INSIGHT INTO THE SPECIFICITY OF THYMIDYLATE SYNTHASE FROM MOLECULAR-DYNAMICS AND FREE-ENERGY PERTURBATION CALCULATIONS

Molecular dynamics and free energy perturbation calculations have been used to calculate the relative free energies of binding of 2'-deoxyur idine 5'-monophosphate (dUMP) and 2'-deoxycytidine S-monophosphate (dC MP) to thymidylate synthase (TS) and two asparagine 229 mutants. Calcu lations qualitatively reproduce experimentally observed dissociation c onstants of the protein-nucleotide complexes. Furthermore, they provid e insight into structural aspects of binding and catalysis of these tw o nucleotides to the protein. The simulations of the wild-type TS comp lexes with dUMP and dCMP support the key role of asparagine 229 in cau sing tighter binding of dUMP than dCMP; repulsion between the base of dCMP and the asparagine 229 side chain reduces the Delta G of binding to the protein from that found in aqueous solution and causes the disp lacement of this nucleotide into a position unsuitable for reaction. T he free energy calculations of the aspartate 229 mutant of TS interact ing with either dUMP or dCMP suggest a synergism between the aspartate 229 side chain and the vicinal histidine 199 in binding. The best agr eement between the calculated and the experimental Delta Delta G of bi nding has been obtained when the aspartate side chain is anionic and t he histidine 199 is either protonated or in its delta H tautomer. Unde r these conditions, dUMP and dCMP are both properly positioned for nuc leophilic attack. In contrast, calculations with a neutral aspartic ac id side chain suggest a strong discriminating power of the neutral 229 side chain in binding the two nucleotides, the preferred one dependin g on which of the two oxygens of the aspartate is protonated. We specu late that protonation of the aspartate 229 side chain can be the key t o rationalizing why the aspartate 229 mutant selectively methylates dC MP. Finally, calculations of the valine 229 mutant demonstrate that su bstitution of the polar asparagine side chain with a hydrophobic resid ue 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 po sitioned for nuclephilic attack than dCMP.