Dynamic Multi-Body Protein Interactions Suggest Versatile Pathways for Copper Trafficking

As part of intracellular copper trafficking pathways, the human copper chaperone Hah1 delivers Cu+ to the Wilson’s Disease Protein (WDP) via weak and dynamic protein–protein interactions. WDP contains six homologous metal binding domains (MBDs) connected by flexible linkers, and these MBDs all can receive Cu+ from Hah1. The functional roles of the MBD multiplicity in Cu+ trafficking are not well understood. Building on our previous study of the dynamic interactions between Hah1 and the isolated fourth MBD of WDP, here we study how Hah1 interacts with MBD34, a double-domain WDP construct, using single-molecule fluorescence resonance energy transfer (smFRET) combined with vesicle trapping. By alternating the positions of the smFRET donor and acceptor, we systematically probed Hah1–MBD3, Hah1–MBD4, and MBD3–MBD4 interaction dynamics within the multidomain system. We found that the two interconverting interaction geometries were conserved in both intermolecular Hah1–MBD and intramolecular MBD–MBD interactions...