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.
Subject(s)
Cisplatin/analogs & derivatives , Cisplatin/chemistry , Metallochaperones/chemistry , Amino Acid Sequence , Antineoplastic Agents/chemistry , Copper/chemistry , Copper Transport Proteins , Hepatocytes/drug effects , Humans , Hydrogen-Ion Concentration , Ions/chemistry , Ligands , Liver/drug effects , Molecular Chaperones , Molecular Sequence Data , Oxidation-Reduction , Peptides/chemistry , Protein Binding , Protein Conformation , Protons , Spectrometry, Mass, Electrospray Ionization , Tandem Mass Spectrometry , Transition Elements/chemistry , Trypsin/chemistry , trans-Golgi Network/chemistryABSTRACT
Expression of the periplasmic protein PcoE of Escherichia coli is induced strongly by cupric salts under the control of the chromosomal copper tolerance system cusRS. Its isolation and study were complicated by de-amidation of Asn 54 and 103 at alkaline pH. Its apo form is essentially unstructured in solution and can be likened to a large unstructured multidentate ligand carrying multiple metal binding sites (15 Met; 10 His; 13 Asp, Glu; 10 Asn; 6 Lys). As expected, it binds multiple soft metal ions Cu(+) and Ag(+) non-cooperatively with the highest affinity for Cu(I) in the picomolar range (K(D)~10(-12) M). Binding of multiple soft ions induced dimerization and formation of some α-helical structure. PcoE also binds the harder metal ions Cu(2+) or Zn(2+) but with lower affinities and in smaller numbers. Cu(II) bound in PcoE is reduced readily to more tightly bound Cu(I). Overall, these properties mean that it is difficult to characterize individual species of defined metal content. Similar properties and difficulties have been reported for the homologous silver-binding protein SilE from Salmonella. However, the properties are consistent with a role for PcoE as a 'metal sponge' acting as a first line of defence against metal toxicity (under the control of the copper tolerance system cusRS) until the copper resistance operon pcoABCD is expressed.
Subject(s)
Copper , Drug Resistance, Bacterial , Escherichia coli Proteins , Gene Expression Regulation, Bacterial , Periplasmic Proteins , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Cations, Divalent/chemistry , Cations, Divalent/metabolism , Cations, Divalent/pharmacology , Copper/chemistry , Copper/metabolism , Copper/pharmacology , Escherichia coli/chemistry , Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Periplasmic Proteins/chemistry , Periplasmic Proteins/metabolism , Protein Binding , Protein Structure, Secondary , Zinc/chemistry , Zinc/metabolismABSTRACT
The chaperone protein CopC from Pseudomonas syringae features high-affinity binding sites (K (D) ~ 10(-13) M) for both Cu(I) (Met-rich) and Cu(II) (His-rich). When presented with these sites in the apoprotein, electrospray ionisation mass spectrometry confirmed that cis-Pt(NH(3))(2)Cl(2) (cisplatin) and the fragments [Pt(II)L](2+) (L is 1,2-diaminoethane, 2,2'-bipyridine) occupied the Cu(I) site specifically in the 1:1 Pt-CopC adducts (purified by cation-exchange chromatography). The cis-Pt(NH(3))(2) fragment was not present in these adducts (the dominant product for cisplatin was Pt-CopC in which all original ligands were displaced), while bidentate ligands L were retained in LPt-CopC adducts. In the context of the Met-rich Cu(I) pump Ctr1 as a significant entry point for cisplatin into mammalian cells, the present work confirms the ability of Met-rich sites in proteins to remove all ligands from cisplatin. It focuses attention on the potential of proteins that are part of the natural copper transport pathways to sequester the drug. These pathways are worthy of further study at the molecular level.
