Molecular Pharmacology, Vol 20, 145-153, Copyright © 1981 by the American Society for Pharmacology and Experimental Therapeutics

¹H and ³¹P NMR Characterization of Two Conformations of the Trimethoprim-NADP⁺-Dihydrofolate Reductase Complex

¹ Division of Molecular Pharmacology, National Institute for Medical Research, London NW7 1AA, England

The ternary complex between Lactobacillus casei dihydrofolate reductase, the coenzyme NADP⁺, and the antibacterial drug trimethoprim was studied by ¹H and ³¹P NMR spectroscopy. The C₂-H resonances of two of the histidine residues of the protein were each split into two signals of approximately half-proton intensity in the ¹H spectrum of this complex. Studies of the temperature-dependence of the lineshape of these histidine signals showed that the splitting is due to the coexistence of approximately equal amounts of two slowly interconverting (6 sec^-1 at 31°) conformational forms of the complex. Two sets of proton resonances from the bound coenzyme were identified by the use of selectively deuterated coenzyme and by transfer of saturation experiments. Conformation I was characterized by nicotinamide proton resonances shifted substantially (0.6-1.1 ppm) to low field from their positions in the free coenzyme, while in Conformation II the changes in chemical shift on binding were much smaller (0.12 ppm). Only a single set of ¹H resonances from the bound trimethoprim was observed in transfer of saturation experiments at 45°, perhaps because of relatively rapid interconversion between the two conformational states at this temperature. However, the addition of NADP⁺ produced a large (1.1 ppm) upfield shift of the 2',6'-proton resonance of trimethoprim relative to its position in the binary complex. In the ³¹P spectrum of the bound coenzyme, two sets of signals were seen for the pyrophosphate phosphorus nuclei. As judged from both the ¹H and ³¹P spectra, the complexes of enzyme, trimethoprim, and NADP⁺ or NHDP⁺ (the hypoxanthine analogue) are mixtures of Conformations I and II, whereas the complexes formed with the thionicotinamide or acetylpyridine analogues of the coenzymes (TNADP⁺ and APADP⁺) are exclusively in Conformation II. The enzyme-methotrexate-NADP⁺ complex is exclusively in Conformation I. Comparing the ³¹P spectra of the enzyme-trimethoprim-TNADP⁺ and enzyme-methotrexate-NADP⁺ complexes, the two conformational states were found to differ in the conformation of the pyrophosphate backbone of the bound coenzyme, as indicated by the ³¹P-¹H and ³¹P-³¹P spin-spin coupling constants. This system appears to be an example of a two-state conformational equilibrium which can be "switched" by the binding of ligands of different structure. The nature of the two conformational states and their implications for structure-activity analysis are discussed.

Note:
ACKNOWLEDGMENTS We are grateful to Gill Ostler and John McCormick for invaluable technical assistance, to Dr. David Matthews for sending us the atomic coordinates derived from his crystallographic work, and to the Oxford Enzyme Group, particularly Iain Campbell and Peter Stiles, for the opportunity to use their 470 MHz spectrometer.