Sequence-specific 1H and 15N resonance assignments for human dihydrofolate reductase in solution.
Dihydrofolate reductase is an intracellular target enzyme for folate antagonists, including the anticancer drug methotrexate. In order to design novel drugs with altered binding properties, a detailed description of protein-drug interactions in solution is desirable to understand the specificity of drug binding. As a first step in this process, heteronuclear three-dimensional NMR spectroscopy has been used to make sequential resonance assignments for more than 90% of the residues in human dihydrofolate reductase complexed with methotrexate. Uniform enrichment of the 21.5-kDa protein with 15N was required to obtain the resonance assignments via heteronuclear 3D NMR spectroscopy since homonuclear 2D spectra did not provide sufficient 1H resonance dispersion. Medium- and long-range NOE's have been used to characterize the secondary structure of the binary ligand-enzyme complex in solution.
Stockman
B J
BJ
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Nirmala
N R
NR
Wagner
G
G
Delcamp
T J
TJ
DeYarman
M T
MT
Freisheim
J H
JH
D000595:Amino Acid Sequence; D001665:Binding Sites; D005492:Folic Acid / Q000378:metabolism; D006801:Humans; D006859:Hydrogen; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008727:Methotrexate / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation; D009587:Nitrogen Isotopes; D011487:Protein Conformation; D012996:Solutions; D013329:Structure-Activity Relationship; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics
D009587:Nitrogen Isotopes; D012996:Solutions; D006859:Hydrogen; D005492:Folic Acid; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate