Tadpole-like Conformations of Huntingtin Exon 1 Are Characterized by Conformational Heterogeneity that Persists regardless of Polyglutamine Length

Abstract

Soluble huntingtin exon 1 (Httex1) with expanded polyglutamine (polyQ) engenders neurotoxicity in Huntington's disease. To uncover the physical basis of this toxicity, we performed structural studies of soluble Httex1 for wild-type and mutant polyQ lengths. Nuclear magnetic resonance experiments show evidence for conformational rigidity across the polyQ region. In contrast, hydrogen-deuterium exchange shows absence of backbone amide protection, suggesting negligible persistence of hydrogen bonds. The seemingly conflicting results are explained by all-atom simulations, which show that Httex1 adopts tadpole-like structures with a globular head encompassing the N-terminal amphipathic and polyQ regions and the tail encompassing the C-terminal proline-rich region. The surface area of the globular domain increases monotonically with polyQ length. This stimulates sharp increases in gain-of-function interactions in cells for expanded polyQ, and one of these interactions is with the stress-granule protein Fus. Our results highlight plausible connections between Httex1 structure and routes to neurotoxicity.

Keywords: Huntington's disease; NMR spectroscopy; hydrogen–deuterium exchange; molecular simulations.

Fig 1. Secondary structure propensities from smFRET reweighted ensembles of Httex1 for five polyQ lengths (15, 23, 37, 43, and 49)

(a-e) Probability that a residue is found in an α-helical stretch of length ≥4 residues (positive) or an extended stretch of ≥2 residues (negative) as defined by the DSSP algorithm [25] for Httex1 of 15Q, 23Q, 37Q, 43Q, and 49Q, respectively. Domains are color-coded as shown. (f) Probability that each region has an α-helical stretch of ≥4 residues for each polyQ length studied.

Fig 2. Httex1 lacks persistent hydrogen bonding with 25Q or 46Q

(a) ¹H,¹⁵N HSQC of Httex1 25Q protein fragment in H₂O versus D₂O based sodium acetate buffer (150 mM; pH 4). Labelled residues are observed after hydrogen-deuterium exchange. (b) Hydrogen-deuterium exchange of Httex1 25Q and 46Q were measured over 10 minutes using mass spectrometry at both pH 7.5 and pH 4. Exchange plateaued at ~80% (due to back-exchange in the protonated solvent during chromatography separation prior to MS). Httex1 was denatured in urea-d₄ (8 M) at pH 7.5, resulting in no further exchange. Hydrogen-deuterium exchange of lysozyme (Lys) was measured as a positive control for protein structure.

Fig 3. Resonance assignment of the Httex1 monomer suggests presence of transient structure

(a) ¹H,¹⁵N HSQC of Httex1 protein fragment (25Q) in sodium acetate buffer (150 mM; pH 4), recorded at 5 °C, indicating N-terminal, polyQ and C-terminal Httex1 backbone amide peaks, with glutamine side chains (boxed). (b) Analysis of propensity of secondary structure based on ΔC_α–ΔC_β values where persistence of positive ΔC_α–ΔC_β differences suggest α-helical structures and persistence of negative ΔC_α–ΔC_β differences are consistent with β-strands. (c) ¹⁵N{¹H}-NOE revealed positive NOE values of 0.4 to 0.6 for N-terminal Httex1 25Q and part of the polyQ region.

Fig 4. Secondary structure propensities from ensembles of HDX Httex1 constructs

(a-b) Probability a residue is found in an α-helical stretch of length ≥4 residues (positive) or an extended stretch of ≥2 residues (negative) as defined by the DSSP algorithm [25] for Httex1 of 25Q and 46Q, respectively. Domains are color-coded as shown.

Fig 5. Local collapse profiles extracted from ensembles of all Httex1 constructs, a representative IDP (Ash1), and a representative folded protein (Ntl9)

Local collapse is defined by the average radius of gyration, R_g, over sliding windows of ten residues divided by the R_g calculated over the same window from an ideal random coil simulation. Values greater than one imply the local conformational properties are more extended than an ideal random coil, whereas values less than one imply the local conformational properties are more compact that an ideal random coil. (a-g) Local collapse profiles for ensembles of Httex1 15Q, 23Q, 25Q, 37Q, 43Q, 46Q, and 49Q, respectively. Window numbers are colored based on what region the position of the fifth residue resides in. Black corresponds to N-terminal residues, red to polyQ resides, cyan to P₁₁ and P₁₀ residues, and blue to residues within the remainder of the C-terminal region. (h) Local collapse profile for ensembles of Ash1 – an IDP that adopts coil-like conformations. (i) Local collapse profile for ensembles of a representative folded protein (Nt19). Error bars correspond to the standard error of the mean calculated over five independent simulations for the Httex1 constructs, ten independent simulations for the Ash1 construct, and three independent simulations for the Ntl9 construct.

Fig 6. The degree of similarity, Φ, between simulated ensembles calculated over fragments of Httex1 (N-terminal domain (a), polyQ domain (b), and C-terminal domain (c)) or over full Httex1 (d) for all polyQ lengths

Low Φ values correspond to high degrees of heterogeneity within the simulated ensembles. The upper dashed line corresponds to the Φ-value associated with a reference folded protein (Ntl9), whereas the lower dashed line corresponds to the Φ-value associated with a reference IDP (Ash1).

Fig 7. Scaling of the polyQ domain as a function of polyQ length and representative snapshots for all Httex1 constructs studied

(a) Scaling of the mean size (<R_g>) of the polyQ domain as a function of polyQ length, N. The line corresponds to the best fit to the equation ln(<R_g>)=vln(N)+ln(R₀) Here, v=0.34 and R₀=2.97 Å. Error bars correspond to the standard error of the mean calculated over five independent simulations. (b) 100 representative snapshots for Httex1 ensembles with wild type polyQ lengths (15, 23, and 25). (c) 100 representative snapshots for Httex1 ensembles with pathogenic polyQ lengths (37, 43, 46, and 49). The N-terminal region is shown in black, the polyQ domain in red, and the C-terminal region in blue. All snapshots are aligned over the polyQ domain and the first 11 residues of the C-terminal region.

Fig 8. Proposed models for the generation of new/enhanced interactions due to expansion of the polyQ domain

The increased surface area of the polyQ domain upon polyQ expansion can lead to an increased number of binding sites on the polyQ domain. (a) At the monomer level, this can lead to enhanced binding between proteins and the polyQ domain. (b) An increased number of binding sites can also engender an increased drive for homotypic oligomerization. For Httex1 constructs with expanded polyQ tracts, homotypic oligomerization can then lead to gain-of-function interactions that arise from multivalent interactions between the Httex1 oligomer and the binding partner [51]. In this case, these interactions do not have to directly involve the polyQ domain.

Fig 9. PolyQ expansion alters binding partners to soluble Httex1 states in cells

Shown is a Volcano plot of binding partners to soluble Httex1 measured from proteomic analysis of GFP-trap immunoprecipitants from Neuro2a cells transfected with Httex1-GFP. Data were acquired using label free MS/MS methods from n=3 replicates.