Pleomorphic Copper Coordination by Alzheimer’s Disease Amyloid-β Peptide

Numerous conflicting models have been proposed regarding the nature of the Cu2+ coordination environment of the amyloid β (Aβ) peptide, the causative agent of Alzheimer’s disease. This study used multifrequency CW-EPR spectroscopy to directly resolve the superhyperfine interactions between Cu2+ and the ligand nuclei of Aβ, thereby avoiding ambiguities associated with introducing point mutations. Using a library of Aβ16 analogues with site-specific 15N-labeling at Asp1, His6, His13, and His14, numerical simulations of the superhyperfine resonances delineated two independent 3N1O Cu2+ coordination modes, {Na D1, O, Nε H6, Nε H13} (component Ia) and {Na D1, O, Nε H6, Nε H14} (component Ib), between pH 6−7. A third coordination mode (component II) was identified at pH 8.0, and simulation of the superhyperfine resonances indicated a 3N1O coordination sphere involving nitrogen ligation by His6, His13, and His14. No differences were observed upon 17O-labeling of the phenolic oxygen of Tyr10, confirming it is not a key oxygen ligand in the physiological pH range. Hyperfine sublevel correlation (HYSCORE) spectroscopy, in conjunction with site-specific 15N-labeling, provided additional support for the common role of His6 in components Ia and Ib, and for the assignment of a {O, Nε H6, Nε H13, Nε H14} coordination sphere to component II. HYSCORE studies of a peptide analogue with selective 13C-labeling of Asp1 revealed 13C cross-peaks characteristic of equatorial coordination by the carboxylate oxygen of Asp1 in component Ia/b coordination. The direct resolution of Cu2+ ligand interactions, together with the key finding that component I is composed of two distinct coordination modes, provides valuable insight into a range of conflicting ligand assignments and highlights the complexity of Cu2+/Aβ interactions.