High pressure NMR reveals active-site hinge motion of folate-bound Escherichia coli dihydrofolate reductase

A high-pressure N-15/H-1 two-dimensional NMR study has been carried out on folate-bound dihydrofolate reductase (DHFR) from Escherichia coli in the pr essure range between 30 and 2000 bar. Several cross-peaks in the N-15/H-1 H SQC spectrum are split into two with increasing pressure, showing the prese nce of a second conformer in equilibrium with the first. Thermodynamic anal ysis of the pressure and temperature dependencies indicates that the second conformer is characterized by a smaller partial molar volume (DeltaV= -25 mL/mol at 15 degreesC) and smaller enthalpy and entropy values, suggesting that the second conformer is more open and hydrated than the first. The spl ittings of the cross-peaks (by similar to1 ppm on N-15 axis at 2000 bar) ar ise from the hinges of the M20 loop, the C-helix, and the F-helix, all of w hich constitute the major binding site for the cofactor NADPH, suggesting t hat major differences in conformation occur in the orientations of the NADP H binding units. The Gibbs free energy of the second, open conformer is 5.2 kJ/mol above that of the first at 1 bar, giving an equilibrium population of about 10%. The second, open conformer is considered to be crucial for NA DPH binding, and the NMR line width indicates that the upper limit for the rate of opening is 20 s(-1) at 2000 bar. These experiments show that high p ressure NMR is a generally useful tool for detecting and analyzing "open" s tructures of a protein that may be directly involved in function.