ИСТИНА

Cooperative effects of ligand binding plays an important role in vast majority of biochemical processes in living cell. In many cases nature of such cooperative effects determined by direct ligand-ligand interaction or through the ligand-induced change the conformation of a protein. Antibacterial drug trimethoprim and coenzyme NADPH have strong positive cooperative effect of binding to bacterial dihydrofolate reductase (DHFR). Lack of cooperative effect between these ligands when they form ternary complex with human DHFR determines selectivity of TMP inhibition of bacterial forms of enzyme and overall effectiveness of the drug. Extensive X-ray studies of DHFR complexes have not allowed offering a single hypothesis that satisfactorily explains the origins of the cooperativity in binding of TMP and NADPH to bacterial DHFR. In our attempts to determine origin of such effect at the molecular level we have used multinuclear NMR spectroscopy. Information on structure, dynamics and specific protein-ligand interactions was obtained for binary and ternary complexes of Lactobacillus casei and human DHFR with TMP and NADPH in solution. Detailed comparisons of structural and dynamic information for the complex where cooperative effect of ligand binding exists with those where there is no such effect were performed. Two main factors that can be responsible for cooperative binding of TMP and NADPH to bacterial enzyme were determined. These include (i) stronger hydrophobic contacts of ligands in ternary complex with L.casei DHFR vs human enzyme and (ii) larger network of hydrogen bond interactions between TMP and bacterial enzyme. The second factor leads to the overall strengthening of hydrogen bonds by cooperative manner in whole protein-ligand complex observed in various NMR experiments. Implications of these results for understanding the molecular recognition processes will be discussed. These results are to be taken into account in search for selectively bound ligands.