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 beta (A beta) 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 A,8, thereby avoiding ambiguities associated with introducing point mutations. Using a library of A,816 analogues with site-specific N-15-labeling at Asp1, His6, His13, and His14, numerical simulations of the superhyperfine resonances delineated two independent 3N1O Cu2+ coordination modes, {N-a(D1), O, N-epsilon(H6), N-epsilon(H13)} (component la) and {N-a(D1), O, N-epsilon(H6), N-epsilon(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 O-17-labeling of the phenolic oxygen of Tyr-10, confirming it is not a key oxygen ligand in the physiological pH range. Hyperfine sublevel correlation (HYSCORE) spectroscopy, in conjunction with site-specific N-15-labeling, provided additional support for the common role of His6 in components Ia and Ib, and for the assignment of a {O, N-epsilon(H6), N-epsilon(H13), N-epsilon(H14)) coordination sphere to component II. HYSCORE studies of a peptide analogue with selective C-13-labeling of Asp1 revealed C-13 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+/A beta interactions.