Structural and dynamic studies reveal that the Ala-rich region of ataxin-7 initiates α-helix formation of the polyQ tract but suppresses its aggregation

Abstract

Ataxin-7 (Atx7) is a disease-related protein associated with the pathogenesis of spinocerebellar ataxia 7, while its polyglutamine (polyQ) tract in N-terminus is the causative source of aggregation and proteinopathy. We investigated the structure, dynamics and aggregation properties of the N-terminal 62-residue fragment of Atx7 (Atx7-N) by biochemical and biophysical approaches. The results showed that the normal Atx7-N with a tract of 10 glutamines (10Q) overall adopts a flexible and disordered structure, but it may contain a short or small population of helical structure in solution. PolyQ expansion increases the α-helical propensity of the polyQ tract and consequently enhances its transformation into β-sheet structures during amyloid aggregation. An alanine-rich region (ARR) just ahead of the polyQ tract forms a local and relatively stable α-helix. The ARR α-helix can initiate and stabilize helical formation of the following polyQ tract, but it may suppress aggregation of the polyQ-expanded Atx7-N both in vitro and in cell. Thus, the preceding ARR segment in Atx7-N may influence the dynamic structure and aggregation property of the polyQ tract and even determine the threshold of the pathogenic polyQ lengths. This study may gain structural and dynamic insights into amyloid aggregation of Atx7 and help us further understand the Atx7 proteinopathy based on polyQ expansion.

Figure 1

PolyQ expansion enhances aggregation of Atx7-N. (A) Domain architecture of Atx7 and its N-terminal sequence. NT, N-terminus; ZnF, zinc finger; SCA7, SCA7 domain. Atx7-N, the N-terminus of Atx7 (residues 1–62); Atx7-N172, the N-terminal 172 residues of Atx7. ARR, alanine-rich region consisting of ARR1 and ARR2 motifs; polyQ, polyglutamine tract; PRR, proline-rich region. (B,C) Supernatant/pellet fractionation assay for aggregation of Atx7_10Q-N (B) and Atx7_33Q-N (C) during incubation. Sup., supernatant; Pel., pellet. The major bands for aggregates are indicated with an arrow, while the SDS-resistant aggregates of large molecular weights are marked with a star. The incubation was carried out at 37 °C with continuous shaking and a protein concentration of 100 μM in a PBS buffer (50 mM phosphate, 50 mM NaCl, pH 7.0). (D) Quantification of the amounts of Atx7_10Q-N and Atx7_33Q-N in supernatant. Data are shown as Means ± SEM (n = 3).

Figure 2

Characterization of the secondary structures of Atx7-N variants by circular dichroism. (A) Far-UV CD spectra of the Atx7-N variants with different polyQ lengths in solution. The spectra were acquired at 25 °C and a protein concentration of 0.2 mg/mL in a PBS buffer (50 mM phosphate, 50 mM NaCl, pH 7.0). (B) Plot of the ellipticity at 222 nm versus polyQ length. Data are shown as molar ellipticities (deg.cm²/dmol). (C) Solid-state CD spectra of the Atx7-N variants with different polyQ lengths. The spectra were acquired on a thin protein film at 25 °C. (D) Plot of the maximal wavelength of the negative peak versus polyQ length. The negative peak shifts from a wavelength of 202 nm to 220 nm with the polyQ expansion.

Figure 3

Structural ensemble analysis for Atx7_10Q-N and Atx7_22Q-N. (A,B) The first 6 structures of Atx7_10Q-N (A) and Atx7_22Q-N (B) in their respective ensembles. The helical portion of ARR2 is labeled in blue, and the other helical regions are in pink. (C) Secondary structure probabilities derived from structural ensemble analysis highlighting the α-helices of Atx7_10Q-N (blue) and Atx7_22Q-N (red) in their polyQ tract regions. The gray and black bars show the random coil probabilities.

Figure 4

HDX experiment showing that Atx7-N forms partially ordered secondary structures in ARR and polyQ regions. (A) Superposition of the HSQC spectra of Atx7_10Q-N before and after HDX. The spectrum (green) was recorded 15 min after HDX, while the spectrum recorded in H₂O (red) was set as a control. The assigned remaining peaks are labeled in the spectrum. The protein concentration for HDX experiment was 100 μM in an NMR buffer (20 mM phosphate, 50 mM NaCl, pH 6.5). (B) Same as (A), Atx7_33Q-N. Additional peaks for unassigned Gln residues are also indicated. (C) Comparison of the exchange rates for the amides of backbone residues. +, the amide of assigned residue with slow HDX. *The amide of unassigned Gln with slow HDX in the polyQ region. The residues are numbered in the sequence with the normal polyQ length (10Q) of Atx7-N.

Figure 5

Secondary-structure prediction based on assigned backbone chemical shifts. (A) Plot of the (ΔCα-ΔCβ) value versus amino-acid sequence for Atx7_10Q-N. (B) SSP score for Atx7_10Q-N. (C) Plot of (ΔCα-ΔCβ) versus amino-acid sequence for the T3N9 mutant of Atx7_10Q-N. (D) SSP score for the T3N9 mutant of Atx7_10Q-N. (E) Comparison of the SSP scores of Atx7_10Q-N (T3N9) and Atx7_22Q-N (T3N9). The helical segments are indicated in the graphs. In SSP program, a score of +1 denotes a well-formed helix while a score of -1 is for the well-formed extended strand.

Figure 6

ARR forms relatively stable α-helix structure but suppresses aggregation of the polyQ tract. (A) Far-UV CD spectra of Atx7_33Q-N and its ARR mutants (A26G, A26P and Δ25–27). All the ARR mutants showed the CD spectra with their shoulders disappeared around 222 nm. (B) Supernatant/pellet fractionation assay for Atx7_33Q-N and its helix-disrupting mutants. The protein concentration was 100 μM in a PBS buffer (50 mM phosphate, 50 mM NaCl, pH 7.0). (C) Time courses showing aggregation of Atx7_33Q-N and its helix-disrupting mutants. The aggregation abilities were represented by the relative amounts of Atx7₃₃-N species in supernatant. Data are shown as Means ± SEM (n = 3).

Figure 7

The ARR helix suppresses aggregation of polyQ-expanded Atx7-N172 in cell. (A) Supernatant/pellet fractionation assay for Atx7_33Q-N172 (residues 1–172) and its helix-disrupting mutants. The HEK 293T cells were transfected with FLAG-tagged Atx7_33Q-N172 or its mutants (A26P and Δ25–26). About 24 h after transfection, the cell lysates were prepared for Western blotting with an anti-FLAG antibody. Ctrl, HEK 293T cell transfected with an empty vector. (B) Quantification of the protein amounts in supernatant and pellet respectively. Data are shown as Means ± SEM (n = 3). *p < 0.05; ***p < 0.001; N.S., no significance.