Functional asymmetry in the lysyl-tRNA synthetase explored by molecular dynamics, free energy calculations and experiment

Hughes, Samantha Jane; Tanner, Julian A.; Hindley, Alison D.; Miller, Andrew D.; Gould, Ian R.

doi:10.1186/1472-6807-3-5

Cited by 24 publications

(6 citation statements)

References 59 publications

(85 reference statements)

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“…The concentration of LysRS2 was determined by active site titration as described previously (24), except that [ 14 C]tyrosine was replaced with [ 14 C]lysine, and reactions were performed for 10 min. Calculations were based upon half of the sites' reactivity for E. coli LysRS (25). B. burgdorferi LysRS1 was prepared as described previously (16).…”

Section: Methodsmentioning

confidence: 99%

Divergence in Noncognate Amino Acid Recognition between Class I and Class II Lysyl-tRNA Synthetases

Levengood¹,

Ataide²,

Roy

et al. 2004

Journal of Biological Chemistry

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Lysine insertion during coded protein synthesis requires lysyl-tRNA Lys , which is synthesized by lysyl-tRNA synthetase (LysRS). Two unrelated forms of LysRS are known: LysRS2, which is found in eukaryotes, most bacteria, and a few archaea, and LysRS1, which is found in most archaea and a few bacteria. To compare amino acid recognition between the two forms of LysRS, the effects of L-lysine analogues on aminoacylation were investigated. Both enzymes showed stereospecificity toward the L-enantiomer of lysine and discriminated against noncognate amino acids with different R-groups (arginine, ornithine). Lysine analogues containing substitutions at other positions were generally most effective as inhibitors of LysRS2. For example, the K i values for aminoacylation of S-(2-aminoethyl)-L-cysteine and L-lysinamide were over 180-fold lower with LysRS2 than with LysRS1. Of the other analogues tested, only ␥-aminobutyric acid showed a significantly higher K i for LysRS2 than LysRS1. These data indicate that the lysinebinding site is more open in LysRS2 than in LysRS1, in agreement with previous structural studies. The physiological significance of divergent amino acid recognition was reflected by the in vivo resistance to growth inhibition imparted by LysRS1 against S-(2-aminoethyl)-L-cysteine and LysRS2 against ␥-aminobutyric acid. These differences in resistance to naturally occurring noncognate amino acids suggest the distribution of LysRS1 and LysRS2 contributes to quality control during protein synthesis. In addition, the specific inhibition of LysRS1 indicates it is a potential drug target.

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Section: Methodsmentioning

confidence: 99%

Divergence in Noncognate Amino Acid Recognition between Class I and Class II Lysyl-tRNA Synthetases

Levengood¹,

Ataide²,

Roy

et al. 2004

Journal of Biological Chemistry

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“…For LysRS, one could measure the effect of Mg 2ϩ on the Lys affinity (predicted here to be small) using an established fluorescence assay (58). For both AspRS and LysRS, we predicted that ATP is always bound with three Mg 2ϩ cations, so that the rate constant, k cat , for the chemical adenylation step should be independent of Mg 2ϩ concentration.…”

Section: Discussionmentioning

confidence: 99%

Molecular Dynamics Simulations Show That Bound Mg2+ Contributes to Amino Acid and Aminoacyl Adenylate Binding Specificity in Aspartyl-tRNA Synthetase through Long Range Electrostatic Interactions

Thompson

Simonson

2006

Journal of Biological Chemistry

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Molecular recognition between the aminoacyl-tRNA synthetase enzymes and their cognate amino acid ligands is essential for the faithful translation of the genetic code. In aspartyl-tRNA synthetase (AspRS), the co-substrate ATP binds preferentially with three associated Mg 2؉ cations in an unusual, bent geometry. The Mg 2؉ cations play a structural role and are thought to also participate catalytically in the enzyme reaction. Co-binding of the ATP⅐Mg 3 2؉ complex was shown recently to increase the Asp/Asn binding free energy difference, indicating that amino acid discrimination is substrate-assisted. Here, we used molecular dynamics free energy simulations and continuum electrostatic calculations to resolve two related questions. First, we showed that if one of the Mg 2؉ cations is removed, the Asp/Asn binding specificity is strongly reduced. Second, we computed the relative stabilities of the three-cation complex and the 2-cation complexes. We found that the 3-cation complex is overwhelmingly favored at ordinary magnesium concentrations, so that the protein is protected against the 2-cation state. In the homologous LysRS, the 3-cation complex was also strongly favored, but the third cation did not affect Lys binding. In tRNAbound AspRS, the single remaining Mg 2؉ cation strongly favored the Asp-adenylate substrate relative to Asn-adenylate. Thus, in addition to their structural and catalytic roles, the Mg 2؉ cations contribute to specificity in AspRS through long range electrostatic interactions with the Asp side chain in both the pre-and post-adenylation states.

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“…23 Subsequent work has had more variable outcomes, although for the most part binding energies could still be regarded as being in reasonable agreement with experiment. [24][25][26] However, these studies have been on, at most, a handful of related inhibitors to a single target. The results presented here represent, to the authors' knowledge, the largest application of MM-PBSA to date and consider both ligand and receptor diversity.…”

Section: Mm-pbsamentioning

confidence: 99%

Can MM‐PBSA calculations predict the specificities of protein kinase inhibitors?

Page

Bates

2006

J Comput Chem

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An application of the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) protocol to the prediction of protein kinase inhibitor selectivity is presented. Six different inhibitors are placed in equivalent orientations in each of six different receptors. Fully solvated molecular dynamics is then run for 1 ns on each of the 36 complexes, and the resulting trajectories scored, using the implicit solvent model. The results show some correlation with experimentally-determined specificities; anomalies may be attributed to a variety of causes, including difficulties in quantifying induced fit penalties and variabilities in normal modes calculations. Decomposing interaction energies on a per-residue basis yields more useful insights into the natures of the binding modes and suggests that the real value of such calculations lies in understanding interactions rather than outright prediction.

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Functional asymmetry in the lysyl-tRNA synthetase explored by molecular dynamics, free energy calculations and experiment

Cited by 24 publications

References 59 publications

Divergence in Noncognate Amino Acid Recognition between Class I and Class II Lysyl-tRNA Synthetases

Divergence in Noncognate Amino Acid Recognition between Class I and Class II Lysyl-tRNA Synthetases

Molecular Dynamics Simulations Show That Bound Mg2+ Contributes to Amino Acid and Aminoacyl Adenylate Binding Specificity in Aspartyl-tRNA Synthetase through Long Range Electrostatic Interactions

Can MM‐PBSA calculations predict the specificities of protein kinase inhibitors?

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