Binding free energies and free energy components from molecular dynamics and Poisson-Boltzmann calculations. Application to amino acid recognition byaspartyl-tRNA synthetase

Specific amino acid binding by aminoacyl-tRNA synthetases (aaRS) is necessa ry for correct translation of the genetic code. Engineering a modified spec ificity into aminoacyl-tRNA synthetases has been proposed as a means to inc orporate artificial amino acid residues into proteins in vine. In a previou s paper, the binding to aspartyl-tRNA synthetase of the substrate Asp and t he analogue Asn were compared by molecular dynamics free energy simulations . Molecular dynamics combined with Poisson-Boltzmann free energy calculatio ns represent a less expensive approach, suitable for examining multiple act ive site mutations in an engineering effort. Here, Poisson-Boltzmann free e nergy calculations for aspartyl-tRNA synthetase are first validated by thei r ability to reproduce selected molecular dynamics binding free energy diff erences, then used to examine the possibility of Asn binding to native and mutant aspartyl-tRNA synthetase. A component analysis of the Poisson-Boltzm ann free energies is employed to identify specific interactions that determ ine the binding affinities. The combined use of molecular dynamics free ene rgy simulations to study one binding process thoroughly, followed by molecu lar dynamics and Poisson-Boltzmann free energy calculations to study a seri es of related ligands or mutations is proposed as a paradigm for protein or ligand design. The binding of Asn in an alternate, "head-to-tail" orientation observed in the homologous asparagine synthetase is analyzed, and found to be more stab le than the "Asp-like" orientation studied earlier. The new orientation is probably unsuitable for catalysis. A conserved active site lysine (Lys198 i n Escherichia coli) that recognizes the Asp side-chain is changed to a leuc ine residue, found at the corresponding position in asparaginyl-tRNA synthe tase. It is interesting that the binding of Asp is calculated to increase s lightly (rather than to decrease), while that of Asn is calculated, as expe cted, to increase strongly, to the same level as Asp binding. Insight into the origin of these changes is provided by the component analyses. The doub le mutation (K198L,D233E) has similar effect, while the triple mutation (K1 98L,Q199E,D233E) reduces Asp binding strongly. No binding measurements are available, but the three mutants are known to have no ability to adenylate Asn, despite the "Asp-like" binding affinities calculated here. in molecula r dynamics simulations of all three mutants, the Asn ligand backbone shifts by 1-2 Angstrom compared to the experimental Asp:AspRS complex, and signif icant side-chain rearrangements occur around the pocket. These could reduce the ATP binding constant and/or the adenylation reaction rate, explaining the lack of catalytic activity in these complexes. Finally, Asn binding to AspRS with neutral K198 or charged H449 is considered, and shown to be less favorable than with the charged K198 and neutral H449 used in the analysis . (C) 2001 Academic Press.