The Effect of (-)-Epigallocatechin-3-Gallate on the Amyloid-β Secondary Structure

Abstract

Amyloid-β (Aβ) is a macromolecular structure of great interest because its misfolding and aggregation, along with changes in the secondary structure, have been correlated with its toxicity in various neurodegenerative diseases. Small drug-like molecules can modulate the amyloid secondary structure and therefore have raised significant interest in applications to active and passive therapies targeting amyloids. In this study, we investigate the interactions of epigallocatechin-3-gallate (EGCG), found in green tea, with Aβ polypeptides, using a combination of in vitro immuno-infrared sensor measurements, docking, molecular dynamics simulations, and ab initio calculations. We find that the interactions of EGCG are dominated by only a few residues in the fibrils, including hydrophobic π-π interactions with aromatic rings of side chains and hydrophilic interactions with the backbone of Aβ, as confirmed by extended (1-μs-long) molecular dynamics simulations. Immuno-infrared sensor data are consistent with degradation of Aβ fibril induced by EGCG and inhibition of Aβ fibril and oligomer formation, as manifested by the recovery of the amide-I band of monomeric Aβ, which is red-shifted by 26 cm^-1 when compared to the amide-I band of the fibrillar form. The shift is rationalized by computations of the infrared spectra of Aβ42 model structures, suggesting that the conformational change involves interchain hydrogen bonds in the amyloid fibrils that are broken upon binding of EGCG.

Figure 1

Illustration of fragmentation procedure for model (A) dimeric and (B) monomeric Aβ. Each fragment contains five amino acids and thus includes all possible intrachain H-bonding observed in a helical structure. The hydrogen-bonding network in (C) monomeric and (D) dimeric fibril (bottom) models is shown. Individual amino acids are shown in different colors. (E) Illustration of the setup for the divide-and-conquer approach and representation of an individual fragment subjected to partial optimization and the subsequent frequency calculation are shown. To see this figure in color, go online.

Figure 2

Experimental IR spectra of monomeric (red) and fibrillar Aβ₄₂ (blue) measured directly after 0 min. The peak of the absorption band of fibrillar Aβ₄₂ at 1628 cm⁻¹ corresponds to the amide-I stretching vibrational mode of β-sheets, which is at 1630 cm⁻¹ (31,51), indicating that Aβ₄₂ fibrils are actually immobilized on the sensor surface. The peak of monomeric Aβ₄₂ at 1654 cm⁻¹ corresponds to the amide-I stretching vibrational mode of helical or coil secondary structure, indicating a nonfibrillar structure. To see this figure in color, go online.

Figure 3

(A) Spectra of fibrillar Aβ₄₂ after immobilization on the immuno-infrared sensor and treatment with EGCG for 1 day. (B) Spectra of preincubated monomeric Aβ with EGCG, which prevents the formation of fibrillar species, are shown. The concentrations of Aβ fibril, monomer, and EGCG in (A) and (B) were 36, 36, and 320 nM, respectively. (C) Spectra corresponding to the evolution of Aβ₄₂ fibrils from monomers in the absence of EGCG are shown. (D) The chemical structure of EGCG is shown. To see this figure in color, go online.

Figure 4

(A) EGCG binding sites (numbered 1–3) in Aβ₄₂ with respective docking scores. Aβ₄₂ is shown in surface representation and colored by residue type: hydrophobic (white), acidic (red), basic (blue), and polar (green). EGCG molecules in each site are shown in van der Waals representation. (B) Interacting residues of Aβ₄₂ with EGCG in each docking pose are shown. To see this figure in color, go online.

Figure 5

Representative figures of EGCG occupying (A) site #1 and site #4, (B) site #2 and site #4, and (C) site #3 and site #4. (D) The relative populations of the docking sites from MD simulation are shown. The populations are grouped by their site numbers. Aβ42 is colored by residue type: hydrophobic (white), acidic (red), basic (blue), and polar (green). EGCG molecules are shown in van der Waals representation. To see this figure in color, go online.

Figure 6

Dimer models of fibrillar A used in the divide-and-conquer approach from four different PDB entries: (A) 2BEG, (B) 5OQV, (C) 2MXU, and (D) 2NAO. (E) Partial disruption of interchain hydrogen bonding observed for one of the structures used for computation is shown. The relevant residues are annotated in red, and the α-carbons of those residues are shown as red spheres. To see this figure in color, go online.

Figure 7

Analysis of the computed IR spectra in the 1600–1700 cm⁻¹ region. (A) Comparison of IR spectra of all the fibril models is shown. (B) A comparison of IR spectra of 2NAO structure with and without the broken interchain hydrogen-bonding residues is shown. To see this figure in color, go online.

Figure 8

Comparison of the computed amide-I band in monomeric (orange) and dimeric Aβ₄₂ (blue) in β-enriched structures. IR spectrum of monomeric Aβ₄₂ (red) is also shown for reference. To see this figure in color, go online.