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

Numerous conflicting models have been proposed regarding the nature of the Cu (2+) coordination environment of the amyloid beta (Abeta) peptide, the causative agent of Alzheimer's disease. This study used multifrequency CW-EPR spectroscopy to directly resolve the superhyperfine interactions between Cu (2+) and the ligand nuclei of Abeta, thereby avoiding ambiguities associated with introducing point mutations. Using a library of Abeta16 analogues with site-specific (15) N-labeling at Asp1, His6, His13, and His14, numerical simulations of the superhyperfine resonances delineated two independent 3N1O Cu (2+) coordination modes, N (a) (D1), O, N (epsilon) (H6), N (epsilon) (H13) (component Ia) 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 (17) O-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 (15) N-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 (13) C-labeling of Asp1 revealed (13) C cross-peaks characteristic of equatorial coordination by the carboxylate oxygen of Asp1 in component Ia/b coordination. The direct resolution of Cu (2+) 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 Cu (2+) /Abeta interactions.