Conformational change of the methionine 20 loop of Escherichia coli dihydrofolate reductase modulates pKa of the bound dihydrofolate.

We evaluate the pK(a) of dihydrofolate (H(2)F) at the N(5) position in three ternary complexes with Escherichia coli dihydrofolate reductase (ecDHFR), namely ecDHFR(NADP(+):H(2)F) in the closed form (1), and the Michaelis complexes ecDHFR(NADPH:H(2)F) in the closed (2) and occluded (3) forms, by performing free energy perturbation with molecular dynamics simulations (FEP/MD). Our simulations suggest that in the Michaelis complex the pK(a) is modulated by the Met20 loop fluctuations, providing the largest pK(a) shift in substates with a "tightly closed" loop conformation; in the "partially closed/open" substates, the pK(a) is similar to that in the occluded complex. Conducive to the protonation, tightly closing the Met20 loop enhances the interactions of the cofactor and the substrate with the Met20 side chain and aligns the nicotinamide ring of the cofactor coplanar with the pterin ring of the substrate. Overall, the present study favors the hypothesis that N(5) is protonated directly from solution and provides further insights into the mechanism of the substrate protonation.