MOLECULAR-DYNAMICS FREE-ENERGY PERTURBATION STUDY ON THE RELATIVE AFFINITIES OF THE BINDING OF REDUCED AND OXIDIZED NADP TO DIHYDROFOLATE-REDUCTASE

The free energy perturbation (FEP) method, implemented within the molecular dynamics (MD) simulation scheme, has been used to investigate the difference between the binding of reduced and oxidized nicotinamide adenine dinucleotide phosphate (NADP) cofactor to Escherichia coli dihydrofolate reductase (DHFR) (binary complex) and E. coli DHFR bound to the substrate dihydrofolate (ternary complex) in aqueous solution. The FEP results for the binary complex predict relative equilibrium binding constants for the reduced and oxidized forms of NADP to E. coli DHFR in good agreement with the available experimental data, suggesting that reduced NADP binds some 10(2) times more strongly than oxidized NADP. The FEP results also predict reduced NADP in the ternary complex with dihydrofolate to bind more strongly than oxidized NADP. However, this differential is calculated to be 10(2)-10(3) times greater than in the binary complex. Although there is no direct experimental information for binding in the active ternary complex with which to compare these results, available results are discussed in the context of the molecular form of active complexes seen kinetically compared with those in the theoretical simulations. The question of the influence of the choice of initial enzyme coordinates and configuration space sampling in these simulations is also discussed. The stronger binding affinity of reduced NADP and the differences between NADP binding strengths computed for the binary and ternary complexes are correlated with solvation effects and structural differences between the complexes. Analysis of the MD structures and available crystallographic data suggests that the positioning of a mobile loop (the "Met-20 loop") plays a key role in determining the relative cofactor binding strengths.