Trimethoprim binding to bacterial and mammalian dihydrofolate reductase: a comparison by proton and carbon-13 nuclear magnetic resonance

Publisher Website
Google Scholar
Abstract
The binding of trimethoprim to dihydrofolate reductase from L1210 mouse lymphoma cells was studied by measuring the changes in chemical shift of nuclei of the ligand that accompanies binding. The 6- and 2'',6''-proton chemical shifts of bound trimethoprim were determined by transfer of saturation experiments; the 2-C chemical shift were determined by using [2-13C]trimethoprim. The changes in proton chemical shift were substantially smaller than those accompanying binding to bacterial dihydrofolate reductase. This difference arose largely from the fact that trimethoprim adopted different conformations when bound to mammalian and to bacterial dihydrofolate reductase. The proton chemical shifts were interpreted in terms of ring-current contributions from the 2 aromatic rings of trimethoprim itself and the nearby aromatic amino acid residues of the enzyme. The latter were located by using the refined crystallographic coordinates of the Lactobacillus casei and Escherichia coli reductases in their complexes with methotrexate, under the assumption that, as indicated by the 13C chemical shifts, the diaminopyrimidine ring of trimethoprim binds in the same way as does the corresponding part of methotrexate. With these assumptions, the conformation of trimethoprim bound to the dihydrofolate reductases from L. casei, E. coli and L1210 cells was calculated. Support for the calculated conformations was obtained from the observation of transferred nuclear Overhauser effects between the 6- and 2'',6''-protons. The calculated conformations of trimethoprim bound to the bacterial and mammalian enzymes differed significantly, by .apprx. 25.degree. in each of the 2 torsion angles, leading to a quite different overall shape for the molecule. These results are compared to the available crystallographic information; the origins of the difference in conformation and its implications for understanding the selectivity of trimethoprim are briefly discussed.

This publication has 1 reference indexed in Scilit: