Cisplatin Affects the Conformation of Apo Form, not Holo Form, of BRCA1 RING Finger Domain and Confers Thermal Stability

Abstract

The breast cancer suppressor protein 1 (BRCA1) has been shown to participate in genomic integrity maintenance. Preclinical and clinical studies have recently revealed that the inactivation of BRCA1 in cancer cells leads to chemosensitivity. Approaching the BRCA1 RING protein as a potentially molecular target for a platinum-based drug might be of interest in cancer therapy. In the present study, the in vitro platination of the BRCA1 RING protein by the anticancer drug cisplatin was observed. The protein contained a preformed structure in the apo form with structural changes and resistance to limited proteolysis after Zn2+ binding. SDS-PAGE and mass-spectrometric analyses revealed that cisplatin preferentially formed monofunctional and bifunctional BRCA1 adducts. Tandem mass spectrometry (MS/MS) of the 656.29(2+) ion indicated that the ion arose from [Pt(NH3)2(OH)]+ bound to the BRCA1 peptide (111)ENNSPEHLK(119). The product-ion spectrum revealed the Pt-binding site on His117. Circular dichroism showed that the apo form, not holo form, of BRCA1 underwent more folded structural rearrangement upon cisplatin binding. Cisplatin-bound protein exhibited an enhanced thermostability by 13 degrees , resulting from the favorably intermolecular cross-links driven by the free energy. Our findings demonstrated the first conformational and thermal evidences for a direct binding of cisplatin to the BRCA1 RING domain and could raise a possibility of selectively targeted treatment of cancer with less toxicity or improved response to conventional regimens.

A DNA Repair Protein BRCA1 as a Potentially Molecular Target for the Anticancer Platinum Drug Cisplatin

“…The BRCA1 RING domain has been found to form favourable intramolecular and intermolecular cross-links caused by cisplatin (Atipairin et al, 2010). Although cisplatin has been demonstrated to induce protein dimerization and has caused perturbations in some protein structures, the secondary structure of the BRCA1 RING domain in the apo-form was maintained and underwent more folded structural rearrangement after increasing cisplatin concentrations as judged by an increase in the negative CD spectra at 208 and 220 nm.…”

“…In order to locate the binding site of cisplatin on the BRCA1 (1-139) protein, in-gel tryptic digestion of the free BRCA1 and the cisplatin-BRCA1 adducts (molar ratio 1:1) were subjected to analysis by LC-MS. A unique fragment ion of 656.29 2+ was obtained from the cisplatin-BRCA1 adduct digests. Tandem mass spectrometric analyses of this fragment ion indicated that the ion arose from (Atipairin et al, 2010).…”

“…Resistance to thermal denaturation of the cisplatin-modifed BRCA1 RING domain might result from the favourably intramolecular and intermolecular crosslinks driven by the free energy (Atipairin et al, 2010).…”

“…After heating to 95°C, the measurement at 20°C was also performed. The CD spectra were plotted between the mean residue ellipticity and wavelength (Atipairin et al, 2010). Fig.…”

“…Samples were incubated in the dark at ambient temperature for 24 h before CD measurements. The CD signals at 208 nm were measured, and the unfolded fraction as a function of temperature was plotted (Atipairin et al, 2010).…”