High-resolution NMR studies of the zinc-binding site of the Alzheimer's amyloid β-peptide

Abstract

Metal binding to the amyloid β-peptide is suggested to be involved in the pathogenesis of Alzheimer's disease. We used high-resolution NMR to study zinc binding to amyloid β-peptide 1-40 at physiologic pH. Metal binding induces a structural change in the peptide, which is in chemical exchange on an intermediate rate, between the apo-form and the holo-form, with respect to the NMR timescale. This causes loss of NMR signals in the resonances affected by the binding. Heteronuclear correlation experiments, 15N-relaxation and amide proton exchange experiments on amyloid β-peptide 1-40 revealed that zinc binding involves the three histidines (residues 6, 13 and 14) and the N-terminus, similar to a previously proposed copper-binding site [Syme CD, Nadal RC, Rigby SE, Viles JH (2004) J Biol Chem279, 18169-18177]. Fluorescence experiments show that zinc shares a common binding site with copper and that the metals have similar affinities for amyloid β-peptide. The dissociation constant Kd of zinc for the fragment amyloid β-peptide 1-28 was measured by fluorescence, using competitive binding studies, and that for amyloid β-peptide 1-40 was measured by NMR. Both methods gave Kd values in the micromolar range at pH 7.2 and 286 K. Zinc also has a second, weaker binding site involving residues between 23 and 28. At high metal ion concentrations, the metal-induced aggregation should mainly have an electrostatic origin from decreased repulsion between peptides. At low metal ion concentrations, on the other hand, the metal-induced structure of the peptide counteracts aggregation. © 2007 The Authors.