Multinuclear NMR characterization of two coexisting conformational states of the Lactobacillus casei dihydrofolate reductase−trimethoprim−NADP+ complex

The complex of Lactobacillus casei dihydrofolate reductase with trimethoprim and NADP' exists in solution as a mixture of approximately equal amounts of two slowly interconverting conformational states [Gronenborn, A., Birdsall, B., Hyde, E. I., Roberts, G. C. K., Feeney, J., & Burgen, A. S . V. (1981) Mol. Pharrnacol. 20, 1451. These have now been further characterized by multinuclear NMR experiments, and a partial structural model has been proposed. 'H NMR spectra at 500 MHz show that the environments of six of the seven histidine residues differ between the two conformations. The characteristic 'H and 31P chemical shifts of nuclei of the coenzyme in the two conformations of the complex are identical in analogous complexes formed with a number of trimethoprim analogues, indicating that the nature of the two conformations is the same in each case. The pyrophosphate 31P resonances have been assigned to the two conformations, and integration of the 31P spectrum shows that the ratio of conformation I to conformation I1 varies from 0.4 to 2.3 in the complexes with the various trimethoprim analogues, the ratio for the trimethoprim complex itself being l .2. Transferred NOE experiments, together with the 'H and 13C Timethopr im [2,4-diamino-5-(3,4,5-trimethoxybenzyl)pyrimidine], a valuable antibacterial drug, is an inhibitor of dihydrofolate reductase, and a substantial effort has been devoted to the study of structureactivity relationships among related 2,4-diaminopyrimidine inhibitors of this enzyme [for a review, see Roth & Cheng (1982)l. With the advent of structural information on trimethoprim binding to the enzyme, both from crystallography (Baker et al., 1981, 1983; Matthews et al., 1983; Stammers et al., 1983) and from NMR spectroscopy (Birdsall et al., 1977, 1983; Cayley et al., 1979; Gronenborn et al., 1981a,b,c; Roberts et al., 1981; Roberts, 1983a), it has become possible to begin to interpret these relationships in molecular detail [see, e.g., Roth & Cheng (1982), Li et al. (1982), Baker et al. (1983), Matthews et al. (1983), Birdsall et al. (1983), Roberts (1983a), and Stuart et al. (1983)l. The binding of the coenzyme (NADP' or NADPH)' to form a ternary complex has a substantial effect not only on the affinity of trimethoprim and other inhibitors but also on the structure-activity relationship (Birdsall et al., 1980a,b; Baccanari et al., 1982; Roberts, 1983a). In some cases, these effects have been shown to be accompanied by changes in protein conformation (Roberts, 1983a; Clore et al., 1984). We From the Division of Physical Biochemistry, National Institute for Medical Research, Mill Hill, London NW7 IAA, U.K. Receiued January 27, 1984. *Recipient of an S.E.R.C. C.A.S.E. Studentship. Present address: Roche Products Ltd., Welwyn Garden City, U.K. Recipient of a Ramsay Memorial Fellowship. Present address: Institute of Organic Chemistry, C.S.I.C., Madrid, Spain. Lister Institute Research Fellow. chemical shifts, indicate that in conformation I1 of the complex the nicotinamide ring of the coenzyme has swung away from the enzyme surface into solution; this is made possible by changes in the conformation of the pyrophosphate moiety. In conformation I, by contrast, the nicotinamide ring remains bound to the enzyme. "C and 15N experiments show that trimethoprim is protonated on N1 in both conformations of the ternary complex. Analysis of the 'H chemical shifts of trimethoprim in terms of ring current effects shows that in conformation I of the ternary complex trimethoprim retains the same conformation as in its binary complex, but 13C, 15N, and 19F [using 2,4-diamino-5-(3,5-dimethoxy-4-fluorobenzyl)pyrimidine] experiments show that the environment of both the pyrimidine ring and benzyl ring is affected by the proximity of the coenzyme. Less information is available about the conformation of the inhibitor in conformation I1 of the complex, but its environment is similar to that in the binary enzyme-inhibitor complex. The implications of the existence of these two conformations of the enzyme for understanding cooperativity in binding between NADP' and trimethoprim are briefly discussed. have shown that the ternary complex of trimethoprim and NADP' with the Lactobacillus casei dihydrofolate reductase exists in solution as a mixture of two conformational states (Gronenborn et al., 1981a,b). These two states, which are approximately equally populated, interconvert slowly (ca. 6 s-' at 30 "C) so that separate resonances from the two are observed for several 'H and 31P nuclei in the complex. We were able to show that the mode of interaction of the nicotinamide ring of the coenzyme with the enzyme is appreciably different in the two states. In addition to the significance of this conformational effect for our understanding of structure-binding constant relationships (Gronenborn et al., 1981b; Roberts, 1983a,b), this complex offers a rare opportunity for detailed characterization of a conformational equilibrium in a protein in solution. We now report further NMR experiments that allow us to propose a structural model for the conformational equilibrium and show that the position of the equilibrium is a sensitive function of ligand structure. A preliminary account of part of this work has been presented (Bevan et al., 1983). Materials and Methods Dihydrofolate reductase was isolated and purified from Lactobacillus casei MTX/R as described previously (Dann et al., 1976). Its concentration was determined by assay of Abbreviations: thioNADP+, thionicotinamide adenine dinucleotide phosphate (oxidized); NMN+, nicotinamide mononucleotide (oxidized); PADPR-OMe, 2'-phosphoadenosine 5'-diphosphoribose methyl & r i b side; APADP+, acetylpyridine adenine dinucleotide phosphate; NOE, nuclear Overhauser effect; EDTA, ethylenediaminetetraacetic acid. 0006-2960/84/0423-4733$01 .SO10