title: Interaction of cisplatin and analogue Pt(en)Cl2 with the copper metallo-chaperone Atox1.
abstract: The human metallo-chaperone protein Atox1 features a high affinity Cu(I) binding site Cys(12)GlyGlyCys(15) (KD = 10(-17.4) M at pH 7.0) and delivers copper to the trans-Golgi network (TGN). Atox1 may participate in the metabolism of the drug cis-Pt(NH3)2Cl2 (cisplatin), either as a component of its delivery to the nucleus or of its loss via transport to the TGN and beyond. The species of stoichiometry [Pt(NH3)2(Atox1)] was the sole adduct of stoichiometry Pt : Atox1 = 1 : 1 detected by mass spectrometry under non-denaturing conditions from solutions containing cisplatin and apo-Atox1. The ions [Atox1 + Pt(NH3)2(2+) + (z - 2)H(+)](z+) (z = 3 to 7) were observed and correspond to different protonation states of the 1 : 1 adduct. Adducts of stoichiometry Pt : Atox1 = 2 : 1 were also detected but 1 : 2 adducts were not detected. The related complex Pt(en)Cl2 (en = 1,2-diaminoethane) behaved similarly. Tandem mass spectrometry experiments using top-down and bottom-up sequencing techniques were carried out, respectively, on the intact platinated protein and on platinated peptides formed from proteolysis by trypsin. A new software programme (PolyCut) designed to analyse the complex high-resolution tandem mass spectra of fragment ions derived from proteins containing transition metal ions was applied to establish the binding site(s) of the platinum atom(s). The analysis, based on the entire isotope patterns, is consistent with the cysteine residues in the Cu(I)-binding sequence Cys(12)GlyGlyCys(15) being the primary coordination site.
authors.affiliation: School of Chemistry and Bio21 Institute, University of Melbourne, Victoria 3010, Australia. chakmsze@gmail.com
authors.forename: Chak Ming
authors.initials: CM
authors.lastname: Sze
authors.forename: Zhenyu
authors.initials: Z
authors.lastname: Shi
authors.forename: George N
authors.initials: GN
authors.lastname: Khairallah
authors.forename: Linda
authors.initials: L
authors.lastname: Feketeová
authors.forename: Richard A J
authors.initials: RA
authors.lastname: O'Hair
authors.forename: Zhiguang
authors.initials: Z
authors.lastname: Xiao
authors.forename: Paul S
authors.initials: PS
authors.lastname: Donnelly
authors.forename: Anthony G
authors.initials: AG
authors.lastname: Wedd
mesh_terms: D000595:Amino Acid Sequence; D000970:Antineoplastic Agents / Q000737:chemistry; D002945:Cisplatin / Q000031:analogs & derivatives* / Q000737:chemistry*; D003300:Copper / Q000737:chemistry; D000079922:Copper Transport Proteins; D022781:Hepatocytes / Q000187:drug effects; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000737:chemistry; D008024:Ligands; D008099:Liver / Q000187:drug effects; D056511:Metallochaperones / Q000737:chemistry*; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D011522:Protons; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D028561:Transition Elements / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry; D021601:trans-Golgi Network / Q000737:chemistry
chemical_list: C474500:ATOX1 protein, human; D000970:Antineoplastic Agents; D000079922:Copper Transport Proteins; D007477:Ions; D008024:Ligands; D056511:Metallochaperones; D018832:Molecular Chaperones; D010455:Peptides; D011522:Protons; D028561:Transition Elements; D003300:Copper; D014357:Trypsin; D002945:Cisplatin