Abstract
The observation of multiple conformations of a functional loop (termed M20) in the Escherichia coli dihydrofolate reductase (ecDHFR) enzyme triggered the proposition that large-scale motions of protein structural elements contribute to enzyme catalysis. The transition of the M20 loop from a closed conformation to an occluded conformation was thought to aid the rate-limiting release of the products. However, the influence of charged species in the solution environment on the observed M20 loop conformations, independent of charged ligands bound to the enzyme, had not been considered. Molecular dynamics simulations of ecDHFR in model CaCl(2) solutions of varying molar ionic strengths I(M) reveal a substantial free energy barrier between occluded and closed M20 loop states at I(M) exceeding the E. coli threshold (∼0.24 M). This barrier may facilitate crystallization of ecDHFR in the occluded state, consistent with ecDHFR structures obtained at I(M) exceeding 0.3 M. At lower I(M) (≤0.15 M), the M20 loop can explore the occluded state, but prefers an open/partially closed conformation, again consistent with ecDHFR structures. Our findings caution against using ecDHFR structures obtained at nonphysiological ionic strengths in interpreting catalytic events or in structure-based drug design.