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

摘要

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 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 Nε in His-13 and His-14, and carboxyl groups in Val-40 as well as in Glu side chains (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 ¹³CεH3-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 inter-molecular contacts and highly concentrated coordination sites due to the in-register parallel β-sheet arrangements.