Interaction of Alzheimer’s Aβ Peptide with an Engineered Binding Protein—Thermodynamics and Kinetics of Coupled Folding–Binding

Abstract

The oligomerization and aggregation of the amyloid-β (Aβ) peptide, a cleavage product of the amyloid precursor protein predominantly 40 or 42 amino acids in length, has been implicated in the pathogenesis of Alzheimer's disease. The identification of Aβ-binding agents, e.g., antibodies or peptides, constitutes a promising therapeutic approach. However, the amount of structural and biophysical data on the underlying Aβ interactions is currently very limited. We have earlier determined the structure of Aβ(1–40) in complex with the affibody protein ZAβ3, a selected binding protein based on a three-helix bundle scaffold (Z domain). ZAβ3 is a dimer of affibody subunits linked via a disulfide bridge involving a selected cysteine mutation at position 28. ZAβ3 binds to the central and C-terminal part of Aβ (residues 17–36), which adopts a β-hairpin conformation in the complex. Here we present a detailed biophysical analysis of the ZAβ3:Aβ(1–40) interaction, employing NMR, circular dichroism spectroscopy, 8-anilino-1-naphthalenesulfonic acid and tyrosine fluorescence, size-exclusion chromatography, thermal denaturation profiles and isothermal titration calorimetry. We conclude that (i) free ZAβ3 is characterized by conformational exchange and the loss of helix 1 of the three-helix bundle scaffold; (ii) a high-energy barrier is associated with the conversion of an initial ZAβ3:Aβ(1–40) recognition complex into the native complex structure, entailing slow binding kinetics; (iii) both Aβ and ZAβ3 fold upon binding, which, e.g., becomes manifest in the binding thermodynamics that feature a large negative change in heat capacity; (iv) the C28-disulfide does not merely afford dimerization, but its impact on the binding interfaces of the affibody subunits and Aβ is a prerequisite for tight binding. The extensive folding coupled to binding observed here likely constitutes an obligate feature of biomolecular interactions involving the central and C-terminal part of Aβ. Options for improvement of ZAβ binding proteins are discussed.