Identification of endogenous ligands bound to bacterially expressed human and E. coli dihydrofolate reductase by 2D NMR

Abstract

Dihydrofolate reductase (DHFR) is a well-studied drug target and a paradigm for understanding enzyme catalysis. Preparation of pure DHFR samples, in defined ligand-bound states, is a prerequisite for in vitro studies and drug discovery efforts. We use NMR spectroscopy to monitor ligand content of human and Escherichia coli DHFR (ecDHFR), which bind different co-purifying ligands during expression in bacteria. An alternate purification strategy yields highly pure DHFR complexes, containing only the desired ligands, in the quantities required for structural studies. Interestingly, ecDHFR is bound to endogenous THF while human DHFR is bound to NADP. Consistent with these findings, a designed "humanized" mutant of ecDHFR switches binding specificity in the cell.

Figure 1

hDHFR is optimally folded and stable when expressed in the presence of folic acid. (A) ¹H-¹⁵N HSQC of hDHFR lysate, expressed without folic acid. (B) ¹H-¹⁵N HSQC of hDHFR lysate, expressed with 2 mM folic acid.

Figure 2

¹H-¹⁵N HSQC spectra for ternary complexes of E. coli DHFR show that ecDHFR can be refolded successfully. Spectra of natively purified protein (black) and refolded HPLC purified protein (red) are shown for: ecDHFR:NADP⁺:FOL (A) and ecDHFR:NADP⁺:THF (B).

Figure 3

NADP co-purifies with hDHFR under native purification conditions. (A) ¹H-¹⁵N HSQCs of hDHFR purified using anion exchange followed by gel filtration, with addition of excess folic acid (black) and with addition of excess NADP⁺ and folic acid (red). (B) ¹H-¹⁵N HSQC spectra of hDHFR purified under native conditions with addition of excess folic acid (black) and hDHFR purified by reversed-phase HPLC and refolded with addition of excess NADP⁺ and folic acid (magenta) are almost identical, showing that hDHFR can be successfully refolded. (C) ¹H-¹⁵N HSQC spectra of hDHFR:NADP⁺:FOL (red) and hDHFR:FOL (blue) prepared by refolding HPLC purified apo-hDHFR with the respective ligands.

Figure 4

ecDHFR:NADPH binary complex is contaminated with bound THF. (A) ¹H-¹⁵N HSQC of ecDHFR purified using anion exchange and gel filtration, with addition of excess NADPH. Several minor peaks are visible, indicating the presence of another species. (B) Overlay of A (black) with ecDHFR:NADPH prepared by HPLC purification (red) and ecDHFR:NADP⁺:THF (cyan). The sample prepared using HPLC purification (red) does not contain the minor peaks observed in A. The minor peaks can be accounted for by the ecDHFR:NADP⁺:THF (cyan) spectrum. The minor peaks arise from contamination with endogenous THF, which remains bound to ecDHFR throughout the anion exchange and gel filtration purification steps.

Figure 5

N23PP/S148A ecDHFR is bound to endogenous NADP after native purification by ion exchange and size exclusion chromatography. (A) ¹H-¹⁵N HSQC of natively purified N23PP/S148A ecDHFR without addition of any ligand (black), showing several more resonances than residues in the protein. Upon addition of NADPH to this sample, the E:NADPH complex is formed (red), in which a clean spectrum of uniform intensity is obtained, with one resonance per residue. The E:NADPH spectrum overlays almost perfectly with a subset of the peaks in the black spectrum, showing that NADP is present in ~50% of the natively purified protein sample without any exogenous ligand added. (B) ¹H-¹⁵N HSQC of natively purified mutant protein with added folate (black) exhibits more resonances than expected for a uniform E:FOL complex. Overlay of this spectrum with E:NADP⁺:FOL (red) shows that endogenously bound NADP is present in the natively purified sample.