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Published online before print May 1, 2007, 10.1110/ps.062724307
Protein Science (2007), 16:1087-1100. Published by Cold Spring Harbor Laboratory Press. Copyright © 2007 The Protein Society
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Conformational change of the methionine 20 loop of Escherichia coli dihydrofolate reductase modulates pKa of the bound dihydrofolate

Ilja V. Khavrutskii, Daniel J. Price, Jinhyuk Lee, and Charles L. Brooks, III

The Scripps Research Institute, Department of Molecular Biology, TPC6, La Jolla, California 92037, USA

(RECEIVED December 14, 2006; FINAL REVISION March 5, 2007; ACCEPTED March 6, 2007)

We evaluate the pKa 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 pKa is modulated by the Met20 loop fluctuations, providing the largest pKa shift in substates with a "tightly closed" loop conformation; in the "partially closed/open" substates, the pKa 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.

Keywords: molecular dynamics; free-energy perturbation; dihydrofolate; pKa shift; conformational change; dihydrofolate reductase; methionine; NADPH; NADP+ ; flexible loop


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