ИСТИНА

Dihydrofolate reductase (DHFR) represents an excellent model for studies of both kinetic and thermodynamic factors that control processes of protein-ligand binding. The enzyme contains two binding sites, one for the substrate (dihydrofolate) or inhibitor (antifolate drugs) and the other for the coenzyme (NADPH). Various aspects of the mechanism of catalysis or enzyme inhibition depend on distinctive features regarding the interactions and motions of these ligands with the surrounding protein residues. Previously we determined high resolution structures for several binary and ternary complexes of Lactobacillus casei DHFR in solution. In particular, we obtained by NMR the 3D structure of the ternary complex of DHFR with the antibacterial drug trimethoprim and NADPH. The large positive cooperative effect of the binding of these two ligands to bacterial DHFR makes TMP a highly selective inhibitor of the bacterial enzyme versus the human form. Using measurements of the NMR relaxation parameters and the proton to deuterium amide exchange rates we also examined the dynamic properties of L.casei DHFR complexes, including protein backbone motions over a broad time scale (from ps to hours). More recently we have used multinuclear NMR methods to study the solution structure and dynamic properties of ternary complex of 13C,15N-labeled human DHFR with trimethoprim and NADPH (m.w. ~21 kDa). The structure determination is now in progress and relaxation measurements have already revealed a much larger mobility of the protein backbone in the human enzyme compared to the bacterial form. Such differences in protein mobility might be related to the observed protein-ligand binding properties. In order to test this hypothesis we have studied protein motions in complexes of L.casei and human DHFR by using molecular dynamics simulations in water. Results of these studies and implications for understanding protein-ligand interactions are discussed.