Cisplatin binds human copper chaperone Atox1 and promotes unfolding in vitro

Abstract

Cisplatin (cisPt), Pt(NH(3))(2)Cl(2), is a cancer drug believed to kill cells via DNA binding and damage. Recent work has implied that the cellular copper (Cu) transport machinery may be involved in cisPt cell export and drug resistance. Normally, the Cu chaperone Atox1 binds Cu(I) via two cysteines and delivers the metal to metal-binding domains of ATP7B; the ATP7B domains then transfer the metal to the Golgi lumen for loading on cuproenzymes. Here, we use spectroscopic methods to test if cisPt interacts with purified Atox1 in solution in vitro. We find that cisPt binds to Atox1's metal-binding site regardless of the presence of Cu or not: When Cu is bound to Atox1, the near-UV circular dichroism signals indicate Cu-Pt interactions. From NMR data, it is evident that cisPt binds to the folded protein. CisPt-bound Atox1 is however not stable over time and the protein begins to unfold and aggregate. The reaction rates are limited by slow cisPt dechlorination. CisPt-induced unfolding of Atox1 is specific because this effect was not observed for two unrelated proteins that also bind cisPt. Our study demonstrates that Atox1 is a candidate for cisPt drug resistance: By binding to Atox1 in the cytoplasm, cisPt transport to DNA may be blocked. In agreement with this model, cell line studies demonstrate a correlation between Atox1 expression levels, and cisplatin resistance.

Fig. 1.

Near-UV CD-monitored titrations of metals to apo/metallated Atox1 (pH 6, 20 °C): (A) 50 μM apo-Atox1 and 50 μM Cu-Atox1 (1∶1); (B) CisPt (up to fivefold excess as indicated) added to 50 μM Cu-Atox1; (C) CisPt (up to fivefold excess as indicated) added to 50 μM apo-Atox1; (D) Cu (up to fivefold excess as indicated) added to 50 μM cisPt-Atox1 (1∶1).

Fig. 2.

(A) Far-UV CD spectra as a function of time (0 h to 14 d) for apo-Atox1 (50 μM) mixed with 5 eq cisPt and incubated at 20 °C. Time points are 0, 5, 8, 24, 29, 32, 48, 73, 144, 168, 240, and 336 h. (B) CD changes at 220 nm (at 20 °C) plotted as a function of time for apo-Atox1 alone, apo-Atox1 (50 μM) mixed with 1 or 5 eq cisPt, and for Cu-Atox1 (50 μM) mixed with 1 or 5 eq cisPt. Also shown are a few time points for 0.5 mM apo-Atox1 mixed with 5 eq cisPt.

Fig. 3.

Interaction between cisPt and apo-Atox1 probed with NMR spectroscopy. ¹H-¹⁵N HSQC NMR spectra of apo-Atox1 (green contours) superimposed on that of a 2∶1 cisPt:Atox1 complex (orange contours). Selected peaks corresponding to the folded cisPt:Atox1 complex (*) and unfolded species (#) are indicated.

Fig. 4.

CisPt-induced unfolding of Atox1. ¹H-¹⁵N HSQC NMR spectra acquired after 4, 8, and 16 h after mixing of a 5∶1 cisPt:apo-Atox1 sample.