ИСТИНА

Cooperative effects of ligand binding to proteins play an important role in many biochemical reactions, including interactions of enzymes with their substrates, cofactors and inhibitors. Both positive and negative cooperative effects can tune enzyme catalytic activity and in some cases determine specificity of ligand binding. For example, the antibacterial drug trimethoprim, an inhibitor of dihydrofolate reductase (DHFR), binds to bacterial enzymes more than 3,000 times more tightly than it does to mammalian forms. Most of this selectivity arises from positive cooperative effect of binding of trimethoprim and NADPH when they form a ternary complex with the bacterial enzyme. We have used NMR spectroscopy in an attempt to determine the molecular origins of the cooperative effects of ligand binding to DHFR. Solution structures were obtained for the binary complexes of Lactobacillus casei DHFR (~18.3 kDa) with trimethoprim and NADPH and for the ternary complex of these ligands. Earlier we also examined details of the dynamic properties of DHFR complexes, including protein backbone motions over a broad time scale (from ps to hours) and rates of ring-flipping in bound inhibitors. Important information about specific interactions between ligand and DHFR residues was obtained from non-empirical quantum mechanical calculations carried out on fragments of the NADPH binding site. Comparison of the results obtained for the bacterial enzyme with results from ongoing studies on related complexes of human DHFR could contribute to understanding the nature of cooperative effects of ligand binding to proteins.