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

## TL;DR

This study reveals how copper binds to serum albumin through a primary site and several secondary sites, offering new insights into copper transport in the blood.

## Contribution

The first X-ray crystal structure of mammalian serum albumin bound to copper is reported, revealing a dynamic multi-site binding model.

## Key findings

- A high-affinity ATCUN site with square-planar geometry was identified as the primary copper-binding site.
- Five secondary copper-binding sites, including histidine-containing sites, were observed.
- EPR and structural simulations showed that multiple sites can be occupied simultaneously, depending on copper concentration.

## Abstract

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.

This work presents the first X-ray crystal structure of a mammalian serum albumin bound to copper(ii), revealing dynamic, multi-site binding that integrates a square-planar ATCUN site with secondary histidine-containing sites.