Structural characterization of the putative DNA-binding domain of CP2c and its relevance to zinc binding

Abstract

The transcription factor CP2c has been recently validated as an oncogenic protein that can serve as a promising target for anticancer therapy. We have recently documented that a recombinant protein corresponding to the putative DNA-binding region (residues 63-244) of CP2c adopted two different conformers, one of which is dominated by zinc binding. However, in the present study, a longer construct encompassing residues 63-302 appeared to form a single structural domain. This domain could be considered to adopt a functionally relevant fold, as the known specific binding of a dodecapeptide to this protein was evident. Hence, the residues 63-302 region rather than 63-244 can be regarded as a natively folded structural domain of CP2c. In addition, it was confirmed that zinc ions can bind to this putative DNA-binding domain of CP2c, which resulted in reduced stability of the protein. In this context, it is suggested that the mode of action of CP2c would resemble that of tumor suppressor p53.

Figure 1. Preparation and biochemical characterization of CP2c(63-302). (A) Schematic diagram showing the putative domain organization of CP2c.² Constructed region in this study and the known binding site of the dodecapeptide are illustrated by arrows. TAD, transactivation domain; BD, basic domain; TD, tetramerization domain. (B) Gel-filtration elution profile of the purified CP2c(63-302). Inset shows SDS-PAGE image for the elution fractions containing the protein. (C) Hydrodynamic size of CP2c(63-302) as estimated by analytical gel-filtration. Black dots indicate the protein standards used. Red dot designates the Kav value of CP2c(63-302), of which apparent molecular weight was calculated using the standard curve.^9,10

Figure 2. Examination of a dodecapeptide binding to CP2c(63-302). (A) Thermal denaturation of CP2c(63-302) (20 μM), monitored by CD at 222 nm, in the absence (black) and presence of the binding peptide (blue and red for 1 and 2 equivalent titration, respectively). Inset shows standard far-UV CD spectra in the absence (black) and presence (green) of 2 equivalents of the peptide. (B) 2D-[¹H/¹⁵N]-TROSY NMR spectra of [¹⁵N]CP2c(63-302) (250 μM), in the absence (black) and presence of 2 equivalents of the binding peptide (green).

Figure 3. Examination of zinc binding to CP2c(63-302). (A) 2D-[¹H/¹⁵N]-TROSY NMR spectra of [¹⁵N]CP2c(63-302) (250 μM), in the absence (black) and presence of 2 equivalents of zinc (red). Inset shows standard far-UV CD spectra in the absence (black) and presence (red) of 2 equivalents of zinc. (B) Thermal denaturation of CP2c(63-302) (20 μM), monitored by CD at 222 nm, in the absence (black) and presence of zinc (molar ratios are indicated for individual colors).