Single-Molecule Mechanochemical Mapping of Hierarchical Cu Coordination in High-Capacity Copper Storage Protein Csp1

Copper is an essential yet potentially toxic transition metal, necessitating precise cellular mechanisms for its storage and regulation. Copper storage proteins (Csp) are pivotal for maintaining cellular copper homeostasis, yet the hierarchical binding sequence through which these proteins accommodate multiple Cu(I) ions remains largely unknown. Here, we applied single-molecule force spectroscopy combined with copper titration and targeted mutagenesis to dissect the Cu(I)-coordination mechanism in bacterial Csp1 at single-molecule resolution. Our approach identifies discrete unfolding intermediates corresponding to specific metalation events, uniquely establishing Cu2 as the primary and highest-affinity binding site under copper-limiting conditions. Subsequently, Cu2 nucleates the formation of a structurally essential tetranuclear copper cluster (Cu1, Cu2, Cu3, Cu4) and an independently stabilized Cu9 site, and then the rest to 13 Cu. These mechanistic insights, inaccessible to traditional ensemble methods, reveal how structural flexibility facilitates ordered metal coordination within a hierarchical network of cysteine-coordinated clusters, each serving as a discrete mechanical checkpoint. This work advances our molecular understanding of copper homeostasis and provides a unique and complementary method applicable to studying metal-binding processes in diverse metalloproteins.