Biochemical and biophysical characterization of ruthenation of BRCA1 RING protein by RAPTA complexes and its E3 ubiquitin ligase activity

RAPTA compounds, ([Ru(η⁶-arene)(PTA)Cl₂], PTA = 1,3,5-triaza-7-phosphaadamantane), have been reported to overcome drug resistance in cisplatin resistant cells. However, the exact mechanism of these complexes is still largely unexplored. In this study, the interaction of some RAPTA compounds with the N-terminal fragment of the BRCA1 RING domain protein was investigated. The binding of the RAPTA compounds to the BRCA1 protein resulted in a release of Zn²⁺ ions in a dose and time dependent manner, as well as thermal alteration of ruthenated-BRCA1 proteins. Electron Transfer Dissociation (ETD) fragmentation mass spectrometry revealed the preferential binding sites of the RAPTA complexes on the BRCA1 zinc finger RING domain at a similar short peptide stretch, Cys²⁴Lys²⁵Phe²⁶Cys²⁷Met²⁸Leu²⁹ and Lys³⁵ (residues 44–49 and 55 on full length BRCA1). Changes in the conformation and binding constants of ruthenium-BRCA1 adducts were established, resulting in inactivation of the RING heterodimer BRCA1/BARD1-mediated E3 ubiquitin ligase function. These findings could provide mechanistic insight into the mode of action of RAPTA complexes for on tested BRCA1 model protein.