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Structural mechanism for specific binding of chemical compounds to amyloid fibrils

Nature Chemical Biology volume 19, pages 1235–1245 (2023)Cite this article

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Abstract

Amyloid fibril is an important pharmaceutical target for diagnostic and therapeutic treatment of neurodegenerative diseases. However, rational design of chemical compounds that interact with amyloid fibrils is unachievable due to the lack of mechanistic understanding of the ligand–fibril interaction. Here we used cryoelectron microscopy to survey the amyloid fibril-binding mechanism of a series of compounds including classic dyes, (pre)clinical imaging tracers and newly identified binders from high-throughput screening. We obtained clear densities of several compounds in complex with an α-synuclein fibril. These structures unveil the basic mechanism of the ligand–fibril interaction, which exhibits remarkable difference from the canonical ligand–protein interaction. In addition, we discovered a druggable pocket that is also conserved in the ex vivo α-synuclein fibrils from multiple system atrophy. Collectively, these findings expand our knowledge of protein–ligand interaction in the amyloid fibril state, which will enable rational design of amyloid binders in a medicinally beneficial way.

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Fig. 1: Binding of chemical ligands to α-syn fibrils.
Fig. 2: Cryo-EM structure of the CCA–α-syn fibril.
Fig. 3: Cryo-EM structure of the EB–α-syn fibril.
Fig. 4: Cryo-EM structures for the ligands binding to the N-pocket of the α-syn fibril.
Fig. 5: Cryo-EM structures of ThT–α-syn and PiB–α-syn fibrils.
Fig. 6: Interligand π–π interactions in ligand-bound α-syn fibrils.

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Data availability

Cryo-EM maps have been deposited in the Electron Microscopy Data Bank (EMDB) under accession nos. EMD-33960 for CCA-bound α-syn fibrils, EMD-33961 for CR-bound α-syn fibrils, EMD-33965 for EB-bound α-syn fibrils, EMD-33966 for ThT-bound α-syn fibrils, EMD-33968 for BF227a-bound α-syn fibrils, EMD-33967 for PiB-bound α-syn fibrils, EMD-33969 for C05-03-bound α-syn fibrils, EMD-33970 for SIL5-bound α-syn fibrils and EMD-33971 for pFTAA-bound α-syn fibrils. The corresponding atomic models have been deposited in the PDB under the following accession nos.: 7YNF for CCA-bound α-syn fibrils, 7YNG for CR-bound α-syn fibrils, 7YNL for EB-bound α-syn fibrils, 7YNM for ThT-bound α-syn fibrils (conformation 1), 7YNN for ThT-bound α-syn fibrils (conformation 2), 7YNP for BF227a-bound α-syn fibrils, 7YNO for PiB-bound α-syn fibrils (conformation 1), 7YNQ for PiB-bound α-syn fibrils (conformation 2), 7YNR for C05-03-bound α-syn fibrils, 7YNS for SIL5-bound α-syn fibrils and 7YNT for pFTAA-bound α-syn fibrils, respectively. The structural models used in the present study are available in the PDB database under accession nos. 6A6B (apo-α-syn fibrils), 6XYO (MSA type I), 6XYP (MSA type II1) and 6XYQ (MSA type II2). Source data are provided with this paper.

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Acknowledgements

We thank the Cryo-Electron Microscopy center at Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry for help with cryo-EM data collection in EB, ThT, BF227a, PiB, C05-03 and SIL5 cases. We thank the Bio-Electron Microscopy Facility of ShanghaiTech University for help with cryo-EM data collection in CCA, CR and pFTAA cases. We thank Y. Sun and Q. Cao for their helpful discussion on cryo-EM data processing. We thank W. Kong from Cytiva and Y. Zhang from the Discovery Technology Platform at the Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University for providing technical support of Biacore 8K. This work was supported by the National Natural Science Foundation of China (grant nos. 82188101 and 32171236 to C.L. and 32170683 to D.L.), the Major State Basic Research Development Program (grant no. 2019YFE0120600 to C.L.), the Science and Technology Commission of Shanghai Municipality (grant nos. 20XD1425000, 2019SHZDZX02 and 22JC1410400 to C.L.) and the Shanghai Pilot Program for Basic Research—Chinese Academy of Science, Shanghai Branch (grant no. CYJ-SHFY-2022-005 to C.L.).

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Author notes

  1. These authors contributed equally: Youqi Tao, Wencheng Xia, Qinyue Zhao.

Authors and Affiliations

  1. Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (Ministry of Education), Shanghai Jiao Tong University, Shanghai, China

    Youqi Tao, Qinyue Zhao, Shenqing Zhang & Dan Li

  2. Zhangjiang Institute for Advanced Study, Shanghai Jiao Tong University, Shanghai, China

    Youqi Tao, Qinyue Zhao, Shenqing Zhang & Dan Li

  3. Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, China

    Wencheng Xia, Huaijiang Xiang, Chao Han, Ying Li, Li Tan & Cong Liu

  4. University of the Chinese Academy of Sciences, Beijing, China

    Wencheng Xia, Huaijiang Xiang, Chao Han, Ying Li, Li Tan & Cong Liu

  5. State Key Laboratory of Chemical Biology, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, China

    Wei Gu, Wenjun Tang & Cong Liu

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Contributions

Y.T., D.L. and C.L. designed the project. Y.T., W.X., Q.Z. and S.Z. prepared cryo-EM samples of ligand-bound α-syn fibrils, and performed cryo-EM data collection and processing. H.X., C.H., W.G., W.T. and L.T. synthesized the chemical compounds. Y.T., Q.Z. and Y.L. performed the high-throughput screening for binders of α-syn fibrils. Y.T. performed the ligand-α-syn fibril-binding assays. S.Z. performed the SPR experiment. All the authors were involved in analyzing the data and contributed to discussion of the paper and editing. Y.T., D.L. and C.L. wrote the paper.

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Correspondence to Dan Li or Cong Liu.

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Nature Chemical Biology thanks Salvador Ventura, Michael Stowell and Myungwoon Lee for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 ELISA-based screening assay for identification of chemical binders of α-syn fibrils.

a, Schematic illustration of the ELISA-based high-throughput screening assay. ELISA plates are coated with heparin. 100 nM of sonicated FLAG-tagged α-syn PFFs are pre−mixed with 20 μM of each compound before added to each well. Bound α-syn PFFs are detected by horse radish peroxidase (HRP) conjugated anti-FLAG antibody (Anti-FLAG-Ab-HRP). b, The binding affinities of heparin to α-syn PFFs. Top, ELISA-based binding saturation assay for heparin-α-syn PFF interaction. ELISA absorbance values are normalized and plotted against FLAG-tagged α-syn PFFs concentrations. Data are shown as mean ± s.d., n = 4 independent samples. Nonlinear regression is used to fit the curve to a binding saturation equation in GraphPad Prism 8.0.2 with R2 0.9966. Bottom, binding affinity (KD) of heparin to α-syn PFFs measured by SPR. Compound concentrations are indicated. c, IC50 measurement of CCA (left), EB (right, top) and CR (right, bottom) against α-syn PFFs-heparin interaction by ELISA. ELISA absorbance values are normalized and plotted against compound concentrations. Data are shown as mean ± s.d., n = 4 independent samples. Nonlinear regression is used to fit each curve to a sigmoidal four parameters [inhibitor]-response (variable slope) equation in GraphPad Prism 8.0.2. with R2 0.9982 for CCA curve, 0.9987 for EB curve, and 0.9995 for CR curve, respectively.

Source data

Extended Data Fig. 2 Binding affinities of ligands to α-syn fibrils measured by fluorescent binding saturation curves.

a, Differential fluorescent spectra of 50 μM ligands in the presence and absence of 50 μM α-syn fibrils. Differential fluorescence signal intensities (Ligandbound - Ligandfree) are shown as mean ± s.d., n = 3 independently prepared samples. The chemical structures of the ligands are shown. b, The fluorescent binding curves of the ligands to α-syn fibrils measured at the excited and maximum emission wavelengths in (a). 1 µM of α-syn fibrils were added. Data are shown as mean ± s.d., n = 3 independent samples. The curves were analyzed by nonlinear regression with a saturation binding equation. c, The fitting results of (b). Binding affinity KD is the concentration of ligand required to reach half-maximal binding, and h is the Hill coefficient. Bmax is the maximal binding signal.

Source data

Extended Data Fig. 3 Binding affinities of ligands to α-syn PFFs measured by surface plasmon resonance (SPR).

For each panel, the chemical structure of ligand is shown on the top. The binding affinities (KD) of ligands to α-syn PFFs were calculated based on the SPR association and dissociation curves.

Extended Data Fig. 4 Cryo-EM 3D reconstruction density maps of the ligand-bound α-syn fibrils.

For each panel, density maps are displayed as a half pitch of side view (left), enlarged top (right, top) and side views (right, bottom). Fibril parameters including the length of half pitch (180° helical turn), twist angle and helical rise are indicated. Extra densities are colored in line with the ligand colors. Chemical structures of the ligands are provided along with the density maps. The thresholds of density maps: CCA, 0.0096; CR, 0.008; EB, 0.013; ThT, 0.0098; BF227a, 0.0045; PiB, 0.0085; C05-03, 0.0045; SIL5, 0.0069; pFTAA, 0.0233.

Extended Data Fig. 5 Ligand densities in the N-pocket, C-pocket, inter-pocket, and on the back-surface around the α-syn fibril.

a, Structure model of the α-syn fibril with all residues and pockets labeled. b, For each ligand-binding pocket/surface around the α-syn fibril, cryo-EM densities and structures are shown and colored in gray. For each ligand, their densities are shown and colored in line with the ligand color. The protein density is restricted to areas within a 2-Å radius of the α-syn model, and combined with the ligand densities displayed with the same threshold: CCA, 0.0096; CR, 0.008; EB, 0.013; ThT, 0.0098; BF227a, 0.0045; PiB, 0.0085; C05-03, 0.0045; SIL5, 0.0069; pFTAA, 0.0233. Chemical structures of the ligands are provided under the ligand names.

Extended Data Fig. 6 Cryo-EM densities and structure models of CCA molecules at different ligand-binding sites of the α-syn fibril.

CCA molecules are shown as sticks. The densities are displayed with a threshold of 0.0096. Alternative orientations of CCA molecules in inter-pocket and back-surfaces are also shown.

Extended Data Fig. 7 Y39 shapes the N-pocket of α-syn fibril.

a, Structure of one layer of apo-α-syn fibril (PBD ID: 6A6B). N-pocket composed of residues 39–46 and 80–83 is highlighted in yellow. b, Two conformations of Y39 observed in the ligand-bound α-syn fibrils. When the side chain of Y39 points outwards (open state), the pocket is in the shape of a narrow deep groove preferable for specific ligand binding. When the side chain of Y39 points inwards (closed state), the pocket is in the shape of a wide shallow groove. Top and side views of N-pocket are shown. Y39 is highlighted in red.

Extended Data Fig. 8 Conserved ligand-binding N-pocket in in vitro and ex vivo α-syn fibrils from MSA.

a, Overlay of the α-syn monomeric structures in the fibrils including BF227a-α-syn fibril (yellow), type I filaments from MSA (red, PDB 6XYO), type II1 filaments from MSA (blue, PDB 6XYP), and type II2 filaments from MSA (green, PDB 6XYQ). α-Syn is shown in ribbons; BF227a is shown in sticks and colored in orange. RMSDs of BF227a-α-syn fibril versus MSA type I is 2.285 Å over 53 C-α atoms (global alignment); BF227a-α-syn fibril versus MSA type II1 is 4.577 Å over 58 C-α atoms; BF227a-α-syn fibril versus MSA type II2 is 4.568 Å over 58 C-α atoms. The N- and C-pockets are indicated by dashed box. The N-pocket is zoomed in in (b) with residue side chains shown in sticks. c, The surface of α-syn fibrils is shown and colored in line with (a). N-pocket of each fibril is framed and enlarged. Y39 is highlighted in black.

Extended Data Fig. 9 Interactions between ThT and the N-pocket of α-syn fibril.

a, π-π interactions between the benzothiazole rings of ThT (top, conformation 1, colored in yellow; bottom, conformation 2, colored in magenta) and Y39 residues (colored in wheat) in three consecutive α-syn molecules (i, i + 1, i + 2) along the fibril. Detailed interaction distances of π planes are labeled. b, Hydrophobic interactions between the methyl groups of ThT (conformation 1, colored in yellow) and the side chains of K80 and V82. The benzene ring of ThT forms cation-π interactions with the side chains of K80. Detailed interaction distances are labeled. c, Summary of the molecular interactions between ThT (conformation 1, left, colored in yellow; conformation 2, right, colored in magenta) and α-syn fibril (black). Interactions are depicted as blue dashed lines.

Extended Data Fig. 10 Interactions between PiB and the N-pocket of α-syn fibril.

a, Interactions of two alternative conformations of PiB (colored in yellow and pink, respectively) with Y39 (colored in wheat) and V82 (colored in light purple) of α-syn. Three layers of α-syn molecules (i, i + 1, i + 2) are shown. Detailed interaction distances are labeled. b, Summary of the molecular interactions between PiB (conformation 1, left; conformation 2, right) and α-syn fibril (black). Interactions are depicted as blue dashed lines. c, π-π stacking of PiB conformations along the fibril axis. Detailed distances and angles are labeled.

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Supplementary Figs. 1–12, Tables 1–2 and Note.

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Statistical source data for Supplementary Fig. 4.

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Source Data Fig. 1

Statistical source data for Fig. 1.

Source Data Extended Data Fig. 1

Statistical source data for Extended Data Fig. 1.

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Statistical source data for Extended Data Fig. 2.

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Tao, Y., Xia, W., Zhao, Q. et al. Structural mechanism for specific binding of chemical compounds to amyloid fibrils. Nat Chem Biol 19, 1235–1245 (2023). https://doi.org/10.1038/s41589-023-01370-x

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