An EPR Study on the Interaction between the Cu(I) Metal Binding Domains of ATP7B and the Atox1 Metallochaperone

Michael Zaccak,Sharon Ruthstein,Zena Qasem,Lada Gevorkyan-Airapetov

doi:10.3390/ijms21155536

Michael Zaccak, Sharon Ruthstein + Show 2 more

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https://doi.org/10.3390/ijms21155536

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Abstract

Copper’s essentiality and toxicity mean it requires a sophisticated regulation system for its acquisition, cellular distribution and excretion, which until now has remained elusive. Herein, we applied continuous wave (CW) and pulsed electron paramagnetic resonance (EPR) spectroscopy in solution to resolve the copper trafficking mechanism in humans, by considering the route travelled by Cu(I) from the metallochaperone Atox1 to the metal binding domains of ATP7B. Our study revealed that Cu(I) is most likely mediated by the binding of the Atox1 monomer to metal binding domain 1 (MBD1) and MBD4 of ATP7B in the final part of its extraction pathway, while the other MBDs mediate this interaction and participate in copper transfer between the various MBDs to the ATP7B membrane domain. This research also proposes that MBD1-3 and MBD4-6 act as two independent units.

Highlights

Copper is an essential trace metal ion, involved in numerous enzymatic and electron transfer reactions of the cell
Probing the Interaction between the metal binding sub-domains (MBDs) of ATP7B and the Metallochaperone Atox1 by Continuous wave (CW)-electron paramagnetic resonance (EPR) at EPR spectroscopy has emerged as an excellent tool for probing protein–protein interaction, since it does not require crystallization and does not depend on protein size
The EPR performed here supports the conclusion that metal binding domain 1 (MBD1)-3 and MBD4-6 act as independent units

Summary

Introduction

Copper is an essential trace metal ion, involved in numerous enzymatic and electron transfer reactions of the cell. Its ability to cycle between two oxidation states can result in the formation of reactive oxygen species that potentially can cause harm and cell death [1,2]. Copper homeostasis is highly controlled in all living species. The main copper import transporter Ctr, and the two P-type ATPase exporters ATP7A and ATP7B, regulate copper concentration [3,4,5]. The metallochaperone Atox is responsible for shuttling copper ions from Ctr to ATP7A/B. ATP7A/B play two roles: (1) transferring copper to the secretory pathway, where Cu(I)

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