Engineering an Mg2+ site to replace a structurally conserved arginine in the catalytic center of histidyl-tRNA synthetase by computer experiments

Arnez, John G.; Flanagan, Karen; Moras, Dino; Simonson, Thomas

doi:10.1002/(sici)1097-0134(19980815)32:3<362::aid-prot11>3.0.co;2-7

Cited by 14 publications

(9 citation statements)

References 39 publications

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“…However, the strong new interaction made with another α‐phosphate oxygen O5′ compensates for this, and the two substrates remain correctly positioned and oriented for reaction. A similar observation has been made in MD simulations of HisRS5 where the α‐phosphate O2A interaction weakens and becomes water‐mediated. As the preliminary criteria for simulation quality have been met, the ND4 simulation has been extended to 1ns and analyzed in greater detail to confirm the model's validity.…”

Section: Resultssupporting

confidence: 77%

“…In the prior models (ND, ND2, ND3) Arg269 enters the active site to stabilize the γ‐phosphate in the absence of a positive charge on His 270. This residue has a high mobility in the crystal structure, and the equivalent residue of HisRS behaved similarly in simulations 5. This noncatalytic arginine may play a role in aiding the diffusion of pyrophosphate out of the active site.…”

Section: Resultsmentioning

confidence: 84%

“…Molecular dynamics (MD) simulations of the wild‐type enzyme in the ATP bound state are a necessary benchmark for the computational mutant calculations. MD simulations have been reported for of other class II synthetases5 and AspRS 6, 7. These studies employed stochastic boundary conditions, whereby only a 20 Å region around one active site was explicitly solvated, and allowed to move freely, with the rest of the protein harmonically restrained to reduce computational effort.…”

Section: Introductionmentioning

confidence: 99%

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Molecular dynamics simulations of LysRS: An asymmetric state

et al. 2005

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We report molecular dynamics simulations of the Escherichia coli Lysyl-tRNA synthetase LysU isoform carried out as a benchmark for mutant simulations in in silico protein engineering efforts. Unlike previous studies of aminoacyl-tRNA synthetases, LysU is modelled in its full dimeric form with explicit solvent. While developing a suitable simulation protocol, we observed an asymmetry that persists despite improvements to the model. This prediction has directly led to experiments that establish a functional asymmetry in nucleotide binding by LysU. The development of a simulation protocol and validation of the model are presented here. The observed asymmetry is described and the role of protein flexibility in developing the asymmetry is discussed. Proteins 2006; 62:649 -662.

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Section: Resultssupporting

confidence: 77%

Section: Resultsmentioning

confidence: 84%

Section: Introductionmentioning

confidence: 99%

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Molecular dynamics simulations of LysRS: An asymmetric state

et al. 2005

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“…The CHARMM22 forcefield was employed for the protein and the AMP moiety of the ligands 121. The linkage between the AMP and aminoacid moieties of the ligands was parameterized as in Ref 122…”

Section: Methodsmentioning

confidence: 99%

Computational protein design with a generalized born solvent model: Application to asparaginyl‐tRNA synthetase

et al. 2011

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Computational Protein Design (CPD) is a promising method for high throughput protein and ligand mutagenesis. Recently, we developed a CPD method that used a polar-hydrogen energy function for protein interactions and a Coulomb/Accessible Surface Area (CASA) model for solvent effects. We applied this method to engineer aspartyl-adenylate (AspAMP) specificity into Asparaginyl-tRNA synthetase (AsnRS), whose substrate is asparaginyl-adenylate (AsnAMP). Here, we implement a more accurate function, with an all-atom energy for protein interactions and a residue-pairwise generalized Born model for solvent effects. As a first test, we compute aminoacid affinities for several point mutants of Aspartyl-tRNA synthetase (AspRS) and Tyrosyl-tRNA synthetase and stability changes for three helical peptides and compare with experiment. As a second test, we readdress the problem of AsnRS aminoacid engineering. We compare three design criteria, which optimize the folding free-energy, the absolute AspAMP affinity, and the relative (AspAMP-AsnAMP) affinity. The sequences and conformations are improved with respect to our previous, polar-hydrogen/CASA study: For several designed complexes, the AspAMP carboxylate forms three interactions with a conserved arginine and a designed lysine, as in the active site of the AspRS:AspAMP complex. The conformations and interactions are well maintained in molecular dynamics simulations and the sequences have an inverted specificity, favoring AspAMP over AsnAMP. The method is not fully successful, since experimental measurements with the seven most promising sequences show that they do not catalyze at a detectable level the adenylation of Asp (or Asn) with ATP. This may be due to weak AspAMP binding and/or disruption of transition-state stabilization.

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“…6 7 The residues composing the nanospace of the active site stabilize the substrates and favor the progress of the reaction by catalysis. 8 Extensive studies about the active site of aaRSs are carried out, principally using crystallographic methods complemented by biochemical methods, spectroscopic studies, computer simulations [33][34][35][36][37][38][39][40] and electronic structure based analysis. [41][42][43][44][45][46] Despite the wealth of information, the understanding of the principles controlling the organization of the active sites is yet to be completed.…”

Section: Introductionmentioning

confidence: 99%

Active Site Nanospace of Aminoacyl tRNA Synthetase: Difference Between the Class I and Class II Synthetases

Dutta¹,

Choudhury²,

Banik³

et al. 2014

j nanosci nanotechnol

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The present work is aimed at understanding the origin of the difference in the molecular organization of the active site nanospaces of the class I and class II aminoacyl tRNA synthetases (aaRSs) which are tunnel-like structures. The active site encloses the cognate amino acid (AA) and the adenosine triphosphate (ATP) to carry out aminoacylation reaction. Comparison of the structures of the active site of the class I and class II (aaRSs) shows that the nanodimensional tunnels are curved in opposite directions in the two classes. We investigated the origin of this difference using quantum mechanical computation of electrostatic potential (ESP) of substrates, surrounding residues and ions, using Atoms in Molecule (AIM) Theory and charge population analysis. We show that the difference is principally due to the variation in the spatial charge distribution of ATP in the two classes which correspond to extended and bent conformations of ATP. The present computation shows that the most feasible pathway for nucleophilic attack to alphaP is oppositely directed for class I and class II aaRSs. The available crystal structures show that the cognate AA is indeed located along the channel favorable for nucleophilic attack as predicted by the ESP analysis. It is also shown that the direction of the channel changes its orientation when the orientation of ATP is changed from extended to a bent like structure. We further used the AIM theory to confirm the direction of the approach of AA in each case and the results corroborate the results from the ESP analysis. The opposite curvatures of the active site nanospaces in class I and class II aaRSs are related with the influence of the charge distributions of the extended and bent conformations of ATP, respectively. The results of the computation of electrostatic potential by successive addition of active site residues show that their roles on the reaction are similar in both classes despite the difference in the organization of the active sites of class I and class II aaRSs. The difference in mechanism in two classes as pointed out in recent study (S. Dutta Banik and N. Nandi, J. Biomol. Struct. Dyn. 30, 701 (2012)) is related with the fact that the relative arrangement of the ATP with respect to the AA is opposite in class I and class II aaRSs as explained in the present work. The charge population difference between the reacting centers (which are the alphaP atom of ATP (q(p)) and the attacking oxygen atom of carboxylic acid group (q(o)), respectively) denoted by delta(q), is a measure of the propensity of nucleophilic attack. The population analysis of the substrate AA shows that a non-negligible difference exists between the attacking oxygens of AA in class I (syn) and in class II (anti) which is one reason for the lower value of delta(q) in class II relative to class I. The population analysis of the AA, ATP, Mg+2 ions and active site residues shows that the difference in delta(q) values of the two classes is substantially reduced. When ions and residues are considered. Thus, the...

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Engineering an Mg2+ site to replace a structurally conserved arginine in the catalytic center of histidyl-tRNA synthetase by computer experiments

Cited by 14 publications

References 39 publications

Molecular dynamics simulations of LysRS: An asymmetric state

Molecular dynamics simulations of LysRS: An asymmetric state

Computational protein design with a generalized born solvent model: Application to asparaginyl‐tRNA synthetase

Active Site Nanospace of Aminoacyl tRNA Synthetase: Difference Between the Class I and Class II Synthetases

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