Utilizing EPR Spectroscopy to Target Structural and Dynamics Changes in the Human ATP7B Protein upon Copper Coordination

Copper is an essential element for living systems because it is a cofactor for many enzymes. Copper is consumed in our body through diet, however, it must be safely transported and regulated in the cell to avoid harmful effects such as oxidative damage.^[1]

It is known that Cu(II) is accumulated in our body and transferred to the cell through the main copper transporter, CTR1. CTR1 reduces it to Cu(I) state, using ascorbate, and then transfer it into the cytoplasmic domain. In cellular environment, Cu(I) is distributed to various pathways, to the Golgi, mitochondria, and SOD.^[2,3] There are two important genes: ATP7B and ATP7A, which encode copper-transporting P-type ATPase, which play central roles in copper transport and homeostasis. These proteins use ATP to transport copper across the membrane against an electrochemical concentration gradient. ATP7A/B receive Cu(I) from the copper chaperone, Atox1, and they are responsible to Cu(I) translocation from the cytosol into the Golgi for later incorporation into various copper enzymes. In addition, under elevated Cu(I) stress, ATP7A/B proteins export Cu(I) from the cell. Genetic defects in ATP7A/B result in copper toxicity and deficiency disorders.^[4]

In this study, we are interested to obtain structural and functional information on one of these transporters, ATP7B. We would like to pinpoint critical residues that are essential both for Cu(I) coordination to ATP7B as well as for proper transfer between Atox1 and ATP7B. We use biochemical methods together with Electron Paramagnetic Resonance (EPR) spectroscopy, to follow changes in structure and dynamics of ATP7B in the presence of Cu(I) and Atox1, as a function of mutagenesis.

[1]. Keller AM, Benítez JJ, Klarin D, Zhong L, Goldfogel M, Yang F, Chen TY, Chen P, J Am Chem Soc 2012, 134, 8934-43.

[2]. Lutsenko S, Curr Opin Chem Biol 2010, 14, 211-7.

[3]. Christopher J. De FeoStephen. G. AllerVinzenz, M. Unger, BioMetals 2007, 20-705.

[4]. Negah Fatemi, Dmitry M. Korzhnev, Algirdas Velyvis, Bibudhendra Sarkar, and Julie D. Forman-Kay, Biochemistry 2010, 49, 8468–8477.