NMR studies of differences in the conformations and dynamics of ligand complexes formed with mutant dihydrofolate reductases

Abstract

Two mutants of Lactobacillus casei dihydrofolate reductase, Trp 21----Leu and Asp 26----Glu, have been prepared by using site-directed mutagenesis methods, and their ligand binding and structural properties have been compared with those of the wild-type enzyme. 1H, 13C, and 31P NMR studies have been carried out to characterize the structural changes in the complexes of the mutant and wild-type enzymes. Replacement of the conserved Trp 21 by a Leu residue causes a decrease in activity of the enzyme and reduces the NADPH binding constant by a factor of 400. The binding of substrates and substrate analogues is only slightly affected. 1H NMR studies of the Trp 21----Leu enzyme complexes have confirmed the original resonance assignments for Trp 21. In complexes formed with methotrexate and the mutant enzyme, the results indicate some small changes in conformation occurring as much as 14 A away from the site of substitution. For the enzyme-NADPH complexes, the chemical shifts of nuclei in the bound coenzyme indicate that the nicotinamide ring binds differently in complexes with the mutant and the wild-type enzyme. There are complexes where the wild-type enzyme has been shown to exist in solution as a mixture of conformations, and studies on the corresponding complexes with the Trp 21----Leu mutant indicate that the delicately poised equilibria can be perturbed. For example, in the case of the ternary complex formed between enzyme, trimethoprim, and NADP+, two almost equally populated conformations (forms I and II) are seen with the wild-type enzyme but only form II (the one in which the nicotinamide ring of the coenzyme is extended away from the enzyme structure and into the solvent) is observed for the mutant enzyme complex. It appears that the Trp 21----Leu substitution has a major effect on the binding of the nicotinamide ring of the coenzyme. For the Asp 26----Glu enzyme there is a change in the bound conformation of the substrate folate. Further indications that some conformational adjustments are required to allow the carboxylate of Glu 26 to bind effectively to the N1 proton of inhibitors such as methotrexate and trimethoprim come from the observation of a change in the dynamics of the bound trimethoprim molecule as seen from the increased rate of the flipping of the 13C-labeled benzyl ring and the increased rate of the N1-H bond breaking.

13C NMR studies of complexes of Escherichia coli dihydrofolate reductase formed with methotrexate and with folic acid

“…This behaviour contrasts with that which we observed earlier for the corresponding complexes formed with the L. casei enzyme where folate binds in at least two forms (see multiple bound signals in Fig. 3b) [13,21], while MTX binds in a single conformation [12,13].…”

“…By contrast, E. coli DHFR occurs o~ly as a single conformation in its complex with folate [7,11]. This behaviour differs from that observed for the corresponding complex formed with Lactobaciflus casei DHFR., which exists as a single conlbrmation in its MTX complex [12], but occurs in at least two conformations in its binary complex with folate and three conformations in the ternary complex with folate and NADP ÷ [4].…”

A kinetic study of wild-type and mutant dihydrofolate reductases from Lactobacillus casei

“…Two major effects of this substitution are on (i) the rate constant for hydride transfer which decreases 100-fold, becoming the rate-limiting step in steady-state turnover, and (ii) the affinities for N A D P H and NADP+ which decrease by =3.5 and =OS kcal mol-', respectively. These results are consistent with high-resolution N M R spectroscopy studies [Birdsall et al (1989) Biochemistry 28, 13531 suggesting the major structural effects of W21 L mutant are on binding of the nicotinamide moiety of the coenzyme. D i h y d r o f o l a t e reductase (5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1 S.1.3) catalyzes the NADPH-dependent reduction of 7,8-dihydrofolate (H2F)' to 5,6,7,8-tetrahydrofolate (H,F).…”

“…For example, the structures of the Escherichia coli, Lactobacillus casei, and chicken liver enzymes have been determined to 1.7 %, for some binary and ternary complexes (Bolin et al, 1982;Filman et al, 1982;Matthews et al, 1985). High-resolution N M R spectroscopy has been used to study complexes of the L. casei, wild-type DHFR, and single-site mutants (W21L and D26E) with substrates, substrate analogues, and coenzymes (Birdsall et al, 1989). In addition, a complete kinetic scheme for wild-type E. coli DHFR has been derived from pre-steady-state and steady-state kinetics (Fierke et al, 1987).…”

NMR Studies of Ligand Binding to Dihydrofolate Reductase

“…[13, 14, 41±45] Thus, although methotrexate (5) and folate (1) have similar structures, differing only at positions C4 and N10, the pteridine ring of 5 binds to DHFR in a different orientation to that of 1 [41,42] and 7,8-dihydrofolate. [13,43,45] This behavior has been confirmed in subsequent X-ray [16] and NMR structural studies, [27,46] where the pterin ring in the DHFR ± folate complex was found to be turned over by about 1808 relative to its orientation in the DHFR ± MTX complex. The finding that such similar molecules can bind in very different ways emphasizes the value of carrying out detailed structural studies in evaluating inhibitor design.…”