CONTRIBUTIONS OF TRYPTOPHAN-24 AND GLUTAMATE-30 TO BINDING LONG-LIVED WATER-MOLECULES IN THE TERNARY COMPLEX OF HUMAN DIHYDROFOLATE-REDUCTASE WITH METHOTREXATE AND NADPH STUDIED BY SITE-DIRECTED MUTAGENESIS AND NUCLEAR-MAGNETIC-RESONANCE SPECTROSCOPY

Previous NMR studies on the ternary complex of human dihydrofolate reductase (hDHFR) with methotrexate (MTX) and NADPH detected six long-lived bound water molecules. Two of the water molecules, WatA and WatB, stabilize the structure of the protein while the other four, WatC, WatD, WatE and WatF, are involved in substrate binding and specificity WatE may also act as a proton shuttle during catalysis. Here, the contributions of individual residues to the binding of these water molecules are investigated by performing NMR experiments on ternary complexes of mutant enzymes, W24F, E30A and E30Q. W24 and E30 are conserved residues that form hydrogen bonds with WatE in crystal structures of DHFR. Nuclear Overhauser effects (NOEs) are detected between WatE and the protein in all the mutant complexes, hence WatE still has a long lifetime bound to the complex when one of its hydrogen-bonding partners is deleted or altered by mutagenesis. The NOEs for WatE are much weaker, however, in the mutants than in wild-type. The NOEs for the other water molecules in and near the active site, WatA, WatC, WatD and WatF, also tend to be weaker in the mutant complexes. Little or no change is apparent in the NOEs for WatB, which is located outside the active site, farthest from the mutated residues. The decreased NOE intensities for the bound water molecules could be caused by changes in the positions and/or lifetimes of the water molecules. Chemical shift and NOE data indicate that the mutants have structures very similar to that of wild-type hDHFR, with possible conformational changes occurring only near the mutated residues. Based on the lack of structural change in the protein and evidence for increased structural fluctuations in the active sites of the mutant enzymes, it is likely that the NOE changes are caused, at least in part, by decreases in the lifetimes of the bound water molecules.