ИСТИНА

Dihydrofolate reductase (DHFR) catalyses the reduction of 7,8-dihydrofolate to 5,6,7,8-tetrahydrofolate using NADPH as coenzyme. The enzyme is of considerable pharmacological interest being the target for a number of clinically useful antibacterial and antineoplastic drugs. One of these is the antibacterial trimethoprim (TMP), which binds to bacterial enzyme about 3,000 times tighter that it does to mammalian enzymes. Most of this increase comes from the high co-operative binding effect between TMP and NADPH when these ligands bind to the bacterial enzyme. It is clearly important to understand the molecular basis for such co-operativity and specificity of binding. We have obtained a high-resolution solution structure for the ternary complex of Lactobacillus casei DHFR (162 a.a., M.W. ~ 18300 Dalton) formed with TMP and NADPH. The position of the ligands in the binding sites of enzyme has been examined and several specific interactions between the ligands and surrounding protein residues have been studied in details. These structural results were complemented by studies of protein dynamics. Protein backbone motions in the binary and ternary complexes of DHFR with TMP and NADPH have been studied using 15N relaxation measurements. Analysis of T1, T2, T1rho and {15N-1H}NOE data measured for apo-form and several complexes of DHFR allowed to obtain an information about fast (pico- to nanoseconds time scale) and slower (millisecond time scale) motions of the protein backbone. Slow (seconds to hours time scale) protein movements were studied using measurement of proton to deuterium exchange rates in amide groups. The information obtained from these studies increases our knowledge of the nature of factors which control the specificity and cooperativity of ligand binding to proteins.