Characterization of the Zn(II) Binding Properties of the Human Wilms’ Tumor Suppressor Protein C-terminal Zinc Finger Peptide

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 pKaeff values of the Cys2His2 ligands bound to Zn(II). The Kd, pKa, and pKaeff values were combined in a minimal, complete equilibrium model to yield the pH-independent formation constant value for Zn(II)–WT1-4, KfML value of 7.5 × 1012 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 KfML 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

Pointing the Zinc Finger on Protein Folding: Energetic Investigation into the Role of the Metal-Ion in the Metal-Induced Protein Folding of Zinc Finger Motifs

Interactions between inorganic metal-ion cofactors and organic protein scaffolds are important for the proper structure and function of metalloproteins. Zinc finger proteins (ZFPs) are an example of proteins with such crucial metal-protein interactions. Incorporation of the Zn(II)-ion into ZFPs allows for their correct folding into structures that can carry out vital biological functions which include gene expression and tumor suppression. In addition, engineered ZFPs have shown to be promising genetic therapeutics in the clinic. And yet, there is still a gap in a quantitative understanding of the energetic contribution of the metal-protein interactions towards the structure and function of these important metalloproteins. Detailed knowledge of the principles that governs such interactions in natural metalloproteins will provide the much-needed insight for the rational construction of novel engineered zinc proteins for promising corrective therapeutics for genetic disorders. In this thesis, the investigation of the role that a specific metal-ion has on the energetics of metal-induced protein folding events is presented. To compare with Zn(II) related studies, an unstructured peptide scaffold, GGG, with Cys4-xHisx (x=0,1,2) metal-binding coordination motifs is utilized to determine the thermodynamics of Co(II) and Pb(II) binding to the three motifs. Furthermore, the difference between Co(II) and Pb(II) affinities to GGG and their affinities to natural ZFPs that undergo metal- induced protein folding events, provides the energetic cost of folding for these natural ZFPs – values that were hithertofore unknown. The data demonstrate that the Co(II)-ion has no binding preference for cysteine thiolate over histidine imidazole, while Pb(II)-ion prefers cysteine thiolate. Each substitution of Cys by His lower the contribution of Pb(II)-binding towards protein stabilization by approximately 2.0 kcal/mol - similarly to previously observed results for Zn(II). Lastly, both metal-ion studies agreed with previous Zn(II) studies, indicating that the cost for apo-ZFPs folding in natural ZFPs is minimal, less than 4.5 kcal/mol