Molecular-Level Examination of Cu2+ Binding Structure for Amyloid Fibrils of 40-Residue Alzheimer's β by Solid-State NMR Spectroscopy

Cu2+ binding to Alzheimer's beta (A beta) peptides in amyloid fibrils has attracted broad attention, as it was shown that Cu ion concentration elevates in Alzheimer's senile plaque and such association of A beta with Cu2+ triggers the production of neurotoxic reactive oxygen species (ROS) such as H2O2. However, detailed binding sites and binding structures of Cu2+ to A beta are still largely unknown for A beta fibrils or other aggregates of A beta In this work, we examined molecular details of Cu2+ binding to amyloid fibrils by detecting paramagnetic signal quenching in ID and 2D high-resolution C-13 solid-state NMR (SSNMR) for full-length 40-residue A beta(1-40). Selective quenching observed in C-13 SSNMR of Cu2+-bound A beta(1-40) suggested that primary Cu2+ binding sites in A beta(1-40) fibrils include N-epsilon in His-13 and His-14 and carboxyl groups in Val-40 as well as in Glu sidechains (Glu-3, Glu-11, and/or Glu-22). C-13 chemical shift analysis demonstrated no major structural changes upon Cu2+ binding in the hydrophobic core regions (residues 18-25 and 30-36). Although the ROS production via oxidization of Met-35 in the presence of Cu2+ has been long suspected, our SSNMR analysis of (C epsilon H3)-C-13-S- in M35 showed little changes after Cu2+ binding, excluding the possibility of Met-35 oxidization by Cu2+ alone. Preliminary molecular dynamics (MD) simulations on Cu2+ A beta complex in amyloid fibrils confirmed binding sites suggested by the SSNMR results and the stabilities of such bindings. The MD simulations also indicate the coexistence of a variety of Cu2+-binding modes unique in A beta fibril, which are realized by both intra- and intermolecular contacts and highly concentrated coordination sites due to the in-register parallel A beta-sheet arrangements.