It is absent in the enzymes from lower organisms such as bacteria and yeast. Here, yeast tyrosyl-tRNA synthetase (TyrRS), which lacks cytokine activity, was used as a model to explore how a human tyrosyl-tRNA synthetase during evolution acquired novel functions beyond aminoacylation. We found that a rationally designed mutant yeast TyrRS(ELR) gained cytokine function. The mutant yeast enzyme gained this function without sacrifice of aminoacylation activity. Therefore, relatively simple alteration of a basic structural motif imparts cytokine activity to a tRNA synthetase while preserving its canonical function. Further work established that mutational switching of a yeast protein to a mammalian-like cytokine was specific to this synthetase and not to just any yeast ortholog of a mammalian cytokine.     

Identification and characterization of human mitochondrial tryptophanyl-tRNA synthetase

A full-length cDNA clone encoding the human mitochondrial tryptophanyl-tRNA synthetase (h(mt)TrpRS) has been identified. The deduced amino acid sequence shows high homology to both the mitochondrial tryptophanyl-tRNA synthetase ((mt)TrpRS) from Saccharomyces cerevisiae and to different eubacterial forms of tryptophanyl-tRNA synthetase (TrpRS). Using the baculovirus expression system, we have expressed and purified the protein with a carboxyl-terminal histidine tag. The purified His-tagged h(mt)TrpRS catalyzes Trp-dependent exchange of PP(i) in the PP(i)-ATP exchange assay. Expression of h(mt)TrpRS in both human and insect cells leads to high levels of h(mt)TrpRS localizing to the mitochondria, and in insect cells the first 18 amino acids constitute the mitochondrial localization signal sequence. Until now the human cytoplasmic tryptophanyl-tRNA synthetase (hTrpRS) was thought to function as the h(mt)TrpRS, possibly in the form of a splice variant. However, no mitochondrial localization signal sequence was ever detected and the present identification of a different (mt)TrpRS almost certainly rules out that possibility. The h(mt)TrpRS shows kinetic properties similar to human mitochondrial phenylalanyl-tRNA synthetase (h(mt)PheRS), and h(mt)TrpRS is not induced by interferon-gamma as is hTrpRS.     

Indolmycin resistance of Streptomyces coelicolor A3(2) by induced expression of one of its two tryptophanyl-tRNA synthetases

Aminoacyl-tRNA synthetases, a family of enzymes essential for protein synthesis, are promising targets of antimicrobials. Indolmycin, a secondary metabolite of Streptomyces griseus and a selective inhibitor of prokaryotic tryptophanyl-tRNA synthetase (TrpRS), was used to explore the mechanism of inhibition and to explain the resistance of a naturally occurring strain. Streptomyces coelicolor A3(2), an indolmycin-resistant strain, contains two trpS genes encoding distinct TrpRS enzymes. We show that TrpRS1 is indolmycin-resistant in vitro and in vivo, whereas TrpRS2 is sensitive. The lysine (position 9) in the enzyme tryptophan binding site is essential for making TrpRS1 indolmycin-resistant. Replacement of lysine 9 by glutamine, which at this position is conserved in most bacterial TrpRS proteins, abolished the ability of the mutant trpS gene to confer indolmycin resistance in vivo. Molecular modeling suggests that lysine 9 sterically hinders indolmycin binding to the enzyme. Tryptophan recognition (assessed by k(cat)/K(M)) by TrpRS1 is 4-fold lower than that of TrpRS2. Examination of the mRNA for the two enzymes revealed that only TrpRS2 mRNA is constitutively expressed, whereas mRNA for the indolmycin-resistant TrpRS1 enzyme is induced when the cells are exposed to indolmycin.     

2.9 A crystal structure of ligand-free tryptophanyl-tRNA synthetase: domain movements fragment the adenine nucleotide binding site

The crystal structure of ligand-free tryptophanyl-tRNA synthetase (TrpRS) was solved at 2.9 A using a combination of molecular replacement and maximum-entropy map/phase improvement. The dimeric structure (R = 23.7, Rfree = 26.2) is asymmetric, unlike that of the TrpRS tryptophanyl-5'AMP complex (TAM; Doublié S, Bricogne G, Gilmore CJ, Carter CW Jr, 1995, Structure 3:17-31). In agreement with small-angle solution X-ray scattering experiments, unliganded TrpRS has a conformation in which both monomers open, leaving only the tryptophan-binding regions of their active sites intact. The amino terminal alphaA-helix, TIGN, and KMSKS signature sequences, and the distal helical domain rotate as a single rigid body away from the dinucleotide-binding fold domain, opening the AMP binding site, seen in the TAM complex, into two halves. Comparison of side-chain packing in ligand-free TrpRS and the TAM complex, using identification of nonpolar nuclei (Ilyin VA, 1994, Protein Eng 7:1189-1195), shows that significant repacking occurs between three relatively stable core regions, one of which acts as a bearing between the other two. These domain rearrangements provide a new structural paradigm that is consistent in detail with the "induced-fit" mechanism proposed for TyrRS by Fersht et al. (Fersht AR, Knill-Jones JW, Beduelle H, Winter G, 1988, Biochemistry 27:1581-1587). Coupling of ATP binding determinants associated with the two catalytic signature sequences to the helical domain containing the presumptive anticodon-binding site provides a mechanism to coordinate active-site chemistry with relocation of the major tRNA binding determinants.     

Evidence for annexin II-S100A10 complex and plasmin in mobilization of cytokine activity of human TrpRS

In mammalian cells, specific aminoacyl-transfer RNA (tRNA) synthetases have cytokine functions that require interactions with partners outside of the translation apparatus. Little is known about these interactions and how they facilitate expanded functions that link protein translation to other cellular pathways. For example, an alternative splice fragment of tryptophanyl-tRNA synthetase (TrpRS) and a similar natural proteolytic fragment are potent angiostatic factors that act through the vascular endothelial-cadherin receptor and Akt signaling pathway. Here we demonstrate mobilization of TrpRS for exocytosis from endothelial cells and the potential for plasmin to activate the cytokine function of the extracellular synthetase. Direct physical evidence showed that the annexin II-S100A10 complex, which regulates exocytosis, forms a ternary complex with TrpRS. Functional studies demonstrate that both annexin II and S100A10 regulate trafficking of TrpRS. Thus, complexes of mammalian tRNA synthetases with seemingly disparate proteins may in general be relevant to understanding how their expanded functions are implemented.     

3种水稻线粒体tRNA~(Trp)突变体的克隆和活力鉴定

 设计并完成了 3种水稻线粒体tRNATrp的突变 ,体外转录并用枯草杆菌和人色氨酰tRNA合成酶 (TrpRS)对tRNATrp及其突变体进行了活力测定 .3种突变体的氨酰化活力比野生型水稻线粒体tRNATrp分别上升了 1 8、1 5和 5倍 .说明A1 U72和G5 C68对于提高线粒体tRNATrp被细胞质TrpRS氨酰化能力的作用并不大 ,细胞质tRNATrp与细胞质TrpRS的识别方式并不适用于线粒体tRNATrp与细胞质TrpRS的相互识别 .研究结果对于了解线粒体tRNATrp和细胞质TrpRS的相互识别及药物设计有重要意义     

Activation of D-tyrosine by Bacillus stearothermophilus tyrosyl-tRNA synthetase: 1. Pre-steady-state kinetic analysis reveals the mechanistic basis for the recognition of D-tyrosine

Tyrosyl-tRNA synthetase (TyrRS) is able to catalyze the transfer of both l- and d-tyrosine to the 3' end of tRNA(Tyr). Activation of either stereoisomer by ATP results in formation of an enzyme-bound tyrosyl-adenylate intermediate and is accompanied by a blue shift in the intrinsic fluorescence of the protein. Single turnover kinetics for the aminoacylation of tRNA(Tyr) by D-tyrosine were monitored using stopped-flow fluorescence spectroscopy. Bacillus stearothermophilus tyrosyl-tRNA synthetase binds d-tyrosine with an 8.5-fold lower affinity than that of l-tyrosine (K (D-Tyr)(d) = 102 microm) and exhibits a 3-fold decrease in the forward rate constant for the activation reaction (k (D-Tyr)(3) = 13 s(-1)). Furthermore, as is the case for l-tyrosine, tyrosyl-tRNA synthetase exhibits "half-of-the-sites" reactivity with respect to the binding and activation of D-tyrosine. Surprisingly, pyrophosphate binds to the TyrRS.d-Tyr-AMP intermediate with a 14-fold higher affinity than it binds to the TyrRS.l-Tyr-AMP intermediate (K (PPi)(d) = 0.043 for TyrRS.d-Tyr-AMP.PP(i)). tRNA(Tyr) binds with a slightly (2.3-fold) lower affinity to the TyrRS.d-Tyr-AMP intermediate than it does to the TyrRS.l-Tyr-AMP intermediate. The observation that the K (Tyr)(d) and k(3) values are similar for l- and d-tyrosine suggests that their side chains bind to tyrosyl-tRNA synthetase in similar orientations and that at least one of the carboxylate oxygen atoms in d-tyrosine is properly positioned for attack on the alpha-phosphate of ATP.     

固定化枯草杆菌色氨酰tRNA合成酶(英文)

为研究ｔＲＮＡＴｒｐ 与色氨酰ｔＲＮＡ合成酶（ＴｒｐＲＳ） 的相互识别及其结构、功能关系, 纯化了枯草杆菌ＴｒｐＲＳ并用溴化氰活化的Ｓｅｐｈａｒｏｓｅ ４Ｂ 将ＴｒｐＲＳ固定化, 固定化ＴｒｐＲＳ的蛋白质回收率为９５ ．５ ％ , 活力回收率为３１．３％ 。研究了固定化ＴｒｐＲＳ的酶学性质, 其热稳定性和贮存稳定性方面均比液相ＴｒｐＲＳ有了较大的提高, 最适温度、最适ｐＨ 均有一定程度的增大, 工作稳定性良好。以固定化ＴｒｐＲＳ为亲和层析介质, 对含有２０ 个核苷酸随机序列、长度为５６ 个核苷酸的单链ＲＮＡ 随机库进行了３ 轮筛选,ＲＮＡ 群体亲和固定化ＴｒｐＲＳ的比例从４ ．３ ％ 上升至１４ ．７ ％ 。筛选得到了与ｔＲＮＡＴｒｐ 氨基酸接受茎类似的ＲＮＡ二级结构。实验结果表明固定化ＴｒｐＲＳ可以作为ＳＥＬＥＸ 亲和层析介质, 进行模拟ｔＲＮＡＴｒｐ 分子的ＲＮＡ 随机库的ＳＥＬＥＸ 筛选。     

Dissociating quaternary structure regulates cell-signaling functions of a secreted human tRNA synthetase

Many tRNA synthetases are homodimers that are catalytically inactive as monomers. An example is the 528-amino acid human tyrosyl-tRNA synthetase, which is made up of an N-terminal catalytic unit (TyrRS(Mini)) and a 164-amino acid C-domain. Although native TyrRS has no known cytokine functions, natural proteolysis of secreted TyrRS releases TyrRS(Mini), which not only has the same aminoacylation activity as native TyrRS but also has strong activity for stimulating migration of polymorphonuclear leukocytes. The migration-stimulating activity is dependent on an ELR tripeptide motif, similar to that in CXC cytokines like IL-8, and also has the familiar bell-shaped concentration dependence seen for CXC cytokines. Here we show that in contrast to IL-8, where the bell-shaped dependence arises from the effects of CXCR1/2 receptor internalization, TyrRS(Mini) does not induce internalization of CXCR1/2. A rationally designed non-associating monomer and a non-dissociating dimer were constructed. With these constructs, the bell-shaped concentration dependence of leukocyte migration was shown to arise from the agonist (for migration) activity of the catalytically inactive monomer and the antagonist activity of the catalytically active dimer. Thus, the dissociating quaternary structure of TyrRS(Mini) regulates two opposing cytokine activities and suggests the possibility of dissociating quaternary structures regulating novel functions of other tRNA synthetases.     

Selective Inhibition of Bacterial Tryptophanyl-tRNA Synthetases by Indolmycin Is Mechanism-based

Indolmycin is a natural tryptophan analog that competes with tryptophan for binding to tryptophanyl-tRNA synthetase (TrpRS) enzymes. Bacterial and eukaryotic cytosolic TrpRSs have comparable affinities for tryptophan (K_m ∼ 2 μm), and yet only bacterial TrpRSs are inhibited by indolmycin. Despite the similarity between these ligands, Bacillus stearothermophilus (Bs)TrpRS preferentially binds indolmycin ∼1500-fold more tightly than its tryptophan substrate. Kinetic characterization and crystallographic analysis of BsTrpRS allowed us to probe novel aspects of indolmycin inhibitory action. Previous work had revealed that long range coupling to residues within an allosteric region called the D1 switch of BsTrpRS positions the Mg²⁺ ion in a manner that allows it to assist in transition state stabilization. The Mg²⁺ ion in the inhibited complex forms significantly closer contacts with non-bridging oxygen atoms from each phosphate group of ATP and three water molecules than occur in the (presumably catalytically competent) pre-transition state (preTS) crystal structures. We propose that this altered coordination stabilizes a ground state Mg²⁺·ATP configuration, accounting for the high affinity inhibition of BsTrpRS by indolmycin. Conversely, both the ATP configuration and Mg²⁺ coordination in the human cytosolic (H_c)TrpRS preTS structure differ greatly from the BsTrpRS preTS structure. The effect of these differences is that catalysis occurs via a different transition state stabilization mechanism in H_cTrpRS with a yet-to-be determined role for Mg²⁺. Modeling indolmycin into the tryptophan binding site points to steric hindrance and an inability to retain the interactions used for tryptophan substrate recognition as causes for the 1000-fold weaker indolmycin affinity to H_cTrpRS.     

The Dual Role of the 2′-OH Group of A76 tRNATyr in the Prevention of d-tyrosine Mistranslation

Aminoacyl-tRNA-synthetases are crucial enzymes for initiation step of translation. Possessing editing activity, they protect living cells from misincorporation of non-cognate and non-proteinogenic amino acids into proteins. Tyrosyl-tRNA synthetase (TyrRS) does not have such editing properties, but it shares weak stereospecificity in recognition of d-/l-tyrosine (Tyr). Nevertheless, an additional enzyme, d-aminoacyl-tRNA-deacylase (DTD), exists to overcome these deficiencies. The precise catalytic role of hydroxyl groups of the tRNA^Tyr A76 in the catalysis by TyrRS and DTD remained unknown. To address this issue, [³²P]-labeled tRNA^Tyr substrates have been tested in aminoacylation and deacylation assays. TyrRS demonstrates similar activity in charging the 2′ and 3′-OH groups of A76 with l-Tyr. This synthetase can effectively use both OH groups as primary sites for aminoacylation with l-Tyr, but demonstrates severe preference toward 2′-OH, in charging with d-Tyr. In both cases, the catalysis is not substrate-assisted: neither the 2′-OH nor the 3′-OH group assists catalysis. In contrast, DTD catalyzes deacylation of d-Tyr-tRNA^Tyr specifically from the 3′-OH group, while the 2′-OH assists in this hydrolysis.     

Interconversion of ATP binding and conformational free energies by tryptophanyl-tRNA synthetase: structures of ATP bound to open and closed,pre-transition-state conformations

Binding ATP to tryptophanyl-tRNA synthetase (TrpRS) in a catalytically competent configuration for amino acid activation destabilizes the enzyme structure prior to forming the transition state. This conclusion follows from monitoring the titration of TrpRS with ATP by small angle solution X-ray scattering, enzyme activity, and crystal structures. ATP induces a significantly smaller radius of gyration at pH=7 with a transition midpoint at approximately 8mM. A non-reciprocal dependence of Trp and ATP dissociation constants on concentrations of the second substrate show that Trp binding enhances affinity for ATP, while the affinity for Trp falls with the square of the [ATP] over the same concentration range ( approximately 5mM) that induces the more compact conformation. Two distinct TrpRS:ATP structures have been solved, a high-affinity complex grown with 1mM ATP and a low-affinity complex grown at 10mM ATP. The former is isomorphous with unliganded TrpRS and the Trp complex from monoclinic crystals. Reacting groups of the two individually-bound substrates are separated by 6.7A. Although it lacks tryptophan, the low-affinity complex has a closed conformation similar to that observed in the presence of both ATP and Trp analogs such as indolmycin, and resembles a complex previously postulated to form in the closely-related TyrRS upon induced-fit active-site assembly, just prior to catalysis. Titration of TrpRS with ATP therefore successively produces structurally distinct high- and low-affinity ATP-bound states. The higher quality X-ray data for the closed ATP complex (2.2A) provide new structural details likely related to catalysis, including an extension of the KMSKS loop that engages the second lysine and serine residues, K195 and S196, with the alpha and gamma-phosphates; interactions of the K111 side-chain with the gamma-phosphate; and a water molecule bridging the consensus sequence residue T15 to the beta-phosphate. Induced-fit therefore strengthens active-site interactions with ATP, substantially intensifying the interaction of the KMSKS loop with the leaving PP(i) group. Formation of this conformation in the absence of a Trp analog implies that ATP is a key allosteric effector for TrpRS. The paradoxical requirement for high [ATP] implies that Gibbs binding free energy is stored in an unfavorable protein conformation and can then be recovered for useful purposes, including catalysis in the case of TrpRS.     

Alternative splicing creates two new architectures for human tyrosyl-tRNA synthetase

Many human tRNA synthetases evolved alternative functions outside of protein synthesis. These functions are associated with over 200 splice variants (SVs), most of which are catalytic nulls that engender new biology. While known to regulate non-translational activities, little is known about structures resulting from natural internal ablations of any protein. Here, we report analysis of two closely related, internally deleted, SVs of homodimeric human tyrosyl-tRNA synthetase (TyrRS). In spite of both variants ablating a portion of the catalytic core and dimer-interface contacts of native TyrRS, each folded into a distinct stable structure. Biochemical and nuclear magnetic resonance (NMR) analysis showed that the internal deletion of TyrRSΔE2–4 SV gave an alternative, neomorphic dimer interface ‘orthogonal’ to that of native TyrRS. In contrast, the internal C-terminal splice site of TyrRSΔE2–3 prevented either dimerization interface from forming, and yielded a predominantly monomeric protein. Unlike ubiquitous TyrRS, the neomorphs showed clear tissue preferences, which were distinct from each other. The results demonstrate a sophisticated structural plasticity of a human tRNA synthetase for architectural reorganizations that are preferentially elicited in specific tissues.