Dihydrofolate reductase: multiple conformations and alternative modes of substrate binding

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Abstract
The complex of Lactobacillus casei dihydrofolate reductase with the substrate folate and the coenzyme NADP+ has been shown to exist in solution as a mixture of three slowly interconverting conformations whose proportions are pH-dependent [Birdsall, B., Gronenborn, A. M., Hyde, E. I., Clore, G. M., Roberts, G. C. K., Feeney, J., [Burgen, A.S.V. (1982) Biochemistry 21, 5831]. The assignment of the resonances of all the aromatic protons of the ligand molecules in all three conformational states of the complex has now been completed by using a variety of NMR methods, particularly two-dimensional exchange experiments. The resonances of the nicotinamide protons of the coenzyme and the pteridine 7-proton of the folate have different chemical shifts in the three conformations, in some cases differing by more than 1 ppm. Comparison of the COSY spectra of the complex at low pH (conformation I) and high pH (conformations IIa and IIb) with that of the enzyme-methotrexate-NADP+ complex shows only slight differences in the conformation of the protein. The pattern of chemical shift changes in the ligand and the protein indicates that the structural differences are localized within the active site of the enzyme. Nucear Overhauser effects (NOEs) are observed between the nicotinamide 5- and 6-protons and the methyl resonance of Thr 45 at both low and high pH, indicating that there is no major movement of the nicotinamide ring. By contrast, NOEs are observed between the pteridine 7-proton and the methyl protons of Leu 19 and Leu 27 in conrmations I and IIa but not in conformation IIb. A model is proposed that can qualitatively account for the observed NOEs and ligand 1H chemical shifts. In this model, the folate pteridine ring in conformations I and IIa is bound in a way similar to that observed for the pteridine ring of methotrexate in the crystal structure of the enzyme-methotrexate-NADPH complex. In conformation IIb, however, it has a quite different orientation in the binding site, related to that in states I and IIa by a rotation of about 180.degree. about an axis approxiametly along the C2.sbd.NH2 bond. This latter orientation would account for the observed stereochemistry of reduction of folate by the enzyme. It thus appears that folate is able to bind to the enzyme both in a productive and in a quite different nonproductive mode, while the inhibitor methotrexate binds only in the nonproductive mode.

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