The stepwise dissociation of the Zn(II)-bound Atox1 homodimer and its energetic asymmetry

The copper chaperone Atox1, which is featured by a peculiar CxxC metal-binding motif, is a protein involved in copper trafficking to the Golgi organelle, allowing the delivery of copper ions to specific target proteins in the secretory pathway. Various experimental studies suggest that this cysteine-containing protein may also be implicated in Zn(II) ion binding, functioning as a regulatory factor. However, despite the relevance of this interaction, the mechanism of zinc ion dissociation from the Atox1 homodimer has not yet been elucidated. We herein devised a simulation workflow based on the combination of parallel bias metadynamics and free energy perturbation theory, to predict the dissociation of the zinc ion from the Atox1 homodimer. The freeenergy simulations successfully predicted the dissociation free energy differences of the process, disclosing a mechanism based on a stepwise dissociation pathway of the zinc ion from the Atox1 homodimer.