Insight into the specificity of Thymidylate synthase from molecular dynamics and free-energy perturbation calculations

Molecular dynamics and free energy pertubation calculations have been used to calculate the relative free energies of binding of 2′-deoxyuridine 5′-monophosphate (dUMP) and 2′-deoxycytidine 5′-monophosphate (dCMP) to thymidylate synthase (TS) and two asparagine 229 mutants. Calculations qualitatively reproduce experimentally observed dissociation constants of the protein-nucleotide complexes. Furthermore, they provide insight into structural aspects of binding and catalysis of these nucleotides to the protein. The simulations of the wild-type TS complexes with dUMP and dCMP support the key role asparagine 229 in causing tighter binding of dUMP than dCMP; repulsion between the base of dCMP support the key role of asparagine 229 side chain reduces the ΔG of binding to the protein from that found in aqueous solution and causes the displacement of this nucleotide into a position unsuitable for reaction. The free energy calculation of the aspartate 229 mutant of TS interacting with either dUMP or dCMP suggest a synergism between the aspartate 229 side chain and the vicinal histidine 199 in binding. The best agreement between the calculated and the experimental ΔΔG of binding has been obtained when the aspartate side chain is anionic and the histidine 199 is either protonated or in its δH tautomer. Under these conditons, dUMP and dCMP are both properly positioned for nucleophilic attack. In contrast, calculations with a neutral aspartic acid side chain suggest a strong discriminating power of the neutral 229 side chain in binding the two nucleotides, the preferred one depending on which of the two oxygens of the aspartate is protonated. We speculate that protonation of the aspartate 229 side chain can be the key to rationalizing why the aspartate 229 mutant selectively methylates dCMP. Finally, calculations of the valine 229 mutant demonstrate that substitution of the polar asparagine side chain with a hydrophobic residue does not result in a significant change in the location of the two nucleotides in the active site, except that dUMP seems to be better positioned for nuclephilic attack than dCMP.