Evidence for two interconverting protein isomers in the methotrexate complex of dihydrofolate reductase from Escherichia coli.
Two-dimensional 1H NMR methods and a knowledge of the X-ray crystal structure have been used to make resonance assignments for the amino acid side chains of dihydrofolate reductase from Escherichia coli complexed with methotrexate. The H7 proton on the pteridine ring of methotrexate was found to have NOEs to the methyl protons of Leu-28 which were assigned by using the L28F mutant. These NOEs indicated that the orientation of the methotrexate pteridine ring is similar in both solution and crystal structures. During the initial assignment process, it became evident that many of the resonances in this complex, unlike those of the folate complex, are severely broadened or doubled. The observation of two distinct sets of resonances in a ratio of approximately 2:1 was attributed to the presence of two protein isomers. At 303 K, NOESY spectra with mixing times of 100 ms did not show interconversion between these isomers. However, exchange cross-peaks were observed in a 700-ms NOESY spectrum at 323 K which demonstrated that these isomers are interconverting slowly on the NMR time scale. Many of the side chains with clearly doubled resonances were located in the beta-sheet and the active site. Preliminary studies on the apoprotein also revealed doubled resonances in the absence of the inhibitor, indicating the existence of the protein isomers prior to methotrexate binding. In contrast to the methotrexate complex, the binary complex with folate and the ternary MTX-NADPH-DHFR complex presented a single enzyme form. These results are proposed to reflect the ability of folate and NADPH to bind predominantly to one protein isomer.
Falzone
C J
CJ
Department of Chemistry, Pennsylvania State University, University Park 16802.
Wright
P E
PE
Benkovic
S J
SJ
D004926:Escherichia coli / Q000201:enzymology*; D006859:Hydrogen; D007536:Isomerism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008727:Methotrexate / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*; D014961:X-Ray Diffraction
D006859:Hydrogen; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate