Specific amino acid recognition by aspartyl-tRNA synthetase studied by free energy simulations
Ημερομηνία
1998Source
Journal of Molecular BiologyVolume
275Issue
5Pages
823-846Google Scholar check
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Εμφάνιση πλήρους εγγραφήςΕπιτομή
Specific amino acid binding by aminoacyl-tRNA synthetases is necessary for correct translation of the genetic code. To obtain insight into the origin of the specificity, the binding to aspartyl-tRNA synthetase (AspRS) of the negatively charged substrate aspartic acid and the neutral analogue asparagine was compared by use of molecular dynamics and free energy simulations. Simulations of the Asn-AspRS complex showed that although Asn cannot bind in the same position as Asp, several possible positions exist 1.5 to 2 Å away from the Asp site. The binding free energy of Asn in three of these positions was compared to that of Asp through alchemical free energy simulations, in which Asp is gradually mutated into Asn in the complex with the enzyme. To correctly account for the electrostatic interactions in the system (including bulk solvent), a recently developed hybrid approach was used, in which the region of the mutation site is treated microscopically, whereas distant protein and solvent are treated by continuum electrostatics. Seven free energy simulations were performed in the protein and two in solution. The various Asn positions and orientations sampled at the Asn endpoints of the protein simulations yielded very similar free energy differences. The calculated Asp→Asn free energy change is 79.8(± 1.5) kcal/mol in solution and 95.1(± 2.8) kcal/mol in the complex with the protein. Thus, the substrate Asp is predicted to bind much more strongly than Asn, with a binding free energy difference of 15.3 kcal/mol. This implies that erroneous binding of Asn by AspRS is highly improbable, and cannot account for any errors in the translation of the genetic code. Almost all of the protein contributions to the Asp versus Asn binding free energy difference arise from an arginine and a lysine residue that hydrogen bond to the substrate carboxylate group and an Asp and a Glu that hydrogen bond to these all four amino acid residues are completely conserved in AspRSs. The protein effectively 'solvates' the Asp side-chain more strongly than water does. The simulations were analyzed to determine the interactions that Asn is able to make in the binding pocket, and which sequence differences between AspRS and the highly homologous AsnRS are important for modifying the amino acid specificity. A double or triple mutation of AspRS that could make it specific for Asn was proposed, and supported by preliminary simulations of a mutant complex.
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