Crystallographic and EPR-based characterisation of Cu²⁺-binding to serum albumin: ATCUN coordination and additional...

Copper homeostasis is essential for mammalian physiology. Serum albumin plays an important role in plasma copper transport and buffering, yet its Cu²⁺ binding sites have remained incompletely characterised. Here we report the first X-ray crystal structure of a mammalian serum albumin, in this case equine albumin, bound to copper(ii). The structure revealed a high-affinity ATCUN site with characteristic square-planar geometry. Additional Cu²⁺ binding was observed at five secondary sites, including sites A and B and other histidine-containing sites (involving either His287, His317 and His509). Continuous-wave EPR spectroscopy further supported a square-planar coordination at the ATCUN site through a low-g spectral feature appearing upon binding of less than one molar equivalent of Cu²⁺. ESEEM and HYSCORE experiments detected nuclear quadrupole interactions and weakly coupled ¹⁴N signals, supporting histidine involvement and increased water coordination at higher Cu²⁺ loading. RIDME-derived distance distributions and structural simulations indicate simultaneous occupancy of multiple sites, with strong evidence for ATCUN and site B, and partial engagement of distal histidines (His287, His317) at elevated Cu²⁺ equivalents. These findings support a dynamic, multi-site binding model in which short-range distances arise from ATCUN and site B co-occupancy, while longer-range peaks reflect contributions from distal histidine sites.