Binding Sites of a Positron Emission Tomography Imaging Agent in Alzheimer's β-Amyloid Fibrils Studied Using 19F Solid-State NMR

Amyloid imaging by positron emission tomography (PET) is an important method for diagnosing neurodegenerative disorders such as Alzheimer's disease. Many C-11- and F-18-labeled PET tracers show varying binding capacities, specificities, and affinities for their target proteins. The structural basis of these variations is poorly understood. Here we employ F-19 and C-13 solid-state NMR to investigate the binding sites of a PET ligand, flutemetamol, to the 40-residue Alzheimer's beta-amyloid peptide (A beta 40). Analytical high-performance liquid chromatography and F-19 NMR spectra show that flutemetamol binds the current A beta 40 fibril polymorph with a stoichiometry of one ligand per four to five peptides. Half of the ligands are tightly bound while the other half are loosely bound. C-13 and N-15 chemical shifts indicate that this A beta 40 polymorph has an immobilized N-terminus, a non-beta-sheet His14, and a non-beta-sheet C-terminus. We measured the proximity of the ligand fluorine to peptide residues using F-19-C-13 and F-19-H-1 rotational-echo double-resonance (REDOR) experiments. The spectra show that three segments in the peptide, (VHH14)-V-12, (VFF20)-V-18, and (VV40)-V-39, lie the closest to the ligand. REDOR-constrained docking simulations indicate that these three segments form multiple binding sites, and the ligand orientations and positions at these sites are similar across different A beta polymorphs. Comparison of the flutemetamol-interacting residues in A beta 40 with the small-molecule binding sites in other amyloid proteins suggest that conjugated aromatic compounds preferentially bind beta-sheet surface grooves lined by aromatic, polar, and charged residues. These motifs may explain the specificity of different PET tracers to different amyloid proteins.