Identification of binding sites of cisplatin to human copper chaperone protein Cox17 by high-resolution FT-ICR-MS

• Published in: Rapid communications in mass spectrometry 2016-08, Vol.30 (S1), p.168-172
• Main Authors: Li, Lijie, Guo, Wei, Wu, Kui, Zhao, Yao, Luo, Qun, Zhang, Qingwu, Liu, Jianan, Xiong, Shaoxiang, Wang, Fuyi
• Format: Article
• Language: English
• Subjects: Adducts; Binding sites; Copper; Fourier transforms; Mass spectrometry; Mitochondria; Proteins; Transportation
• ISSN: 0951-4198; 1097-0231
• DOI: 10.1002/rcm.7645

Rationale Cox17 is a key copper chaperone protein responsible for delivery of cuprous ions to mitochondria and has been demonstrated to be involved in the anticancer action of cisplatin. However, the binding sites of the drug to the protein have not yet been directly identified. Methods The recombinant protein apo‐Cox172s‐s, the functional state of Cox17 transferring Cu(I), was reacted with an excess of cisplatin to produce platinated Cox17 adducts, of which the platination sites were identified by high‐resolution Fourier transform ion cyclotron tandem mass spectrometry (FT‐ICR‐MS/MS) through electron capture dissociation (ECD). Results Primary FT‐ICR‐MS showed that mono‐platinated Cox17 adducts were the main products, and top‐down MS/MS results indicated that cisplatin bound to the Cys26 or Cys27 residue which is the binding site of cuprous ions in apo‐Cox172s‐s. Conclusions This is the first report for identification of the main binding sites of cisplatin to Cox17 by top‐down high‐resolution mass spectrometry, providing direct evidence for the competitive coordination with Cox17 of cisplatin and cuprous ions. These findings will also be helpful to understand further how Cox17 facilitates cisplatin accumulation in mitochondria, and how cisplatin disturbs the transportation of cuprous ions. Copyright © 2016 John Wiley & Sons, Ltd.