Protein Interactions that Enable Safe and Efficient Copper Ion Transport in the Human Cytoplasm

Although copper (Cu) is an essential metal for most living organisms, highlevels and free such ions are toxic. In humans, after cellular uptake via themembrane-bound importer Ctr1, Cu is transported to targets by cytoplasmicCu chaperones: Atox1 delivers Cu to membrane-bound P1B-type ATPases(i.e., ATP7B or Wilson disease protein) in the Golgi (secretory path; here,most Cu-dependent enzymes are loaded with Cu) whereas CCS delivers Cuspecifically to cytoplasmic superoxide dismutase. In contrast to bacterial andyeast homologs, ATP7B has six similar metal-binding domains protrudinginto the cytoplasm: possibly, conformational changes among these regulateoverall ATP7B activity. To reveal underlying molecular mechanisms as wellas thermodynamic and kinetic driving forces for human Cu transport - fromthe cell membrane to the Golgi - our strategy involves a range of complementary biophysical experiments on purified proteins, domain constructs and engineered variants. From our studies, we have discovered that (a) the cytoplasmic C-terminus of Ctr1 binds Cu through its HCH motif with a moderate affinity that allows for Cu delivery to Atox1, (b) Atox1 can interact with CCS and exchange Cu implying cross-reactivity between cytoplasmic chaperones, (c) transfer of Cu from Atox1 to metal-binding domains in ATP7B proceedsthrough Cu-bridged hetero-protein dimers displaying enthalpy-entropycompensation, (d) conformational changes and domain-domain interactionswithin ATP7B depend on Cu loading status and minute changes in solvent conditions, and (e), in addition to its cytoplasmic chaperone activity, Atox1 may have functionality in the nucleus as it interacted with several DNA-binding proteins in a yeast two-hybrid screen