期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 284, 期 30, 页码 20079-20089出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M109.018010
关键词
-
资金
- Canadian Institutes of Health Research
- Natural Sciences and Engineering Research Council of Canada (NSERC)
Methotrexate is a slow, tight-binding, competitive inhibitor of human dihydrofolate reductase (hDHFR), an enzyme that provides key metabolites for nucleotide biosynthesis. In an effort to better characterize ligand binding in drug resistance, we have previously engineered hDHFR variant F31R/Q35E. This variant displays a > 650-fold decrease in methotrexate affinity, while maintaining catalytic activity comparable to the native enzyme. To elucidate the molecular basis of decreased methotrexate affinity in the doubly substituted variant, we determined kinetic and inhibitory parameters for the simple variants F31R and Q35E. This demonstrated that the important decrease of methotrexate affinity in variant F31R/Q35E is a result of synergistic effects of the combined substitutions. To better understand the structural cause of this synergy, we obtained the crystal structure of hDHFR variant F31R/Q35E complexed with methotrexate at 1.7-angstrom resolution. The mutated residue Arg-31 was observed in multiple conformers. In addition, seven native active-site residues were observed in more than one conformation, which is not characteristic of the wild-type enzyme. This suggests that increased residue disorder underlies the observed methotrexate resistance. We observe a considerable loss of van der Waals and polar contacts with the p-aminobenzoic acid and glutamate moieties. The multiple conformers of Arg-31 further suggest that the amino acid substitutions may decrease the isomerization step required for tight binding of methotrexate. Molecular docking with folate corroborates this hypothesis.
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