Crystal Structure of Tryptophanyl-tRNA Synthetase Complexed with Adenosine-5′ Tetraphosphate: Evidence for Distributed Use of Catalytic Binding Energy in Amino Acid Activation by Class I Aminoacyl-tRNA Synthetases (original) (raw)
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Journal of Molecular Biology, 2003
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 ¼ 7 with a transition midpoint at , 8 mM. 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 (, 5 mM) that induces the more compact conformation. Two distinct TrpRS:ATP structures have been solved, a high-affinity complex grown with 1 mM ATP and a low-affinity complex grown at 10 mM 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 6.7 Å . 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 (2.2 Å ) 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 a and g-phosphates; interactions of the K111 side-chain with the g-phosphate; and a water molecule bridging the consensus sequence residue T15 to the b-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.
2014
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 tran-sition midpoint at,8 mM. 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 (,5 mM) that induces the more compact conformation. Two distinct TrpRS:ATP structures have been solved, a high-affinity complex grown with 1 mM ATP and a low-affinity complex grown at 10 mM ATP. The former is isomorphous with unliganded TrpRS and the Trp complex from mono-clinic crystals. React...
Protein Science, 2008
The crystal structure of ligand-free tryptophanyl-tRNA synthetase~TrpRS! was solved at 2.9 Å using a combination of molecular replacement and maximum-entropy map0phase improvement. The dimeric structure~R ϭ 23.7, R free ϭ 26.2! is asymmetric, unlike that of the TrpRS tryptophanyl-59AMP 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 aA-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.
Computational Studies of Tryptophanyl-tRNA Synthetase: Activation of ATP by Induced-Fit
Journal of Molecular Biology, 2006
Catalysis of amino acid activation by Bacillus stearothermophilus tryptophanyl-tRNA synthetase involves three allosteric states: (1) Open; (2) closed pre-transition state (PreTS); and (3) closed products (Product). The interconversions of these states entail significant domain motions driven by ligand binding. We explore the application of molecular dynamics simulations to investigate ligand-linked conformational stability changes associated with this catalytic cycle. Multiple molecular dynamics trajectories (5 ns) for 11 distinct liganded and unliganded monomer configurations show that the PreTS conformation is unstable in the absence of ATP, reverting within ∼600 ps nearly to the Open conformation. In contrast, Open and Product state trajectories were stable, even without ligands, confirming the previous suggestion that catalysis entails destabilization of the protein conformation, driven by ATP-binding energies developed as the PreTS state assembles during induced-fit. The simulations suggest novel mechanistic details associated with both induced-fit (Open-PreTS) and catalysis (PreTS-Product). Notably, Mg 2+-ATP interactions are coupled to interactions between ATP and active-site lysine side-chains via mechanisms that cannot be captured under the molecular mechanics approximations, and which therefore require restraining potentials for stable simulation. Simulations of Mg 2+ •ATP-bound PreTS complexes with restraining potentials and with a virtual K111A mutant confirm that these coupling interactions are necessary to sustain the PreTS conformation and, in turn, provide a new model for how the PreTS conformation activates ATP for catalysis. These results emphasize the central role of the PreTS state as a high-energy intermediate structure along the catalytic pathway and suggest that Mg 2+ and the KMSKS loop function cooperatively during catalysis.
Structure, 2007
B. stearothermophilus tryptophanyl-tRNA synthetase catalysis proceeds via high-energy protein conformations. Unliganded MD trajectories of the Pre-Transition-state complex with Mg 2+ •ATP and the (post) transition-state analog complex with adenosine tetraphosphate relax rapidly in opposite directions, the former regressing, the latter progressing along the structural reaction coordinate. The two crystal structures (RMSD 0.7 Å) therefore lie on opposite sides of a conformational free energy maximum as the chemical transition state forms. SNAPP analysis illustrates the complexity of the associated long-range conformational coupling. Switching interactions in four non-polar core regions are locally isoenergetic throughout the transition. Different configurations, however, propagate their effects to unfavorable, longer-range interactions at the molecular surface. Designed mutation shows that switching interactions enhance the rate, perhaps by destabilizing the ground state immediately before the transition state and limiting non-productive diffusion before and after the chemical transition state, thereby reducing the activation entropy. This paradigm may apply broadly to energytransducing enzymes.
Structure, 1995
Background: Tryptophanyl-tRNA synthetase (TrpRS) catalyzes activation of tryptophan by ATP and transfer to tRNATrP, ensuring translation of the genetic code for tryptophan. Interest focuses on mechanisms for specific recognition of both amino acid and tRNA substrates. Results: Maximum-entropy methods enabled us to solve the TrpRS structure. Its three parts, a canonical dinucleotide-binding fold, a dimer interface, and a helical domain, have enough structural homology to tyrosyl-tRNA synthetase (TyrRS) that the two enzymes can be described as conformational isomers. Structurebased sequence alignment shows statistically significant genetic homology. Structural elements interacting with the activated amino acid, tryptophanyl-5'AMP, are almost exactly as seen in the TyrRS:tyrosyl-5'AMP complex. Unexpectedly, side chains that recognize indole are also highly conserved, and require reorientation of a 'specificity-determining' helix containing a conserved aspartate to assure selection of tryptophan versus tyrosine. The carboxy terminus, which is disordered and therefore not seen in TyrRS, forms part of the dimer interface in TrpRS. Conclusions: For the first time, the Bayesian statistical paradigm of entropy maximization and likelihood scoring has played a critical role in an X-ray structure solution. Sequence relatedness of structurally superimposable residues throughout TrpRS and TyrRS implies that they diverged more recently than most aminoacyl-tRNA synthetases. Subtle, tertiary structure changes are crucial for specific recognition of the two different amino acids. The conformational isomerism suggests that movement of the KMSKS loop, known to occur in the TyrRS transition state for amino acid activation, may provide a basis for conformational coupling during catalysis.
Journal of Molecular Biology, 1996
A semi-conserved tryptophan residue of Bacillus subtilis tryptophanyl-tRNA synthetase (TrpRS) was previously asserted to be an essential University of Ottawa, 451 residue and directly involved in tRNA Trp binding and recognition. The Smyth Road, Ottawa crystal structure of the Bacillus stearothermophilus TrpRS tryptophanyl-5'-Ontario, Canada K1H 8M5 adenylate complex (Trp-AMP) shows that the corresponding Trp91 is 2 Department of Biochemistry buried and in the dimer interface, contrary to the expectations of the earlier and Biophysics CB 7260 assertation. Here we examine the role of this semi-conserved tryptophan University of North Carolina residue using fluorescence spectroscopy. B. subtilis TrpRS has a single at Chapel Hill, Chapel Hill tryptophan residue, Trp92. 4-Fluorotryptophan (4FW) is used as a NC 27599-7260, USA non-fluorescent substrate analog, allowing characterization of Trp92 fluorescence in the 4-fluorotryptophanyl-5'-adenylate (4FW-AMP) TrpRS 3 Department of Biochemistry complex. Complexation causes the Trp92 fluorescence to become quenched University of Toronto by 70%. Titrations, forming this complex under irreversible conditions, Toronto, Ontario, Canada show that this quenching is essentially complete after half of the sites are M5S 1A8 filled. This indicates that a substrate-dependent mechanism exists for the 4 The Department of inter-subunit communication of conformational changes. Trp92 fluor-Chemistry and Biochemistry escence is not efficiently quenched by small solutes in either the apo-or University of Windsor complexed form. From this we conclude that this tryptophan residue is not Windsor, Ontario, Canada solvent exposed and that binding of the Trp92 to tRNA Trp is unlikely. N9B 3P4 Time-resolved fluorescence indicates conformational heterogeneity of B. subtilis Trp92 with the fluorescence decay being best described by three discrete exponential decay times. The decay-associated spectra (DAS) of the apo-and complexed-TrpRS show large variations of the concentration of individual fluorescence decay components. Based on recent correlations of these data with changes in the local secondary structure of the backbone containing the fluorescent tryptophan residue, we conclude that changes observed in Trp92 time-resolved fluorescence originate primarily from large perturbations of its local secondary structure. The quenching of Trp92 in the 4FW-AMP complex is best explained by the crystal structure conformation, in which the tryptophan residue is found in an a-helix. The amino acid residue cysteine is observed clearly within the quenching radius (3.6 Å) of the conserved tryptophan residue. These tryptophan and cysteine residues are neighbors, one helical turn
2015
Abstract: The correct amino acid sequence of E. coli isoleucyl-tRNA synthetase (IleRS) was established by means of peptide mapping by MALDI mass spectrometry, using a set of four endoproteases (trypsin, LysC, AspN and GluC). Thereafter, the active site of IleRS was mapped by affinity labeling with reactive analogs of the substrates. For the ATP binding site, the affinity labeling reagent was pyridoxal 5'-diphospho-5'-adenosine (ADP-PL), whereas periodate-oxidized tRNAIle, the 2',3'-dialdehyde derivative of tRNAIle was used to label the binding site for the 3'-end of tRNA on the syn-thetase. Incubation of either reagent with IleRS resulted in a rapid loss of both the tRNAIle aminoacylation and isoleucine-dependent isotopic ATP-PPi exchange activities. The stoichiometries of IleRS labeling by ADP-PL or tRNAIleox corre-sponded to 1 mol of reagent incorporated per mol of enzyme. Altogether, the oxidized 3'-end of tRNAIle and the pyridoxal moiety of the ATP ...
The 2 Å crystal structure of leucyl-tRNA synthetase and its complex with a leucyl-adenylate analogue
The EMBO Journal, 2000
Leucyl-, isoleucyl-and valyl-tRNA synthetases are closely related large monomeric class I synthetases. Each contains a homologous insertion domain of~200 residues, which is thought to permit them to hydrolyse (`edit') cognate tRNA that has been mischarged with a chemically similar but non-cognate amino acid. We describe the ®rst crystal structure of a leucyl-tRNA synthetase, from the hyperthermophile Thermus thermophilus, at 2.0 A Ê resolution. The overall architecture is similar to that of isoleucyl-tRNA synthetase, except that the putative editing domain is inserted at a different position in the primary structure. This feature is unique to prokaryote-like leucyl-tRNA synthetases, as is the presence of a novel additional exibly inserted domain. Comparison of native enzyme and complexes with leucine and a leucyladenylate analogue shows that binding of the adenosine moiety of leucyl-adenylate causes signi®cant conformational changes in the active site required for amino acid activation and tight binding of the adenylate. These changes are propagated to more distant regions of the enzyme, leading to a signi®cantly more ordered structure ready for the subsequent aminoacylation and/or editing steps.