Dihydrofolate reductases (DHFR's) are enzymes with important functions in mammalian and bacterial metabolism. Designing folate antagonist drugs has wide applications for treating diseases, such as certain types of cancer and pneumacystis infection, the major cause of death in AIDS patients. The size of human DHFR is beyond the limit of what has been handled with NMR so far. However, advances in isotope labeling and multidimensional NMR appear to make high quality structure determination of such large or even larger proteins possible. This is challenging but would open new avenues for understanding enzyme function on a structural level. We will investigate structural and dynamic properties that underlie the interaction of dihydrofolate reductase (DHFR) with the anti-folate drug methotrexate. The basis of the study will be complete 1H, 13C, and 15N resonance assignments. This will require uniform and selective enrichment of DHFR with 13C and 15N. Extensive use of 2D, 3D and 4D NMR will be made to resolve problems of spectral overlap. For the determination of the solution structure, inter-proton distances and homonuclear and heteronuclear coupling constants will be measured. Availability of 15N and 13C labeled protein will be crucial for the success of this research. The anti-folate binding site will be of primary interest. The geometry of the methotrexate binding site will be probed by using isotope-editing techniques to elucidate interactions with amino acid side chains, and by measuring heteronuclear coupling constants to determine the conformation of the bound ligand. 13C and 15N relaxation parameters will be determined to investigate dynamic properties of apoenzyme, binary (DHFR-NADPH or DHFR-methotrexate), and ternary (DHFR-NADPH-methotrexate) complexes. Ligand-induced conformational changes will be determined to identify ligand-protein interactions responsible for including the conformational change. Single amino acid mutations in the methotrexate binding site will be investigated in a similar fashion. Structural and dynamic differences among the mutant proteins will be correlated in terms of structure-function relationships underlying the interaction of methotrexate with DHFR.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
1R01CA054513-01A1
Application #
3199063
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
1991-08-01
Project End
1994-07-31
Budget Start
1991-08-01
Budget End
1992-07-31
Support Year
1
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Harvard University
Department
Type
Schools of Medicine
DUNS #
082359691
City
Boston
State
MA
Country
United States
Zip Code
02115