Protofilament Structure and Supramolecular Polymorphism of Aggregated Mutant Huntingtin Exon 1

Abstract

Huntington's disease is a progressive neurodegenerative disease caused by expansion of the polyglutamine domain in the first exon of huntingtin (HttEx1). The extent of expansion correlates with disease progression and formation of amyloid-like protein deposits within the brain. The latter display polymorphism at the microscopic level, both in cerebral tissue and in vitro. Such polymorphism can dramatically influence cytotoxicity, leading to much interest in the conditions and mechanisms that dictate the formation of polymorphs. We examine conditions that govern HttEx1 polymorphism in vitro, including concentration and the role of the non-polyglutamine flanking domains. Using electron microscopy, we observe polymorphs that differ in width and tendency for higher-order bundling. Strikingly, aggregation yields different polymorphs at low and high concentrations. Narrow filaments dominate at low concentrations that may be more relevant in vivo. We dissect the role of N- and C-terminal flanking domains using protein with the former (htt^NT or N17) largely removed. The truncated protein is generated by trypsin cleavage of soluble HttEx1 fusion protein, which we analyze in some detail. Dye binding and solid-state NMR studies reveal changes in fibril surface characteristics and flanking domain mobility. Higher-order interactions appear facilitated by the C-terminal tail, while the polyglutamine forms an amyloid core resembling those of other polyglutamine deposits. Fibril-surface-mediated branching, previously attributed to secondary nucleation, is reduced in absence of htt^NT. A new model for the architecture of the HttEx1 filaments is presented and discussed in context of the assembly mechanism and biological activity.

Keywords: Huntington's disease; MAS ssNMR; TEM; amyloid; supramolecular assembly.

Fig. 1.. Huntingtin exon 1 and MBP-based fusion protein.

Primary and secondary structure schematics of wild type HttEx1 (a) and mutant HttEx1 (b) showing the htt^NT domain (orange; α-helix), polyQ domain (green; intrinsically disordered), and the proline rich domain (PRD, blue; intrinsically disordered with PPII helices). Mutant Q44-HttEx1 contains an expanded polyQ domain. (c) Top: Primary structure schematic of the employed MBP-HttEx1 fusion protein, with C-terminal His tag marked (black). Bottom: HttEx1 monomer and free MBP are released from the MBP-HttEx1 fusion protein by proteolytic cleavage using Factor Xa (FXa) protease. (d) Model of the previously determined fibril architecture of single-filament narrow (~ 6 nm wide) HttEx1 fibrils formed at 37 °C, with a single monomer highlighted in yellow. An example of local dynamic domains is depicted in red. The His tag is not shown. Panel (d) is adapted from Lin, H. K., Boatz, J. C., Krabbendam, I. E., Kodali, R., Hou, Z., Wetzel, R., Dolga, A. M., Poirier, M. A., van der Wel, P. C. A. Fibril polymorphism affects immobilized non-amyloid flanking domains of huntingtin exon1 rather than its polyglutamine core, Nature Communications 85462. Copyright (2017) Lin, H. K., et. al. [36]

Fig. 2.. Q44-HttEx1 fibril polymorphism is dependent on the monomer concentration and molar ratio of fusion protein to protease.

(a-c) Histograms depicting a range of widths observed by negative stain TEM of Q44-HttEx1 fibrils, with one representative image shown per sample. Fibrils were prepared at 37 °C following cleavage of (a) 14.3 μM (62.5:1 FP:P), (b) 28.6 μM (62.5:1 FP:P), and (c) 98.9 μM (42.5:1 FP:P) MBP-Q44-HttEx1 by FXa. FP:P indicates the molar ratio of MBP-Q44-HttEx1 to FXa. See Fig. S1 for additional TEM images.

Fig. 3.. Morphological analysis of Q44-HttEx1 fibrils.

(a) Wide (> 16 nm) fibrils formed from 78.8 μM MBP-Q44-HttEx1; 170:1 FP:P. Left: fibril with two branch points (fibril border highlighted yellow). (b) Fibril area displaying striations that correspond to a supramolecular multifilament structure. High gray values mark regions with increased levels of staining. Top right: projected grey scale values summed along the fiber length, as a function of the fiber diameter, based on the aligned fiber section shown below. (c) Fibril bundle composed of intermediate (> 9 nm and < 16 nm) width fibrils, formed from 10.1 μM MBP-Q44-HttEx1; 510:1 FP:P. A band pass filter has been applied to the image to balance contrast. (d-e) Fibrils from a single sample of aggregated 28.6 μM MBP-Q44-HttEx1 (62.5:1 FP:P). (d) Although the majority of fibrils observed in this sample were categorized as narrow, TEM-based width analysis shows there is heterogeneity of fibril widths within the sample. Shown are aligned fiber sections and corresponding grey scale projections as in panel (b). (e) Fibril width can vary along the long axis of a single fibril.

Fig. 4.. Morphology of ΔN15-Q44-HttEx1 fibrils that lack most of htt^NT.

(a) Schematic diagram of the employed cleavage reaction, in which trypsin cleavage liberates ΔN15-Q44-HttEx1 after a rapid multistep cleavage process [Fig. S6]. (b) The formation of Q44-HttEx1 (red) and ΔN15-Q44-HttEx1 (blue) amyloid fibrils over time tracked by ThT fluorescence, normalized to the maximum fluorescence of the predicted curves (nucleated elongation) for each. Data is fit to nucleated elongation (solid), secondary nucleation dominated (dotted), and fragmentation dominated (dashed) aggregation kinetics models using AmyloFit [60]. Right: magnified inset of the lag phase. (c) Normalized Q44-HttEx1 (red) and ΔN15-Q44-HttEx1 (blue) monomer concentrations over time following cleavage by FXa and trypsin, respectively, measured by SDS-PAGE and HPLC [36]. The HPLC assays were performed in parallel to the ThT fluorescence assays. (d) Average ThT fluorescence in the lag and plateau phase for Q44-HttEx1 (red) and ΔN15-Q44-HttEx1 (blue). The average fluorescence of a control sample containing MBP-Q44-HttEx1 only was subtracted for each. The lag phase was measured at 50 and 60 minutes for ΔN15-Q44-HttEx1 (n = 3) and Q44-HttEx1 (n = 2), respectively. The plateau phase was measured at 4 and 3 days for ΔN15-Q44-HttEx1 (n = 3) and Q44-HttEx1 (n = 2) respectively. (e) Distribution of widths measured for ΔN15-Q44-HttEx1 fibrils, outfitted with uniform ¹³C,¹⁵N labels for ssNMR. The average width is between 11–12 nm (right), however a wide distribution of widths was observed (left). (f) Wide ΔN15-Q44-HttEx1 fibrils (~ 18 – 19 nm). Right: evidence of striations consistent with a supramolecular multifilament structure.

Fig. 5.. MAS SSNMR comparison of ΔN15-Q44-HttEx1 and Q44-HttEx1 fibrils.

(a) Top: 1D cross polarization (CP) ¹³C spectrum of Q44-HttEx1 [36] and (middle) ΔN15-Q44-HttEx1, and (bottom) their overlaid comparison, normalized to the peak maxima for Q44-HttEx1. Spectral differences are consistent with the absence in the latter sample of the partly immobilized htt^NT seen in HttEx1 fibrils. (b) From top to bottom: ¹³C INEPT of Q44-HttEx1, ΔN15-Q44-HttEx1, and overlaid. The CP spectra feature signals from rigid and partly immobilized parts of the structure, while the INEPT data show only highly flexible residues. The spectra of Q44-HttEx1 are reprinted with permission from Lin et al. (2017) Nature Communications [36].

Fig. 6.. 2D ssNMR analysis of ΔN15-Q44-HttEx1 and Q44-HttEx1 fibrils.

(a-b) Overlay of 2D CP/DARR ssNMR spectra for U-¹³C,¹⁵N ΔN15-Q44-HttEx1 (blue) and Q44-HttEx1 (red) [36], obtained at 13 kHz MAS and 8 ms DARR mixing. In these CP spectra, the signals from immobilized parts of the protein assembly are visible. Panel (b) shows assignment of preserved peaks, while (a) shows assignments for peaks absent in ΔN15-HttEx1 fibrils. (c-d) Analogous ¹³C-¹³C INEPT-TOBSY ssNMR spectra of the same samples, reflecting signals of flexible residues. (e) 1D slices from a series of 2D TEDOR spectra with marked mixing times, for Gln N-Cα and Pro N-Cα peaks. Differences in maximum transfer times indicate mobility differences between Gln, Pro_PPII and Pro_RC. (f) Normalized CP/PDSD buildup profiles for proline residues with random coil (RC; filled circles) and PPII-helical (filled diamonds) structure in PRD of Q44-HttEx1 [36], and (g) ΔN15-Q44-HttEx1 fibrils. Lower buildup curves are indicative of increased mobility, showing that the random-coil prolines are more dynamic than the PPII helices and that the PRD in ΔN15-Q44-HttEx1 fibrils retains higher mobility than in Q44-HttEx1 fibrils. Q44-HttEx1 spectra are reprinted with permission from Lin et al. (2017) Nature Communications [36].

Fig. 7.. Model building of Q44-HttEx1 fibril structure and assembly.

(a) Hypothetical model of a single-filament ‘narrow’ fibril and a multi-filament ‘wide’ fibril. (b) Based on TEM widths, each filament core contains several β-sheets with the depth of the sheet stack approximately the same as the β-strand length. Shown is a model of β-sheet stacking within a filament (see also Supplementary Fig. S10), with each sheet represented as a unique color, showing how approximately 9 stacked β-sheets make up a 6–7nm filament core.(c) Secondary nucleation events on the filament surface initiate the formation of protofilaments that can elongate in parallel (middle) or branch sideways while remaining associated with the template filament (right). Color coding in panels (a,c) is: green: polyQ b-strand core; blue: PRD, with blue cylinders being PPII helices; orange cylinder: htt^NT segment.