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 β (Aβ) 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 Aβ with Cu²⁺ triggers the production of neurotoxic reactive oxygen species (ROS) such as H₂O₂. However, detailed binding sites and binding structures of Cu²⁺ to Aβ are still largely unknown for Aβ fibrils or other aggregates of Aβ. In this work, we examined molecular details of Cu²⁺ binding to amyloid fibrils by detecting paramagnetic signal quenching in 1D and 2D high-resolution ¹³C solid-state NMR (SSNMR) for full-length 40-residue Aβ(1-40). Selective quenching observed in ¹³C SSNMR of Cu²⁺-bound Aβ(1-40) suggested that primary Cu²⁺ binding sites in Aβ(1-40) fibrils include N_ε 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). ¹³C 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 ¹³C_εH₃-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⁺-Aβ 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 Aβ fibril, which are realized by both intra- and intermolecular contacts and highly concentrated coordination sites due to the in-register parallel β-sheet arrangements.