Molecular Mechanism for the (−)-Epigallocatechin Gallate-Induced Toxic to Nontoxic Remodeling of Aβ Oligomers

Abstract

(-)-Epigallocatechin gallate (EGCG) effectively reduces the cytotoxicity of the Alzheimer's disease β-amyloid peptide (Aβ) by remodeling seeding-competent Aβ oligomers into off-pathway seeding-incompetent Aβ assemblies. However, the mechanism of EGCG-induced remodeling is not fully understood. Here we combine N andH dark-state exchange saturation transfer (DEST), relaxation, and chemical shift projection NMR analyses with fluorescence, dynamic light scattering, and electron microscopy to elucidate how EGCG remodels Aβ oligomers. We show that the remodeling adheres to a Hill-Scatchard model whereby the Aβ(1-40) self-association occurs cooperatively and generates Aβ(1-40) oligomers with multiple independent binding sites for EGCG with a K ∼10-fold lower than that for the Aβ(1-40) monomers. Upon binding to EGCG, the Aβ(1-40) oligomers become less solvent exposed, and the β-regions, which are involved in direct monomer-protofibril contacts in the absence of EGCG, undergo a direct-to-tethered contact shift. This switch toward less engaged monomer-protofibril contacts explains the seeding incompetency observed upon EGCG remodeling and suggests that EGCG interferes with secondary nucleation events known to generate toxic Aβ assemblies. Unexpectedly, the N-terminal residues experience an opposite EGCG-induced shift from tethered to direct contacts, explaining why EGCG remodeling occurs without release of Aβ(1-40) monomers. We also show that upon binding Aβ(1-40) oligomers the relative positions of the EGCG B and D rings change with respect to that of ring A. These distinct structural changes occurring in both Aβ(1-40) oligomers and EGCG during remodeling offer a foundation for understanding the molecular mechanism of EGCG as a neurotoxicity inhibitor. Furthermore, the results reported here illustrate the effectiveness of DEST-based NMR approaches in investigating the mechanism of low-molecular-weight amyloid inhibitors.

Formation of pH‐Resistant Monodispersed Polymer–Lipid Nanodiscs

“…Them agnitude of the observed quadrupolar coupling is dependent on the orientation of the quaternary ammonium C b À Nb ond vector with respect to the magnetic field direction (see the Supporting Information). The observed quadrupolar coupling magnitude of about 7.6 kHz arises from the choline group of DMPC and confirms the uniaxial orientation of nanodiscs with the lipid bilayer normal perpendicular to the magnetic field direction in agreement with 31 PNMR data, as reported for other polymer nanodiscs. [18] Theo bserved quadrupolar couplings of amagnitude of about 2.9 kHz and about 14 kHz are from the quaternary ammonium group of the SMA-QA polymer.T he presence of these two distinct doublets suggests that SMA-QA polymer belt completely surrounds the lipid bilayer in ah ighly ordered fashion and with orientations as shown in Figure 4e,w hich is similar to the orientations of detergent molecules in aligned bicelles.…”

“…[19] Additional experiments using paramagnetic lanthanide ions to tilt the orientation of lipid macro-nanodiscs would reveal the exact orientations of the polymer molecules.A se xpected, the 14 NN MR spectrum of small nanodiscs (ca. 10 nm diameter) showed asingle narrow peak at the isotropic chemical shift value (0 ppm) suggesting the fast tumbling nature of isotropic nanodiscs in agreement with 31 PNMR data. Them ajor disadvantages of SMA and other polymers used to form nanodiscs is their poor stability towards pH and metal ions.…”

“…[14,[21][22][23][24] Because of these unique properties of SMA-QA polymer nanodiscs, SMA-QA is ar obust membrane mimetic tool that offers significant advantages over all currently reported nanodisc systems,a nd therefore we foresee as ignificant expansion in the applicability of nanodisc technology.Weexpect direct and immediate impacts in the structural biology studies of those membrane proteins that are sensitive to pH and divalent metal ions [25][26][27][28][29] and amyloid proteins that self-assemble at the membrane interface. [30,31]…”

“…a)31 Pand b)14 NNMR spectra of magnetically aligned large-size(ca. a)31 Pand b)14 NNMR spectra of magnetically aligned large-size(ca.…”

“…SMA-QA nanodiscs exhibit magnetic alignment and remarkable tolerance to pH and divalent metal ions. a)31 Pand b)14 NNMR spectra of magnetically aligned large-size(ca. 30 nm diameter) nanodiscs made from DMPC:SMA-QA( 1:0.25 w/w).…”