Characterization of the Zn(II) binding properties of the human Wilms' tumor suppressor protein C-terminal zinc finger peptide

Abstract

Zinc finger proteins that bind Zn(II) using a Cys2His2 coordination motif within a ββα protein fold are the most abundant DNA binding transcription factor domains in eukaryotic systems. These classic zinc fingers are typically unfolded in the apo state and spontaneously fold into their functional ββα folds upon incorporation of Zn(II). These metal-induced protein folding events obscure the free energy cost of protein folding by coupling the protein folding and metal-ion binding thermodynamics. Herein, we determine the formation constant of a Cys2His2/ββα zinc finger domain, the C-terminal finger of the Wilms' tumor suppressor protein (WT1-4), for the purposes of determining its free energy cost of protein folding. Measurements of individual conditional dissociation constants, Kd values, at pH values from 5 to 9 were determined using fluorescence spectroscopy by direct or competition titration. Potentiometric titrations of apo-WT1-4 followed by NMR spectroscopy provided the intrinsic pKa values of the Cys2His2 residues, and corresponding potentiometric titrations of Zn(II)-WT1-4 followed by fluorescence spectroscopy yielded the effective pKa(eff) values of the Cys2His2 ligands bound to Zn(II). The Kd, pKa, and pKa(eff) values were combined in a minimal, complete equilibrium model to yield the pH-independent formation constant value for Zn(II)-WT1-4, Kf(ML) value of 7.5 × 10(12) M(-1), with a limiting Kd value of 133 fM. This shows that Zn(II) binding to the Cys2His2 site in WT1-4 provides at least -17.6 kcal/mol in driving force to fold the protein scaffold. A comparison of the conditional dissociation constants of Zn(II)-WT1-4 to those from the model peptide Zn(II)-GGG-Cys2His2 over the pH range 5.0 to 9.0 and a comparison of their pH-independent Kf(ML) values demonstrates that the free energy cost of protein folding in WT1-4 is less than +2.1 kcal/mol. These results validate our GGG model system for determining the cost of protein folding in natural zinc finger proteins and support the conclusion that the cost of protein folding in most zinc finger proteins is ≤+4.2 kcal/mol, a value that pales in comparison to the free energy contribution of Zn(II) binding, -17.6 kcal/mol.

Scheme 1. Equilibrium Model for Zn(II)–WT1-4 Formation

Figure 1

(Left) Molecular model of the Zn(II)–WT1-4 complex rendered in PyMOL and (Right) free energy diagram of zinc finger protein folding.

Figure 2

Steady-state fluorescence emission spectra of 22 μM apo-WT1-4 (dotted line) and 22 μM Zn(II)–WT1-4 (solid line) in pH 7.0 buffer (20 mM HEPES, 100 mM KCl). Each sample was excited through a 5 nm slit at 280 nm, the tryptophan λ_max value, and the fluorescence emission was collected through a 2.5 nm slit.

Figure 3

Direct titration of Zn(II)Cl₂ in unbuffered aqueous solution at pH 7.0 into 22 μM WT1-4 buffered at pH 5.25 (20 mM HEPES, 100 mM KCl) followed by fluorescence spectroscopy. Spectra are shown for the addition of 0.00, 0.22, 0.67, 0.99, 1.81, 2.71, 4.07, 6.78, 8.59, and 12.20 equiv of Zn(II) added, with the others were omitted for clarity. The increase in emission intensity at 355 nm observed upon Zn(II) binding is fit in the inset to a Zn(II)–WT1-4 conditional dissociation constant, K_d, value of 9.9 μM at pH 25.

Figure 4

Competition titration of Zn(II)Cl₂ in unbuffered aqueous solution at pH 7.0 into an aqueous solution containing 22 μM WT1-4 and 110 μM HEDTA buffered at pH 9.0 (20 mM Tris, 100 mM KCl) followed by fluorescence spectroscopy. Spectra are shown for the addition of 0.00, 0.90, 2.03, 2.94, and 4.29 equiv of Zn(II) added, with the others omitted for clarity. Under these conditions, a fit to the plot of fluorescence at 357 nm vs equivalents of Zn(II) added to peptide using eq 6 gives a competition constant value of 3.31 between WT1-4 and HEDTA. Because the K_d of Zn(II)–HEDTA at pH 9.0 is 20.1 fM, the resulting Zn(II)–WT1-4 dissociation constant at pH 9.0 is 66.5 fM.

Figure 5

Kinetics of Zn(II) removal from 22 μM Zn(II)–WT1-4 by 100 μM EDTA buffered at pH 6.65 (20 mM MES, 100 mM KCl) followed by the decrease in fluorescence emission intensity at 355 nm. The fluorescence emission intensity drops from an initial value of 145.67 au to 78.45 au in 15 min. Under similar conditions, Sénèque and Latour measured an equilibration time for Zn(II)–CP1-CCHH of 1600 min.

Figure 6

pH titration of the Zn(II) binding residues in WT1-4 followed by NMR spectroscopy. The pH titration curves are fit to single-proton pK_a values of 8.0 (○, Cys⁵), 7.9 (□, Cys¹⁰), 6.2 (■, His²³), and 6.9 (●, His²⁷).

Figure 7

pH titration of 17 μM Zn(II)–WT1-4 followed by fluorescence spectroscopy. The decrease in tryptophan fluorescence emission intensity at 355 nm as the pH is lowered by addition of microliter aliquots of 0.1 N HCl is due to protonation/dissociation of the Zn(II) bound thiolate/imidazole ligands. The pH titration data is best fit to an equilibrium model involving two separate protonation events, a two-proton event with a pK_a1,2^eff value of 5.2 and a cooperative two-proton event with a pK_a3,4^eff value of 2.6.

Figure 8

Speciation diagram of the Zn(II)–WT1-4 metal–ligand complex depicting the diimidazole–dithiolate zinc species (solid line), Zn(II)–WT1-4, the diimidiazole–dithiol zinc species (dashed line), Zn(II)–WT1-4–2H⁺, and the diimidazolium–dithiol species (dotted line), Zn(II)–WT1-4–4H⁺. The diagram was generated on the basis of the protonation behavior of the Zn(II)–WT1-4 complex in Figure 7.

Figure 9

EXAFS Fourier transform for Zn(II)–WT1-4 (solid line) and best fit (open diamonds), modeled as a Cys₂His₂ coordination sphere. Inset: Percent improvement (P_i) vs composition plot for Zn(II)–WT1-4 (open symbols, black line) compared to those for characterized model peptides (gray lines, as labeled).

Figure 10

pH dependence of the conditional dissociation constant of Zn(II) complexation by WT1-4, shown as a plot of the logarithm of the association constant vs solution pH. The equilibrium binding model employed to fit the data yields a pH-independent formation constant, K_f^ML value, of 7.5 × 10¹² M^–1, or a limiting dissociation constant of 133 fM, which corresponds to a reaction free energy of −17.6 kcal mol^–1.