Multiple Conformers in Active Site of Human Dihydrofolate Reductase F31R/Q35E Double Mutant Suggest Structural Basis for Methotrexate Resistance

Jordan P Volpato,Brahm J Yachnin,Jonathan Blanchet,Vanessa Guerrero,Lucie Poulin,Elena Fossati,Albert M Berghuis,Joelle N Pelletier

doi:10.1074/jbc.m109.018010

Jordan P Volpato, Brahm J Yachnin + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m109.018010

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Abstract

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-A 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.

Highlights

Late in a NADPH-dependent manner. 5,6,7,8-Tetrahydrofolate is a cofactor in purine and thymidylate biosynthesis, which are essential metabolites in cell division and proliferation
We report the structure of the highly MTX-resistant, doubly substituted hDHFR variant F31R/Q35E bound to MTX
We have shown that this variant of hDHFR allows rapid and efficient selection of hematopoietic cells, offering the potential to address this issue

Summary

EXPERIMENTAL PROCEDURES

Construction of Vectors hDHFR F31R/Q35E-pET24, hDHFR F31R-pQE32, and hDHFR Q35E-pQE32—The hDHFR F31R/ Q35E gene was amplified by PCR using the following primer set: forward (5Ј-CACACACCATATGGTTGGTTCGCTAAACTG-3Ј, NdeI restriction site in italics) and reverse (5ЈGTTCTGAGGTCATTACTGG-3Ј, external primer) from the hDHFR F31R/Q35E-pQE32 template [18]. The amplified gene was subcloned in the modified pET24 vector [29] between the NdeI and HindIII restriction sites using T4 DNA ligase, and the ligation mixture was transformed into electrocompetent BL21(DE3) cells. The kcat values were determined in the presence of saturating substrate concentrations (100 ␮M each of DHF and NADPH) in 1-cm cells according to kcat ϭ Vmax/[E]. KmDHF values were obtained using 10-cm cells containing 1 nM enzyme, 10 ␮M NADPH and a range of DHF concentrations (0.05 ␮M to 10 ␮M). HDHFR activity was monitored in MATS buffer, pH 7.6, in the presence of 100 ␮M each of NADPH and DHF. MTX and NADPH were prepared as described previously [18] and were added at a final concentration of 2 mM each (5-fold molar excess) to the protein sample. Clusters were evaluated according to total binding energies calculated by Autodock 4, and the minimal energy conformation within the lowest energy cluster was retained for comparison with crystal structures

RESULTS

19 Ϯ 4 3100 Ϯ 1600

DISCUSSION