Meiering E M, Wagner G
Department of Biological Chemistry, Harvard Medical School, Boston, MA 02115, USA.
J Mol Biol. 1995 Mar 24;247(2):294-308. doi: 10.1006/jmbi.1994.0140.
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 using 15N-resolved, three-dimensional ROESY-HMQC and NOESY-HSQC spectra 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. 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 in 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 caused by a change in the position of water molecule, it may also be caused by an increase 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 complex. WatF forms hydrogen bonds bridging the cofactor and the protein in crystal structures of hDHFR.
利用在25℃和8℃下采集的15N分辨的三维ROESY-HMQC和NOESY-HSQC谱，研究了人二氢叶酸还原酶（hDHFR）与甲氨蝶呤（MTX）的二元复合物以及hDHFR与MTX和NADPH的三元复合物中长寿命结合水分子的位置。在二元复合物中检测到与蛋白质NH基团有NOE作用的5个结合水分子，在三元复合物中检测到6个结合水分子。对hDHFR晶体结构的检查表明，结合水分子在复合物中发挥结构和功能作用。位于活性位点之外的两个水分子WatA和WatB，在二元和三元复合物中有相似的NOE。这些水分子在hDHFR晶体结构中通过多个氢键连接环和/或二级结构元件，从而稳定酶的三级结构。活性位点中的两个水分子WatC和WatD，在两种复合物中也有相似的NOE。在hDHFR晶体结构中，WatC通过与配体和蛋白质形成氢键参与MTX结合，而WatD通过与WatC和蛋白质形成氢键来稳定WatC。第三个活性位点水分子WatE，在三元复合物中的NOE明显强于二元复合物。二元和三元复合物中WatE结合的差异对于理解DHFR的机制很重要，因为这个水分子被认为参与底物质子化。虽然WatE的NOE强度增加可能是由于水分子位置的变化引起的，但也可能是由于其寿命增加，因为辅因子结合后活性位点的结构波动减小。在三元复合物中可能观察到另一个水分子WatF的NOE，但在二元复合物中未观察到。在hDHFR晶体结构中，WatF形成连接辅因子和蛋白质的氢键。
