Huntingtin exon 1 fibrils feature an interdigitated β-hairpin-based polyglutamine core

Abstract

Polyglutamine expansion within the exon1 of huntingtin leads to protein misfolding, aggregation, and cytotoxicity in Huntington's disease. This incurable neurodegenerative disease is the most prevalent member of a family of CAG repeat expansion disorders. Although mature exon1 fibrils are viable candidates for the toxic species, their molecular structure and how they form have remained poorly understood. Using advanced magic angle spinning solid-state NMR, we directly probe the structure of the rigid core that is at the heart of huntingtin exon1 fibrils and other polyglutamine aggregates, via measurements of long-range intramolecular and intermolecular contacts, backbone and side-chain torsion angles, relaxation measurements, and calculations of chemical shifts. These experiments reveal the presence of β-hairpin-containing β-sheets that are connected through interdigitating extended side chains. Despite dramatic differences in aggregation behavior, huntingtin exon1 fibrils and other polyglutamine-based aggregates contain identical β-strand-based cores. Prior structural models, derived from X-ray fiber diffraction and computational analyses, are shown to be inconsistent with the solid-state NMR results. Internally, the polyglutamine amyloid fibrils are coassembled from differently structured monomers, which we describe as a type of "intrinsic" polymorphism. A stochastic polyglutamine-specific aggregation mechanism is introduced to explain this phenomenon. We show that the aggregation of mutant huntingtin exon1 proceeds via an intramolecular collapse of the expanded polyglutamine domain and discuss the implications of this observation for our understanding of its misfolding and aggregation mechanisms.

Keywords: Huntington's disease; amyloid; amyloid disease; protein aggregation; solid-state NMR.

Fig. 1.

Huntingtin exon1 construct design and fibril formation. (A) Sequence of the used MBP-htt exon1 fusion protein. The exon1 sequence, factor Xa cleavage site, and location of the fibrils’ rigid amyloid core (16) are indicated. (B–E) Negatively stained TEM as a function of time after factor Xa release of unlabeled exon1. (B) Uncleaved htt exon1 MBP fusion protein. (C) Oligomers observed 1 h after cleavage. (D) By 3 h, fibrils have begun to form. (E) After 25 h, fibrils have grown and oligomers are no longer visible on the grid. (F) Mature [U-¹³C,¹⁵N]-labeled fibrils prepared for ssNMR. (Scale bars: 200 nm.)

Fig. 2.

MAS NMR on the polyQ core of mature huntingtin exon1 fibrils prepared at room temperature. (A) Two-dimensional ¹³C-¹³C spectrum shows the two sets of Gln peaks (type a and b) that account for the rigid amyloid core (red and blue lines). (B) An intraresidue NCACX spectrum connects ¹³C signals to their own backbone ¹⁵N. (C) An interresidue NCOCX spectrum connects the ¹³C signals to the ¹⁵N backbone shift of the next Gln. (D) Overlay of B and C. (E) The identical NCACX and NCOCX spectra show that connected Gln always have the exact same chemical shifts. No direct backbone connections between a and b are observed.

Fig. 3.

PolyQ amyloid structure and dynamics by MAS NMR. (A and B) Intersections (circled) of experimental NCCN data (horizontal lines) with the theoretical dependence on ψ (black line). Shaded areas indicate the SE. (C) Consensus of experimental backbone angles for polyQ amyloid, based on chemical shift analysis (diamonds) and ψ-angle measurements (colored lines and shaded areas) for conformers a (red) and b (blue). (D) Simulated HCCH curves for distinct χ₂ side-chain angles. (E) Experimental χ₂-sensitive HCCH data (C^β/C^γ) for conformers a (red diamonds) and b (blue triangles), along with the theoretical χ₂ = 180° curve (solid line). (F) χ₁-sensitive HCCH data, with simulated curves for χ₁ = −65° and 55° (lines). (G) R_1ρ and R₁ ¹³C relaxation for backbone and side chains of a Gln in polyQ amyloid, measured at 60 kHz MAS. (H and I) Nitrogen-15 R₁ and R_1ρ values for Gln in polyQ amyloid (right) and in GB1 protein crystals (left), showing a striking difference for the side-chain N^ε.

Fig. 4.

Intramolecular and intermolecular β-strand–β-strand interactions within the htt exon1 core. (A) Extended (250 ms) ¹³C-¹³C mixing 2D spectrum on exon1 fibrils with complete ¹³C labeling. (B) Enlargement showing strong cross-peaks between the a and b Gln conformers. (C) Analogous 2D spectrum on diluted (26%) ¹³C-labeled fibrils (Materials and Methods), which also shows significant cross-peaks between the a and b signals. (D–F) One-dimensional slices extracted from the 2D spectra for polarization transferred from C^α, C′, and C^δ carbons of each Gln conformer (circled labels). Peaks due to transfer to the other β-strand type are marked (color-coded labels). These spectra were obtained at 800 MHz (¹H) and with 13 kHz MAS. (G) Normalized volumes for cross peaks between the a and b strands in the mixed fibrils (C).

Fig. 5.

PolyQ amyloid structure. (A) X-ray powder diffraction on hydrated K₂Q₃₁K₂ fibrils shows the cross-β dimensions of polyQ amyloid. (B) The cross-β dimensions reflect repeat distances between β-strands (4.7 Å) and between β-sheets (8.4 Å). (C) The intraprotein ¹³C-¹³C contacts between a and b Gln backbones are too short to occur between sheets and are therefore between β-strands (color-coded by type) within a β-sheet. (D) β-strand structures that fulfill the torsion angle constraints, close proximity of side-chain C^δ to the backbone, and allow hydrogen bonding of both backbones and side chains. Extended side chains form a steric zipper interface to allow the 8.4-Å sheet-to-sheet distance. (E) The a and b strands are mutually compatible, but a–a or b–b interactions are not possible.

Fig. 6.

Stochastic polyQ β-sheet assembly mechanism. (A) Peak doubling is seen for a single-labeled Gln in the amyloid core of a fibrillar htt N-terminal fragment (15). The specific Gln (Q19) is distributed between both β-strand types, shown with their schematic ssNMR spectra. (B) Schematic fibril core containing β-sheets with 2n β-strands (Top). Elongation maintains the alternating β-strand pattern, but can be initiated by different segments of the polyQ domain, which then causes the N-terminal labeled Gln (circles) to end up in both β-strand types (Bottom). (C) During nucleation, an a–b β-strand assembly can be formed in two ways, yielding related but distinct β-hairpin structures. The circles mark a single labeled Gln nearer the N terminus.