Copper(II) Binding to Amyloid-β Fibrils of Alzheimer’s Disease Reveals a Picomolar Affinity: Stoichiometry and Coordination Geometry Are Independent of Aβ Oligomeric Form

Cu2+ ions are found concentrated within senile plaques of Alzheimer’s disease patients directly bound to amyloid-β peptide (Aβ) and are linked to the neurotoxicity and self-association of Aβ. The affinity of Cu2+ for monomeric Aβ is highly disputed, and there have been no reports of affinity of Cu2+ for fibrillar Aβ. We therefore measured the affinity of Cu2+ for both monomeric and fibrillar Aβ(1−42) using two independent methods: fluorescence quenching and circular dichroism. The binding curves were almost identical for both fibrillar and monomeric forms. Competition studies with free glycine, l-histidine, and nitrilotriacetic acid (NTA) indicate an apparent (conditional) dissociation constant of 10−11 M, at pH 7.4. Previous studies of Cu-Aβ have typically found the affinity 2 or more orders of magnitude weaker, largely because the affinity of competing ligands or buffers has been underestimated. Aβ fibers are able to bind a full stoichiometric complement of Cu2+ ions with little change in their secondary structure and have coordination geometry identical to that of monomeric Aβ. Electron paramagnetic resonance studies (EPR) with Aβ His/Ala analogues suggest a dynamic view of the tetragonal Cu2+ complex, with axial as well as equatorial coordination of imidazole nitrogens creating an ensemble of coordination geometries in exchange between each other. Furthermore, the N-terminal amino group is essential for the formation of high-pH complex II. The Aβ(1−28) fragment binds an additional Cu2+ ion compared to full-length Aβ, with appreciable affinity. This second binding site is revealed in Aβ(1−42) upon addition of methanol, indicating hydrophobic interactions block the formation of this weaker carboxylate-rich complex. A Cu2+ affinity for Aβ of 1011 M−1 supports a modified amyloid cascade hypothesis in which Cu2+ is central to Aβ neurotoxicity.