Detection of Long-lived Bound Water Molecules in Complexes of Human Dihydrofolate Reductase with Methotrexate and NADPH

Abstract

The locations of long-lived bound water molecules in the binary complex of human dihydrofolate reductase (hDHFR) with methotrexate (MTX) and the ternary complex of hDHFR with MTX and NADPH have been investigated using15N-resolved, three-dimensional ROESY-HMQC and NOESY-HSQC spectra acquired at 25°C and 8°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. Inspection of crystal structures of hDHFR reveals that the bound water molecules perform structural and functional roles in the complexes. Two water molecules located outside the active site, WatA and WatB, have similar NOEs in the binary and ternary complexes. These water molecules from multiple hydrogen bonds bridging loops and/or secondary structural elements in crystal structures of hDHFR and so stabilize the tertiary fold of the enzyme. Two water molecules in the active site, WatC and WatD, also have similar NOEs in both complexes. In crystal structures of hDHFR, WatC is involved in MTX binding by forming hydrogen bonds to the ligand and protein, while WatD stabilizes WatC by hydrogen bonding to it and the protein. A third active-site water molecule, WatE, has a markedly stronger NOE in the ternary complex than the binary complex. Differences in the binding of WatE in the binary and ternary complexes are important for understanding the mechanism of DHFR, since this water molecule is believed to be involved in substrate protonation. Although the increased NOE intensity for WatE could be causd by a change in the position of water molecule, it may also be caused by an increase in its lifetime, since structural fluctuations in th 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 complex. WatF forms hydrogen bonds bridging the cofactor and the protein crystal structures of hDHFR.f2f2Abbreviations used: DHFR, dihydrofolate reductase; MTX, methotrexate; MTXγT, methotrexate-γ-tetrazole; H2folate, 7,8-dihydrofolate; H4folate, 5,6,7,8-tetrahydrofolate; HSQC, heteronuclear single quantum coherence; TSP, sodium trimethylsilylpropionate; TOCSY, total correlation spectroscopy; ROESY, rotating frame Overhauser effect spectroscopy; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; GARP, globally optimized alternating-phase rectangular pulses; WALTZ, wideband alternating-phase low-power technique for zero residual splitting; 3D, three- dimensional; 2D, two-dimensional