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.
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 Mg2+ cations in an unusual, bent geometry. The Mg2+ cations play a structural role and are thought to also participate catalytically in the enzyme reaction. Co-binding of the ATP x Mg3(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 Mg2+ 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 tRNA-bound AspRS, the single remaining Mg2+ cation strongly favored the Asp-adenylate substrate relative to Asn-adenylate. Thus, in addition to their structural and catalytic roles, the Mg2+ cations contribute to specificity in AspRS through long range electrostatic interactions with the Asp side chain in both the pre- and post-adenylation states.
Thompson
Damien
D
Laboratoire de Biochimie, CNRS, UMR7654, Department of Biology, Ecole Polytechnique, 91128 Palaiseau, France.
Simonson
Thomas
T
D000249:Adenosine Monophosphate / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D049148:Aminoacylation; D019843:Archaeal Proteins / Q000737:chemistry / Q000378:metabolism; D001216:Asparagine / Q000378:metabolism*; D001226:Aspartate-tRNA Ligase / Q000737:chemistry* / Q000378:metabolism; D001224:Aspartic Acid / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D002413:Cations, Divalent / Q000378:metabolism; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D008274:Magnesium / Q000737:chemistry* / Q000378:metabolism; D019711:Pyrococcus / Q000201:enzymology; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism; D055672:Static Electricity; D013379:Substrate Specificity; D013816:Thermodynamics
D019843:Archaeal Proteins; D002413:Cations, Divalent; D001224:Aspartic Acid; D000249:Adenosine Monophosphate; D001216:Asparagine; D000255:Adenosine Triphosphate; D012343:RNA, Transfer; D001226:Aspartate-tRNA Ligase; D008274:Magnesium