Trimethoprim binding to Lactobacillus casei dihydrofolate reductase: a carbon-13 NMR study using selectively carbon-13-enriched trimethoprim

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Abstract
We have measured the 13C chemical shifts for trimethoprim molecules selectively enriched with 13C at the 2-, 4-, 5-, 6-, and 7-positions and the p-OCH3 position in their complexes with Lactobacillus casei dihydrofolate reductase in the presence and absence of coenzyme analogues. The C2 carbon shifts indicate that the pyrimidine ring is protonated at N1 in all the complexes of trimethoprim with the enzyme and coenzymes and in each case the pyrimidine ring is binding in a similar way to that of the corresponding part of methotrexate in the enzyme-methotrexate complex. The C6 carbon of trimethoprim shows a large upfield shift in all complexes (3.51 to 4.70 ppm) but no shift in the complex of 2,4-diaminopyrimidine with the enzyme: these shifts probably arise from steric interactions between the C1'' and C2'' carbons and the H6 proton, which approach van der Waals contact in the folded conformation adopted by trimethoprim when bound to the enzyme. The large shift observed for C6 in all complexes indicates that the basic folded conformation is present in all of them. A comparison of the 13C shifts in the enzyme-trimethoprim-NADPH complex with those in the enzyme-trimethoprim binary complex shows substantial changes even for carbons such as C6 and p-OCH3 (0.46 and-0.36 ppm, respectively), which are remote from the coenzyme: these are caused by ligand-induced conformational changes that may involve displacement of the helix containing residues 42-49. In the ternary complex with NADP+, the two conformational states previously described are further characterized: separate signals are seen for conformations I and II for the C2, C4, C5, C6, and C7 carbons. One set of chemical shifts has values similar to those measured in the binary complex with trimethoprim and also to those in the ternary complex with the methyl .beta.-riboside of 2''-phosphoadenosine 5''-(diphosphoribose); these are assigned to conformation II of the complex. The complex of [m-methoxy-13C]brodimoprim [[3''-methoxy-13C]-or [5''-methoxy-13C]-2,4-diamino-5-(3'',5''-dimethoxy-4''-bromobenzyl)pyrimidine] with the enzyme shows two equal-intensity 13C signals at 274 K, which coalesce to a single absorption when the temperature is raised to 287 K. This two-site exchange between nuclei at the C3'' and C5'' positions has been characterized in terms of "ring flipping", and the rate of this process can be estimated to be 65 .+-. 8 s-1 at 287 K. For the ternary complex with NADP+, the 13C spectrum showed the same coalescence temperature (287 K) as observed for the binary complex: at this temperature the ternary complex is predominantly in form II (75%).

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