The structural flexibility of the human copper chaperone Atox1: Insights from combined pulsed EPR studies and computations.

Metallochaperones are responsible for shuttling metal ions to target proteins. Thus, a metallochaperone's structure must be sufficiently flexible both to hold onto its ion while traversing the cytoplasm and to transfer the ion to or from a partner protein. Here, we sought to shed light on the structure of Atox1, a metallochaperone involved in the human copper regulation system. Atox1 shuttles copper ions from the main copper transporter, Ctr1, to the ATP7b transporter in the Golgi apparatus. Conventional biophysical tools such as X-ray or NMR cannot always target the various conformational states of metallochaperones, owing to a requirement for crystallography or low sensitivity and resolution. Electron paramagnetic resonance (EPR) spectroscopy has recently emerged as a powerful tool for resolving biological reactions and mechanisms in solution. When coupled with computational methods, EPR with site-directed spin labeling and nanoscale distance measurements can provide structural information on a protein or protein complex in solution. We use these methods to show that Atox1 can accommodate at least four different conformations in the apo state (unbound to copper), and two different conformations in the holo state (bound to copper). We also demonstrate that the structure of Atox1 in the holo form is more compact than in the apo form. Our data provide insight regarding the structural mechanisms through which Atox1 can fulfill its dual role of copper binding and transfer.

Ctr1 Intracellular Loop Is Involved in the Copper Transfer Mechanism to the Atox1 Metallochaperone.

Understanding the human copper cycle is essential to understand the role of metals in promoting neurological diseases and disorders. One of the cycles controlling the cellular concentration and distribution of copper involves the copper transporter, Ctr1; the metallochaperone, Atox1; and the ATP7B transporter. It has been shown that the C-terminus of Ctr1, specifically the last three amino acids, HCH, is involved in both copper coordination and the transfer mechanism to Atox1. In contrast, the role of the intracellular loop of Ctr1, which is an additional intracellular segment of Ctr1, in facilitating the copper transfer mechanism has not been investigated yet. Here, we combine various biophysical methods to explore the interaction between this Ctr1 segment and metallochaperone Atox1 and clearly demonstrate that the Ctr1 intracellular loop (1) can coordinate Cu(I) via interactions with the side chains of one histidine and two methionine residues and (2) closely interacts with the Atox1 metallochaperone. Our findings are another important step in elucidating the mechanistic details of the eukaryotic copper cycle.

SOD mimetic activity and antiproliferative properties of a novel tetra nuclear copper (II) complex.

The search for novel anticancer therapeutic agents is an urgent and important issue in medicinal chemistry. Here, we report on the biological activity of the copper-based bioinorganic complex Cu4 (2,4-di-tert-butyl-6-(1H-imidazo- [1, 10] phenanthrolin-2-yl)phenol)4]·10 CH3CN (2), which was tested in rat L6 myotubes, mouse NSC-34 motor neurone-like cells, and HepG-2 human liver carcinoma. Upon 96 h incubation, 2 exhibited a significant cytotoxic effect on all three types of cells via activation of two cell death mechanisms (apoptosis and necrosis). Complex 2 exhibited better potency and efficacy than the canonical cytotoxic drug cisplatin. Moreover, during shorter incubations, complex 2 demonstrated a significant SOD mimetic activity, and it was more effective and more potent than the well-known SOD mimetic TEMPOL. In addition, complex 2 was able to interact with DNA and, cleave DNA in the presence of sodium ascorbate. This study shows the potential of using polynuclear redox active compounds for developing novel anticancer drugs through SOD-mimetic redox pathways.

Probing the structural flexibility of the human copper metallochaperone Atox1 dimer and its interaction with the CTR1 c-terminal domain.

Both the essentiality and the toxicity of copper in human, yeast, and bacteria cells require precise mechanisms for acquisition, intimately linked to controlled distribution, which have yet to be fully understood. This work explores one aspect in the copper cycle, by probing the interaction between the human copper chaperone Atox1 and the c-terminal domain of the copper transporter, CTR1, using electron paramagnetic resonance (EPR) spectroscopy and circular dichroism (CD). The data collected here shows that the Atox1 keeps its dimer nature also in the presence of the CTR1 c-terminal domain; however, two geometrical states are assumed by the Atox1. One is similar to the geometrical state reported by the crystal structure, while the latter has not yet been constructed. In the presence of the CTR1 c-terminal domain, both states are assumed; however, the structure of Atox1 is more restricted in the presence of the CTR1 c-terminal domain. This study also shows that the last three amino acids of the CTR1 c-terminal domain, HCH, are important for maintaining the crystal structure of the Atox1, allowing less structural flexibility and improved thermal stability of Atox1.