ИСТИНА

The high-resolution 3D structures of two binary complexes of L.casei DHFR with TMP and NADPH have been determined and compared to the structures of the ternary complexes of L.casei and human DHFR with NADPH and TMP. The overall protein topology is identical in all the studied complexes: 8-stranded beta-sheet core surrounded by four alpha-helices. Protein structure is changed very slightly between complexes formed with bacterial enzyme. This indicates on the little conformational change of DHFR upon the ligand binding. The following structural differences have been found: 1) conformational changes of the loop 12-22 and the ligands upon their binding; 2) tighter hydrophobic interactions between TMP and NADPH in the L.casei DHFR ternary complex comparing to the human DHFR ternary complex; 3) stronger network of H-bonds in the bacterial complex. Protein backbone dynamics studies of DHFR complexes using the measurements of 15N relaxation rates and 15N{1H} heteronuclear Overhauser effects show that although the mobility of the backbone in the fast time-scale (ps-ns) is almost unaffected upon the binding of the ligands. For slow time scale (ms) motions, significant decrease of conformational mobility upon the ligand binding to protein was detected. Human DHFR is undergoing more intensive conformational motions than L.casei enzyme in slow time scale (ms) as follows from the comparison of two ternary complexes formed with TMP and NADPH. The nature of cooperative binding of TMP and NADPH to bacterial form of enzyme and therefore selectivity of TMP binding can be explained by the additive contributions of structural and dynamical factors rather than by the single effect.