The amyloid-Congo red interface at atomic resolution.

Amyloids are b-sheet-rich proteinaceous aggregates that are a pathological hallmark of a number of human diseases. They can replicate by seeded nucleation and propagate as prions, and may even exert important physiological activities. Amyloids are universally defined by their stainability with Congo red and the resulting green birefringence. Yet, remarkably, the binding mechanism, geometry, and fine structure of the Congo red/amyloid complex are not known. By using solid-state NMR spectroscopy we have characterized, at atomic resolution, the binding interface between Congo red and amyloid fibrils formed by the prion domain of the fungal HET-s protein. The dye binds highly sitespecifically by interacting with residues flanking a groove in the vicinity of a b-arc. The three-dimensional (3D) structure of the fibril is strongly conserved upon the binding of Congo red. Remarkably, a single point mutation, designed according to the binding information, provides an artificial amyloid structurally indistinguishable from HET-s but not stainable by Congo red. The methods used require no isotope labeling of the small molecule and can be used to characterize the interaction of a broad range of dyes, drugs, and tracers with amyloids or other insoluble proteins. Congo red (Figure S1 in the Supporting Information) is a small molecule that reacts specifically with amyloids and has been used since the 1920s as the analytical “gold standard” for amyloid characterization and diagnostics. Despite its binding specificity and its antiamyloidogenic properties, the use of Congo red as a molecular amyloid tracer in vivo and as an antiamyloid drug has been hindered by its high toxicity and poor pharmacokinetics. Contradictory reports exist on the nature of the interaction between Congo red and amyloid fibrils, but the following facts are generally agreed upon: Congo red binds roughly stochiometrically, 9] and the bound form displays a characteristic red-shift in its absorbance maximum, and shows dichroism and birefringence. 10] The negatively charged sulfate groups of Congo red are believed to be relevant for binding to two sites separated by a distance of approximately 20 . In the absence of atomic-resolution structure information, the binding mode is still debated. The long axis of the dye has been postulated to orient parallel or perpendicular to the fibril axis, and the dye binds as a monomer, oligomer, or micelle, or intercalated between the b-sheets. Hydrogen bonds and hydrophobic, aromatic, and ionic interactions have been assumed to contribute to the binding of Congo red to amyloid fibrils. Also, different side chains from residues such as histidine, arginine, and lysine have been suspected to promote congophilia. Atomic-resolution structures of amyloids, which have become available recently, enable studies of the interplay between these proteins and their binding partners in detail. HET-s(218–289), the prion-forming domain of the HET-s prion from the filamentous fungus Podospora anserina, is presently one of the structurally most precisely defined amyloids. To identify the protein surface that interacts with binding partners by NMR spectroscopy, we set out to detect characteristic chemical-shift perturbations (CSP) upon binding by comparing two-dimensional (2D) C–C correlation spectra (proton-driven spin diffusion, PDSD) of stained and unstained [C,N]-labeled fibrils. Since CSPs can be allosteric, we also performed polarization-transfer (PT) experiments which directly probe the spatial proximity of ligand and protein by exploiting the pronounced distance dependence (r ) of the dipolar interaction. Previously published PT approches have proven difficult for our system as they require isotope-labeled Congo red. We have therefore devised a method relying on polarization transfer from ligand H atoms to protein C atoms, which uses standard [H,C,N]-isotope-labeled protein but does not require isotopically labeled ligand. In such samples, the protons from Congo red are the unique polarization source in H–C polarization-transfer experiments, and the C signals detected identify residues in spatial proximity to Congo red (< 4 ). For enhanced spectral resolution, the C atoms are detected in a two-dimensional correlation experiment (dipolar recoupling enhanced by amplitude modulation, DREAM). The main results of the NMR experiments are summarized in Figure 1; details are given in Figures S2–S4 in the [*] A. K. Sch tz, A. Soragni, Dr. M. Ernst, Prof. B. H. Meier Physikalische Chemie, ETH Z rich, 8093 Z rich (Switzerland) E-mail: beme@ethz.ch