Backbone dynamics in dihydrofolate reductase complexes: Role of loop flexibility in the catalytic mechanism

To elucidate the influence of local motion of the polypeptide chain on the catalytic mechanism of an enzyme, we have measured N-15 relaxation data for Escherichia coli dihydrofolate reductase in three different complexes, rep resenting different stages in the catalytic cycle of the enzyme. NMR relaxa tion data were analyzed by the model-free approach, corrected for rotationa l anisotropy, to provide insights into the backbone dynamics. There are sig nificant differences in the backbone dynamics in the different complexes. C omplexes in which the cofactor binding site is occluded by the Met20 loop d isplay large amplitude motions on the picosecond/nanosecond time scale for residues in the Met20 loop, the adjacent betaF-betaG loop and for residues 67-69 in the adenosine binding loop. Formation of the closed Met20 loop con formation in the ternary complex with folate and NADP(+), results in attenu ation of the motions in the Met20 loop and the betaF-betaG loop but leads t o increased flexibility in the adenosine binding loop. New fluctuations on a microsecond/millisecond time scale are observed in the closed E:folate:NA DP+ complex in regions that form hydrogen bonds between the Met20 and the b etaF-betaG loops. The data provide insights into the changes in backbone dy namics during the catalytic cycle and point to an important role of the Met 20 and betaF-betaG loops in controlling access to the active site. The high flexibility of these loops in the occluded conformation is expected to pro mote tetrahydrofolate-assisted product release and facilitate binding of th e nicotinamide ring to form the Michaelis complex. The backbone fluctuation s in the Met20 loop become attenuated once it closes over the active site, thereby stabilizing the nicotinamide ring in a geometry conducive to hydrid e transfer. Finally, the relaxation data provide evidence for long-range mo tional coupling Im between the adenosine binding loop and distant regions o f the protein.