Cu(I) and Cu(II) binding by hyperactive variants of the human CTR1 N-terminus: Insights from cellular and model...

Copper (Cu) trafficking requires precise control of Cu(II)/Cu(I) redox chemistry to enable cellular acquisition while preventing toxicity. Human Cu transporter 1 protein (hCTR1) mediates cellular Cu uptake, yet key molecular details governing its extracellular Cu acquisition remain unresolved. Although hCTR1 is a Cu(I)-selective pore, extracellular Cu may be presented by carriers in the Cu(II) oxidation state. The N-terminal ectodomain of hCTR1 contains adjacent high-affinity Cu(II) and Cu(I)-binding sites that are proposed to facilitate Cu(II) reduction prior to transport. To investigate the role of residues in mediating Cu uptake, we combined cellular screening for hCtr1 mutations that confer enhanced Cu uptake phenotypes with spectroscopic studies of model peptides. We identified four site-specific substitutions within the distal ectodomain (D2N, M7T, G8E, and M9T) that increase cellular Cu uptake. Using model peptides corresponding to the first 14 residues of hCTR1 (hCTR_1-14; sequence MDHSHHMGMSYMDS-am), we evaluated the effect of each substitution on the Cu-binding affinity, coordination structure, and redox kinetics. The mutations occur in residues comprising a high-affinity amino terminal Cu(II)-Ni(II)-binding (ATCUN) motif and an adjacent Met-rich MXM sequence. Despite the gain-of-function phenotypes, spectroscopic and competition studies on model peptides reveal no detectable changes in Cu(I) affinity or Cu coordination structures for any variant. However, the substitution of D2 to N within the Cu(II)-binding ATCUN motif measurably altered Cu(II) affinity and reduction rate. This finding demonstrates that D2 can modulate ATCUN stability and redox behavior, potentially tuning Cu(II) reduction and transfer during Cu uptake. The implications of these findings for understanding mammalian CTR1 proteins are discussed.