DETECTION OF LONG-LIVED BOUND WATER-MOLECULES IN COMPLEXES OF HUMAN DIHYDROFOLATE-REDUCTASE WITH METHOTREXATE AND NADPH

The locations of long-lived bound water molecules in the binary comple x of human dihydrofolate reductase (hDHFR) with methotrexate (MTX) and the ternary complex of hDHFR with MTX and NADPH have been investigate d using N-15-resolved, three-dimensional ROESY-HMQC and NOESY-HSQC spe ctra acquired at 25 degrees C and 8 degrees C. NOEs with NH groups of the protein are detected for five bound water molecules in the binary complex and six bound water molecules in the ternary complex. Inspecti on of crystal structures of hDHFR reveals that the bound water molecul es perform structural and functional roles in the complexes. Two water molecules located outside the active site, WatA and WatB, have simila r NOEs in the binary and ternary complexes. These water molecules from multiple hydrogen bonds bridging loops and/or secondary structural el ements in crystal structures of hDHFR and so stabilize the tertiary fo ld of the enzyme. Two water molecules in the active site, WatC and Wat D, also have similar NOEs in both complexes. In crystal structures of hDHFR, WatC is involved in MTX binding by forming hydrogen bonds to th e ligand and protein, while WatD stabilizes WatC by hydrogen bonding t o it and the protein. A third active-site water molecule, WatE, has a markedly stronger NOE in the ternary complex than in the binary comple x. Differences in the binding of WatE in the binary and ternary comple xes are important for understanding the mechanism of DHFR, since this water molecule is believed to be involved in substrate protonation. Al though the increased NOE intensity for WatE could be caused by a chang e in the position of water molecule, it may also be caused by an incre ase in its lifetime, since structural fluctuations in the active site are decreased upon cofactor binding. NOEs for one other water molecule , WatF, may be observed in the ternary complex but not the binary comp lex. WatF forms hydrogen bonds bridging the cofactor and the protein i n crystal structures of hDHFR.