THE INFLUENCE OF STARTING COORDINATES IN FREE-ENERGY SIMULATIONS OF LIGAND-BINDING TO DIHYDROFOLATE-REDUCTASE

Molecular dynamics (MD) simulation combined with free energy perturbation (FEP) methods have been used to study the key structural differences and relative free energies for the binding of 6-methyl-N5-deazapterin (N8 protonated) and the 8-substituted compound, 6,8-dimethyl-N5-deazapterin (N3 protonated), to dihydrofolate reductase (DHFR). The free energy changes have been calculated using a variety of initial X-ray coordinates derived from bacterial and vertebrate (including human) DHFRs, and both with and without the reduced cofactor nicotinamide adenine dinucleotide (NADPH) bound. Given a sufficiently long simulation time for the FEP calculations (ca. 200 ps), an structures obtained after mutating 6,8-methyl-N5-deazapterin to 6-methyl-N5-deazapterin exhibited hydrogen bond formation between a backbone carbonyl group of DHFR and H(N8) of 6-methyl-N5-deazapterin, analogous to that found in the X-ray crystal structure of N5-deazafolate (N8 protonated) bound to human DHFR. However, both simulation and experiment suggest this additional H-bonding does not greatly enhance thermodynamic stability, with experiment indicating at most a factor of 2 difference in the relative affinities of the two ligand cations for vertebrate DHFR. Moreover, a binding differential of 10 in favour of the protonated 8-substituted compound is found experimentally for bacterial DHFR. The MD/FEP calculations suggest that the relative cost of ligand desolvation may largely cancel the lowering of free energy obtained in the active site, resulting in predicted binding differences within the range indicated by the vertebrate and bacterial DHFR experiments. However, the theoretical free energy changes could not be obtained with the accuracy required for the rationalization of the observed species dependence. While sampling difficulties are known to be inherent in MD simulation methodologies, these studies with several initial coordinate sets have demonstrated the contribution of coordinate choice to this problem. The results indicate that for demanding protein-ligand binding problems such as this one, the accuracy of the method may be no better than +/- 2 kcal/mol.