Structural Polymorphism of Alzheimer's β-Amyloid Fibrils as Controlled by an E22 Switch: A Solid-State NMR Study - PubMed (original) (raw)

Structural Polymorphism of Alzheimer's β-Amyloid Fibrils as Controlled by an E22 Switch: A Solid-State NMR Study

Matthew R Elkins et al. J Am Chem Soc. 2016.

Abstract

The amyloid-β (Aβ) peptide of Alzheimer's disease (AD) forms polymorphic fibrils on the micrometer and molecular scales. Various fibril growth conditions have been identified to cause polymorphism, but the intrinsic amino acid sequence basis for this polymorphism has been unclear. Several single-site mutations in the center of the Aβ sequence cause different disease phenotypes and fibrillization properties. The E22G (Arctic) mutant is found in familial AD and forms protofibrils more rapidly than wild-type Aβ. Here, we use solid-state NMR spectroscopy to investigate the structure, dynamics, hydration and morphology of Arctic E22G Aβ40 fibrils. (13)C, (15)N-labeled synthetic E22G Aβ40 peptides are studied and compared with wild-type and Osaka E22Δ Aβ40 fibrils. Under the same fibrillization conditions, Arctic Aβ40 exhibits a high degree of polymorphism, showing at least four sets of NMR chemical shifts for various residues, while the Osaka and wild-type Aβ40 fibrils show a single or a predominant set of chemical shifts. Thus, structural polymorphism is intrinsic to the Arctic E22G Aβ40 sequence. Chemical shifts and inter-residue contacts obtained from 2D correlation spectra indicate that one of the major Arctic conformers has surprisingly high structural similarity with wild-type Aβ42. (13)C-(1)H dipolar order parameters, (1)H rotating-frame spin-lattice relaxation times and water-to-protein spin diffusion experiments reveal substantial differences in the dynamics and hydration of Arctic, Osaka and wild-type Aβ40 fibrils. Together, these results strongly suggest that electrostatic interactions in the center of the Aβ peptide sequence play a crucial role in the three-dimensional fold of the fibrils, and by inference, fibril-induced neuronal toxicity and AD pathogenesis.

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Figures

Figure 1

Fibrillization of wild-type, Arctic and Osaka Aβ40 peptides. (A) Osaka and Arctic Aβ40 peptides form fibrils much more rapidly than the wild-type (WT) peptide. Kinetics of fibril formation (0.2 mg/ml in 10 mM sodium phosphate buffer, pH 7.2) was measured by the amyloid-binding dye ThT (10 μM). n=5, mean±SEM. (B) Negative stain TEM images of WT and mutant Aβ40 fibrils. Arctic Aβ40 fibrils display significant twists with variable periodicities and more pronounced entanglement than wild-type and Osaka fibrils. Scale bar = 100 nm. (C) Mutant fibrils have altered stability to denaturation by GdnHCl. Fibrils were incubated with increasing concentrations (1 – 6 M) of GdnHCl and then ultracentrifuged at 100,000 × g to pellet any fibrils that resisted denaturation, which were resolved by SDS-PAGE and Coomassie stain.

Figure 2

(a) Amino acid sequences of the three Aβ40 peptides. Isotopically labeled residues are shown in red. (b–c) 1D 13C (b) and 15N (c) CP-MAS spectra of Arctic, Osaka, and wild-type Aβ40 fibrils. Spectra were measured on 800 and 900 MHz spectrometers at 273 K under 16 kHz MAS. Assignments obtained from 2D spectra are indicated. All three fibrils show narrow intrinsic linewidths, but the number of peaks and the chemical shifts differ significantly.

Figure 3

2D correlation spectra of Arctic Aβ40 (E22G) fibrils. (a) 2D 13C-13C DARR spectrum with 50 ms mixing. For simplicity, assignments are given with lower-case letters for various carbons, and F19 aromatic carbons are designated by “r”. ssb denotes spinning sidebands. (b) 2D N(CA)CX 15N-13C correlation spectrum. Two to four sets of chemical shifts (denoted by subscripts I – IV) are identified for all labeled residues, indicating extensive structural polymorphism. Ellipses guide the eye for chemical shift multiplicity. The DARR spectrum was processed using a QSINE window function with SSB = 2.5, and were plotted with Topspin parameters lev0 = 4, toplev = 90 and nlev = 16. 2D N(CA)CX spectrum was processed using a Gaussian window function with LB = −10 Hz and GB = 0.05, and was plotted using lev0 = 5, toplev = 80 and nlev =16.

Figure 4

Comparison of the chemical shifts of the three Aβ40 fibrils. (a–c) 2D 13C-13C DARR spectra with 50 ms mixing for (a) Arctic Aβ40 (E22G), (b) wild-type Aβ40, and (c) Osaka Aβ40 (E22Δ). (d–f) 2D 15N-13C correlation spectra for (d) Arctic Aβ40 (E22G), (e) wild-type Aβ40, and (f) Osaka Aβ40 (E22Δ). A single set of chemical shifts is observed for Osaka Aβ40, the wild-type fibril shows modest polymorphism from I32 to V36, while the Arctic Aβ40 fibril has the largest number of polymorphs. All DARR spectra were processed using QSINE window functions with SSB = 2.5 and plotted using lev0 = 4, toplev = 90 and nlev = 16. 2D 15N-13C correlation spectra were processed using LB = −10 Hz and GB = 0.05, and were plotted using lev0 = 5, toplev = 90 and nlev = 12.

Figure 5

Per-residue average chemical shift differences between different Aβ fibrils, calculated using equation 1. 13C chemical shifts were referenced to DSS for the calculation. (a) Chemical shift differences of Arctic conformer I from published wild-type Aβ40 and Aβ42 data. (b) Chemical shift differences of wild-type Aβ40 studied here from published wild-type Aβ40 and Aβ42 data. (c) Chemical shift differences between Osaka DMSO and Osaka NaOH studied here. (d) Chemical shift differences of Osaka DMSO from recombinant Osaka Aβ40. The very small differences indicate that the synthetic Osaka fibrils have the same structure as fibrils obtained from recombinant peptide . (e) 2D heat map representing the average pairwise Pearson product-moment correlation coefficients between the chemical shifts of Aβ fibrils. Eleven different Aβ fibrils, including five studied here and six literature cases, are compared. The white cross in each row indicates the strain with the highest correlation coefficient. Arctic conformer I has the highest correlation with WT-42 2MXU. PDB codes are given where available to simplify notation. WT-42 corresponds to the study of Colvin et al , Osaka 2MVX corresponds to the study of Schutz et al , WT 2M4J is the study of Lu et al , WT 2LMP/Q is that of Paravastu et al , and WT 2LMN/O is that of Petkova et al .

Figure 6

Mobilities of Aβ40 fibrils. (a) Representative 13C-1H dipolar dephasing curves for Arctic, wild-type and Osaka DMSO Aβ40 fibrils. Best-fit couplings and the resulting order parameters (SCH) are given in each panel. All fibrils show mostly immobilized backbones but significant sidechain motions. The data were obtained at 298 K under 7 kHz MAS. (b) Representative 13C-detected 1H T1ρ relaxation data of the three Aβ40 fibrils. Top row: Arctic E22G Aβ40 shows longer T1ρ than wild-type and Osaka Aβ40 at 298 K. Second to fourth rows: temperature dependence of the 1H T1ρ. Arctic Aβ40 shows shorter T1ρ at lower temperature, while wild-type and Osaka Aβ40 exhibit longer T1ρ’s at lower temperature. These indicate that the Arctic Aβ40 sidechains have faster microsecond motions than the other two fibrils.

Figure 7

Different hydration behaviors of the three Aβ40 fibrils. (a–c) 13C-detected water-to-protein 1H spin diffusion spectra at 100 ms and 4 ms for (a) Arctic (E22G) Aβ40, (b) wild-type Aβ40, and (c) Osaka (E22Δ) Aβ40. The Osaka fibrils show the highest initial intensity. The Arctic initial intensity distribution deviates from the equilibrium intensity more than the wild-type peptide fibrils. (d–f) Water-to-protein 1H spin diffusion buildup curves for (d) Arctic Aβ40, (e) wild-type Aβ40, and (f) Osaka Aβ40. Vertical dashed line and shaded horizontal bars guide the eye for the magnetization equilibration times and the initial buildup rates. Osaka Aβ40 has the shortest equilibration time, indicating the highest hydration, while Arctic Aβ40 has the largest distribution of buildup rates.

Figure 8

Inter-residue contacts of the Ab40 fibrils from 1.5 s mixing 2D 13C-13C PDSD spectra. In the aromatic region, Arctic Ab40 (E22G) shows several F19–I32 cross peaks for conformer I while Osaka Ab40 (E22D) shows F19–L34 and F19–V36 cross peaks. The unresolved aromatic carbons of F19 are denoted as F19r.

Figure 9

Comparison of four published structures of wild-type and mutant Aβ fibrils. The sidechains of residues labeled in this study are shown as red and green sticks. Key charged residues that are known to be important for the three-dimensional fold of the peptides are shown in blue. (a) Two-fold in vitro structure of wild-type Aβ40 (PDB: 2LMN) . (b) Three-fold in vitro structure of wild-type Aβ40 (PDB: 2LMQ) . (c) Osaka Aβ40 (E22Δ) structure . (d) Wild-type Aβ42 structure (PDB: 2MXU) . The shortest F19–I32 and F19–L34 distances are indicated in the structures. Note that the distances in (a) and (b) are intermolecular, while distances in (c) and (d) are intramolecular.

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Structural Polymorphism of Alzheimer's β-Amyloid Fibrils as Controlled by an E22 Switch: A Solid-State NMR Study - PubMed (original) (raw)