Oxaliplatin Binding to Human Copper Chaperone Atox1 and Protein Dimerization.
Copper trafficking proteins have been implicated in the cellular response to platinum anticancer drugs. We investigated the reaction of the chaperone Atox1 with an activated form of oxaliplatin, the third platinum drug to reach worldwide approval. Unlike cisplatin, which contains monodentate ammines, oxaliplatin contains chelated 1,2-diaminocyclohexane (DACH), which is more resistant to displacement by nucleophiles. In solution, one or two {Pt(DACH)(2+)} moieties bind to the conserved CXXC metal-binding motif of Atox1; in the latter case the two sulfur atoms likely bridging the two platinum units. At longer reaction times, a dimeric species is formed whose composition, Atox12·Pt(2+)2, indicates complete loss of the diamine ligands. Such a dimerization process is accompanied by partial unfolding of the protein. Crystallization experiments aiming at the characterization of the monomeric species have afforded, instead, a dimeric species resembling that already obtained by Boal and Rosenzweig in a similar reaction performed with cisplatin. However, while in the latter case there was only one Pt-binding site (0.4 occupancy) made of four sulfur atoms of the CXXC motifs of the two Atox1 chains in a tetrahedral arrangement, we found, in addition, a secondary Pt-binding site involving Cys41 of the B chain (0.25 occupancy). Moreover, both platinum atoms have lost their diamines. Thus, there appears to be little relationship between what is observed in solution and what is formed in the solid state. Since full occupancy of the tetrahedral cavity is a common feature of all Atox1 dimeric structures obtained with other metal ions (Cu(+), Cd(2+), and Hg(2+)), we propose that in the case of platinum, where the occupancy is only 0.4, the remaining cavities are occupied by Cu(+) ions. Experimental evidence is reported in support of the latter hypothesis. Our proposal represents a meeting point between the initial proposal of Boal and Rosenzweig (0.4 Pt occupancy) and the reinterpretation of the original crystallographic data put forward by Shabalin et al. (1 Cu occupancy), and could apply to other cases.
Institute of Crystallography, Consiglio Nazionale delle Ricerche , via Amendola 122/o, 70126 Bari, Italy.
Benny D
BD
Belviso
Department of Chemistry, University of Bari "A. Moro" , via E. Orabona 4, 70125 Bari, Italy.
Angela
A
Galliani
Department of Chemistry, University of Bari "A. Moro" , via E. Orabona 4, 70125 Bari, Italy.
Alessia
A
Lasorsa
Institute of Crystallography, Consiglio Nazionale delle Ricerche , via Amendola 122/o, 70126 Bari, Italy.|Department of Economics, University of Foggia , Via A. Gramsci 89/91, 71122 Foggia, Italy.
Valentina
V
Mirabelli
Institute of Crystallography, Consiglio Nazionale delle Ricerche , via Amendola 122/o, 70126 Bari, Italy.
Rocco
R
Caliandro
Department of Chemistry, University of Bari "A. Moro" , via E. Orabona 4, 70125 Bari, Italy.
Fabio
F
Arnesano
Department of Chemistry, University of Bari "A. Moro" , via E. Orabona 4, 70125 Bari, Italy.
Giovanni
G
Natile
D001665:Binding Sites; D003300:Copper / Q000737:chemistry / Q000378:metabolism*; D000079922:Copper Transport Proteins; D018360:Crystallography, X-Ray; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D056511:Metallochaperones / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D018832:Molecular Chaperones; D009944:Organoplatinum Compounds / Q000737:chemistry / Q000378:metabolism*; D000077150:Oxaliplatin; D013057:Spectrum Analysis / Q000379:methods
C474500:ATOX1 protein, human; D000079922:Copper Transport Proteins; D056511:Metallochaperones; D018832:Molecular Chaperones; D009944:Organoplatinum Compounds; D000077150:Oxaliplatin; D003300:Copper