Conformational change of the methionine 20 loop of a of the bound dihydrofolate

We evaluate the pK_a of dihydrofolate (H₂F) at the N⁵ position in three ternary complexes with Escherichia coli dihydrofolate reductase (ecDHFR), namely ecDHFR(NADP⁺:H₂F) in the closed form (1), and the Michaelis complexes ecDHFR(NADPH:H₂F) 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 N⁵ is protonated directly from solution and provides further insights into the mechanism of the substrate protonation.