ИСТИНА

Human dihydrofolate reductase is a 21.3 kDa (186 amino acids) enzyme that catalyses the NADPH-dependent reduction of folate and 7,8-dihydrofolate to 5,6,7,8-tetrahydrofolate (H4Fol). Since H4Fol is an important cofactor in the biosynthesis of purines and amino acids, DHFR has proved to be an excellent target for antifolate drugs that act by inhibiting the enzyme in parasitic or malignant cells. The antibacterial effectiveness of one of such drug, trimethoprim is due to its significant increased binding to the bacterial enzyme compared with the binding to the vertebrate form. This specificity of TMP binding is mainly driven by the strong positive co-operative binding effect between inhibitor (trimethoprim) and the cofactor (NADPH) when they bind to bacterial DHFR. Only a small cooperative binding effect is observed in the case of these ligands binding to human enzyme. Information on the structure and dynamics of complexes of Lactobacillus casei DHFR with TMP and NADPH was obtained earlier, and in order to explore the origins of the specificity and cooperativiy of binding of these two ligands to the bacterial enzyme, similar information must be obtained for the complex of human DHFR. Essentially complete 1H, 15N and 13C resonance assignments were obtained for the protein and ligand signals from the ternary complex of human DHFR with TMP and NADPH. A high resolution solution structure of the complex was calculated using the distance, torsion angle and chemical shift restraints. Positions of both ligands were defined using a set of distance restraints obtained in 15N and 13C-filtered NOESY experiments. Information on the protein backbone dynamics was obtained from T1, T2 and 15N{1H}-NOE experiments carried out at three magnetic fields. Differences in structure and dynamics between bacterial and vertebrate forms of DHFR complexes relevant to the inhibitor binding selectivity will be reported.