Abstract

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.