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The structure of pathogenic huntingtin exon 1 defines the bases of its aggregation propensity

Nature Structural & Molecular Biology volume 30, pages 309–320 (2023)Cite this article

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Abstract

Huntington’s disease is a neurodegenerative disorder caused by a CAG expansion in the first exon of the HTT gene, resulting in an extended polyglutamine (poly-Q) tract in huntingtin (httex1). The structural changes occurring to the poly-Q when increasing its length remain poorly understood due to its intrinsic flexibility and the strong compositional bias. The systematic application of site-specific isotopic labeling has enabled residue-specific NMR investigations of the poly-Q tract of pathogenic httex1 variants with 46 and 66 consecutive glutamines. Integrative data analysis reveals that the poly-Q tract adopts long α-helical conformations propagated and stabilized by glutamine side chain to backbone hydrogen bonds. We show that α-helical stability is a stronger signature in defining aggregation kinetics and the structure of the resulting fibrils than the number of glutamines. Our observations provide a structural perspective of the pathogenicity of expanded httex1 and pave the way to a deeper understanding of poly-Q-related diseases.

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Fig. 1: NMR analyses of H46 and comparison with H16.
Fig. 2: NMR analyses of H66 and comparison with H46.
Fig. 3: A structural model of pathogenic and nonpathogenic httex1 from the synergistic integration of NMR and SAXS data.
Fig. 4: NMR analysis of H46 side chains.
Fig. 5: Insights into the conformational landscape of httex1 with molecular dynamics simulations.
Fig. 6: Effect of the α-helical stability on the aggregation properties of huntingtin.
Fig. 7: Scheme illustrating the structural influences within nonpathogenic and pathogenic httex1 and their respective modes of interaction.

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Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. The accession codes for the SAXS data (SASDB) are SASDQR8 (H16) and SASDQS8 (H46) and the ensembles have been deposited in the Protein Ensemble Database under the accession codes PED00223 (H16) and PED00224 (H46). The 3D structure of sfGFP was downloaded from the PDB (PDB 3LVA). Source data are provided with this paper.

Code availability

The in-house script to analyze fluorescence images and disordered chain building program will be made available from the corresponding author on reasonable request.

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Acknowledgements

We thank G. Otting (Australian National University, Canberra, Australia) for providing the BL21 (DE3) Star::RF1-CBD3 strain. This work was supported by the European Research Council under the European Union’s H2020 Framework Programme (2014–2020)/ERC grant agreement no. 648030 and Labex EpiGenMed, an Investissements d’avenir program (grant no. ANR-10-LABX-12-01) awarded to P.B., grant no. ANR-17-CE11-0022-01 awarded to N.S. and grant no. ANR-19-PI3A-0004 awarded to J.C. The Centre for Structural Biology (CBS) is a member of France-BioImaging (FBI) and the French Infrastructure for Integrated Structural Biology, two national infrastructures supported by the French National Research Agency (grant nos. ANR-10-INBS-04-01 and ANR-10-INBS-05, respectively). A.U. is supported by a grant from the Fondation pour la Recherche Médicale (grant no. SPF20150934061). D.S. acknowledges a grant from the Métropole Européenne de Lille (PUSHUP). G. Levy (Université de Lille) is thanked for help with sample preparation and the 19F-NMR experiments. This work benefited from the high-performance computing resources of CSUC and the CALMIP supercomputing center under the allocations 2016-P16032 and 2021-P21043. The 600 MHz spectrometer for 19F-NMR measurements is funded by the Nord Region Council, CNRS, Institut Pasteur de Lille, the European Community (European Regional Development Fund, ERDF), the French Ministry of Research and the Université de Lille and by the CTRL CPER cofunded by the European Union with the ERDF, by the Hauts-de-France Regional Council (contract no. 17003781), Métropole Européenne de Lille (contract no. 2016_ESR_05) and the French State (contract no .2017-R3-CTRL-Phase1). We thank the SWING beamline at the SOLEIL synchrotron, Saint-Aubin, France (proposal 20181386), and P12 beamline at PETRAIII, Hamburg, Germany, for beamtime allocation to the project and assistance during data collection.

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Authors and Affiliations

  1. Centre for Structural Biology, University of Montpellier, INSERM, CNRS, Montpellier, France

    Carlos A. Elena-Real, Amin Sagar, Annika Urbanek, Matija Popovic, Anna Morató, Alejandro Estaña, Aurélie Fournet, Christine Doucet, Xamuel L. Lund, Luca Costa, Frédéric Allemand, Pierre-Emmanuel Milhiet, Alessandro Barducci, Nathalie Sibille & Pau Bernadó

  2. LAAS-CNRS, University of Toulouse, CNRS, Toulouse, France

    Alejandro Estaña & Juan Cortés

  3. Institute of Laue Langevin, Grenoble, France

    Xamuel L. Lund

  4. The Chemistry and Synthesis Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Rockville, MD, USA

    Zhen-Dan Shi & Rolf E. Swenson

  5. SWING Beamline, Synchrotron SOLEIL, Gif sur Yvette, France

    Aurélien Thureau

  6. Institute for Advanced Chemistry of Catalonia (IQAC), CSIC, Barcelona, Spain

    Ramon Crehuet

  7. Univ. Lille, INSERM, CHU Lille, Institut Pasteur de Lille, U1167 - RID-AGE - Risk Factors and Molecular Determinants of Aging-Related Diseases, Lille, France

    Davy Sinnaeve

  8. CNRS, EMR9002, Integrative Structural Biology, Lille, France

    Davy Sinnaeve

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  1. Carlos A. Elena-Real
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Contributions

P.B. conceived the project. C.A.E.-R., A.U., A.S., C.D., R.C., A.B., D.S., N.S. and P.B. designed experiments. C.A.E.-R., A.U., A.S., M.P., A.M., A.E., A.F., C.D., X.L.L., Z.-D.S., L.C., A.T., F.A., R.C. and D.S. performed experiments. R.E.S., P.-E.M., A.B., J.C., N.S. and P.B. supervised experiments. C.A.E.-R., A.U., A.S. and P.B. wrote the paper with the help of all the coauthors.

Corresponding author

Correspondence to Pau Bernadó.

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The authors declare no competing interests.

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Nature Structural & Molecular Biology thanks Burkhard Bechinger and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Comparison of H46 and H16 by NMR.

(a) Overlay of the 15N-HSQC spectra of fully labeled H16 (red) with selectively labeled samples of H46 (blue). (b) Zoom of the 15N-HSQC and 13C-HSQC overlay of H16 and H46 SSIL spectra showing the poly-Q NH, NH and Cα regions of Q18, Q20 and Q21.

Extended Data Fig. 2 Comparison of H66 with H16 and H46 by NMR.

(Left) Overlay of the 15N-HSQC spectra of fully labeled H66 (green) with fully labeled H46 (blue). Black arrows indicate peaks corresponding to glutamates, which were not labeled in the H66 sample. (Right) Comparison of the SCS profiles of H16 (red), H46 (blue) and the values measured for Q56 and Q76 in H66 (green).

Extended Data Fig. 3 SAXS analyses of H46 and H16.

The plots of average intensity vs. frame number obtained from SEC–SAXS for H16 (a) and H46 (b) The insets show all−vs-all χ2 comparison for the frames selected for further processing. The small values of χ2 show that the selected frames were very similar to each other. (c) Pairwise distance distribution functions, P(r), obtained for H16 (red) and H46 (blue) by indirect Fourier transformation of the SAXS data. (d) and (e) show the Rg distribution of the pool (gray, filled) and the selected sub-ensemble by EOM for H16 (red) and H46 (blue), respectively.

Extended Data Fig. 4 Side chain NMR scanning.

(a) Cβ-Hβ and (b) Cγ-Hγ zooms of the 13C-HSQC spectra for all glutamines scanned in H46. (c) Zoom of the 15N-HSQC spectra showing the side chain NεH2 region. The color code is equivalent to the one used in Figs. 1 and 3 in the main text.

Extended Data Fig. 5 Labeling of httex1 with fluorinated glutamines.

(a) Enzymatic loading of suppressor tRNACUA with canonical (Gln) or 2 S,4R-fluoroglutamine (4F-Gln). Upper and lower bands correspond to loaded and unloaded suppressor tRNACUA, respectively. A negative control of empty tRNACUA is shown in the third lane. (b) Averaged fluorescence endpoint derived from n = 2 independent cell-free suppression reactions of H16 with a stop codon at position Q18 or Q21 when titrating with increasing concentrations of both Gln-tRNACUA and 4F-Gln-tRNACUA. “+” indicates a positive control, a cell-free reaction of H16 without any amber stop codon. Data are presented as mean values + /- standard deviation.

Source data

Extended Data Fig. 6 Helicity propagation in H46 MD trajectories.

(a) Plot showing the number of times (in log scale) an α-helix expands 1 to 5 residues towards the C-terminus (+1 to +5) or the N-terminus (-1 to -5) from each of the residues of N17 and poly-Q. For all the positions (1 to 5), the number of events is greater in direction of the C-terminus than the N-terminus, indicating a higher propensity of helix propagation from N- to C-terminus. The comparison of the number of events for expansion of a helix by 1, 2 and 3 residues towards the N- or C-terminus is presented in (b) for further clarity. These plots show that in httex1 helical segments are more prone to expand towards the C-terminus than towards the N-terminus. Additionally, the preference for directional growing increases with the extent of propagation, that is, the relative difference in the number of events where a helix is propagated three residues towards the C-terminus and the N-terminus is larger than propagation the of two residues, which is in turn larger than the propagation of a single residue.

Extended Data Fig. 7 Quantum chemistry calculations of the 19F chemical shifts.

(A) Box plots of the 19F CS when the F-Gln was incorporated in Q20 (blue) or in Q21 (orange) for n = 50 distinct conformations belonging to the following families: (left, bifurcated) an α-helical structure with either a S16-Q20 or a F17-Q21 bifurcated hydrogen bond, (middle, non-bifurcated) having a canonical α-helical structure in the S16-Q21 segment without a bifurcated hydrogen bond, and (right, random coil) not displaying any specific secondary structure. A statistically significant difference was observed between the 19F CS distributions for Q20 and Q21 obtained from conformations with a bifurcated hydrogen bond between S16-Q20 and F17-Q21, respectively (p = 0.003). Q20 and Q21 19F chemical shifts were also statistically distinct when both residues were involved in an α-helix (p = 0.0023). No statistical difference was observed in the random coil scenario (p = 1.0). The boxplots use the default settings in the seaborn statistical plotting library. The centre of each box represents the median. The box limits represent the interquartile range, that is Q3-Q1. “Whiskers” extend to points that lie within 1.5 interquartile ranges of the lower and upper quartile. Observations that fall outside this range are displayed independently. The test used was the two-sided T-test for 2 independent samples with the same population variance with the Bonferroni correction, as implemented in the statannotation python library (https://github.com/trevismd/statannotations). (B) Cartoon displaying the distance between the fluorine atom when placed in Q21 and the center of the phenylalanine ring. (C) Influence of the distance on the computed 19F CSs of F-Q21 when adopting an α-helical conformation in the presence (blue) or absence (red) of a bifurcate hydrogen bond. Horizontal lines display the largest 19F CS values for the different scenarios. (D) Distance distribution between the fluorine atom when placed in Q21 and the center of the phenylalanine ring for the bifurcated and non-bifurcated hydrogen bonds computed from conformations extracted from the GaMD trajectory. In both cases, a large population of conformations placed the fluorine atom in very close proximity (<4 Å) of F17 aromatic ring.

Extended Data Fig. 8 NMR investigation of the N17 mutants exhibiting a different poly-Q helical propensity while preserving its length.

Uniformly labeled and SSIL samples of LKGG- and LLLF-H46 mutants were produced and analyzed by NMR. On one hand, LKGG-H46 glutamine 15N-HSQC signals (blue) collapsed in a broad, high-intensity, downfield-shifted peak, proving a substantial loss of helicity in comparison with the wild-type H46 (in gray) (a). Interestingly, this broad peak did not overlap with the positions corresponding to fully unstructured glutamines, which were shifted further downfield. This indicates that poly-Q, even when disconnected from the flanking region, contains a small intrinsic propensity for helical conformations, in agreement with our MD simulations. On the other hand, the 15N-HSQC spectrum of fully labeled LLLF-H46 (green) displayed a more dispersed density of glutamine peaks and an additional upfield density compared with the wild-type (in gray), pointing to a helicity increase of the poly-Q tract (b). The detailed analysis of NH, NεH2 and Cα signals of Q18, Q20 and Q21 SSIL samples from both mutants confirmed the decrease in structuration of LKGG-H46 and the increase in helicity in LLLF-H46, in comparison with the wild-type form (in gray) (a,b and c). Importantly, Q21 in LLLF-H46 displayed signatures of two conformations as also observed for the wild-type. This residue exhibited two NHε correlation peaks, suggesting the formation of a stronger bifurcated hydrogen bond than in the wild-type. Unfortunately, a single Cα peak, corresponding to the less helical conformation, was observed for Q21 in this mutant, suggesting an unfavorable exchange regime for the NMR detection.

Extended Data Fig. 9 AFM investigation of huntingtin fibrils.

(a) Fluorescence microscopy (upper panels) and AFM (middle and lower panels) images of 2-day-old fibrils of H46, LLLF-H46 and LKGG-H46. Each fluorescence image corresponds to the average of 150 pictures. (b) AFM images of 5-day-old fibrils of the three H46 variants. White squares indicate the zoom region displayed in panels below. At least 6 fluorescence and AFM images for each H46 variant and time point were extracted from two large fields (8.13μm x 8.13μm). Images displayed in (a) and (b) are representative of the ensemble of them.

Supplementary information

Supplementary Information

Supplementary Figs. 1–6, Notes 1 and 2, Discussion and Tables 1–4.

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Source data

Source Data Extended Data Fig. 5

Unprocessed urea PAGE gel to monitor the loading of F-Gln onto tRNA.

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Elena-Real, C.A., Sagar, A., Urbanek, A. et al. The structure of pathogenic huntingtin exon 1 defines the bases of its aggregation propensity. Nat Struct Mol Biol 30, 309–320 (2023). https://doi.org/10.1038/s41594-023-00920-0

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