Bridging Mechanical Properties with Atomic Structures of Polymorphic α-Synuclein Fibrils by Single-Molecule Analysis and Molecular Dynamics Simulations

Abstract

α-Synuclein (α-syn) forms structurally distinct fibril polymorphs with various pathological activities in different subtypes of synucleinopathies, such as Parkinson's disease (PD). As a unique proteinaceous polymer, the mechanical property of α-syn fibril is a primary determinant of its neurotoxicity, immunogenicity, and seeding and transmission capacity. Nevertheless, how genetic mutations in α-syn fibrils cause varied polymer behaviors remains largely unknown. Using optical tweezers, we quantitatively characterize the mechanical properties of three α-syn fibril variants at the single-molecule level. We find that wild-type α-syn fibrils are generally more sustainable to an axial disruption force than those formed by the disease-causing E46K and A53T α-syn mutants, whereas their heterogeneous elastic properties manifest similarity. Based on the molecular dynamics simulations, the β-sheet motif and the interface between the two protofilaments dominate in stabilizing the fibril structure. Additionally, single-molecule and simulation analysis consistently reveal the force-driven α-syn protein unfolding without a fibril break. Due to the flexible periphery, these subtle structural changes become more pronounced with the E46K fibril. The structure–mechanics relationship of α-syn fibrils built in this work sheds new light on the fibril assembly and disassembly mechanism and the mutant-associated pathogenesis in PD.

Keywords

molecular simulations / optical tweezers / Parkinson's disease / single molecule / α-Syn fibril

References

[1] J. A. Hardy and G. A. Higgins, “Alzheimer's Disease: The Amyloid Cascade Hypothesis,” Science 256 (1992): 184.

[2] T. Iwatsubo, A. Odaka, N. Suzuki, H. Mizusawa, N. Nukina, and Y. Ihara, “Visualization of Aβ42(43) and Aβ40 in Senile Plaques With End-Specific Aβ Monoclonals: Evidence That an Initially Deposited Species is Aβ42(43),” Neuron 13 (1994): 45.

[3] M. G. Spillantini, M. L. Schmidt, V. M. Y. Lee, J. Q. Trojanowski, R. Jakes, and M. Goedert, “α-Synuclein in Lewy bodies,” Nature 388 (1997): 839.

[4] C. Soto, “Unfolding the Role of Protein Misfolding in Neurodegenerative Diseases,” Nature Reviews Neuroscience 4 (2003): 49.

[5] M. Neumann, D. M. Sampathu, L. K. Kwong, et al., “Ubiquitinated TDP-43 in Frontotemporal Lobar Degeneration and Amyotrophic Lateral Sclerosis,” Science 314 (2006): 130.

[6] C. Ballatore, V. M. Y. Lee, and J. Q. Trojanowski, “Tau-Mediated Neurodegeneration in Alzheimer's Disease and Related Disorders,” Nature Reviews Neuroscience 8 (2007): 663.

[7] D. M. Wilson, M. R. Cookson, L. Van Den Bosch, H. Zetterberg, D. M. Holtzman, and I. Dewachter, “Hallmarks of Neurodegenerative Diseases,” Cell 186 (2023): 693.

[8] L. Meda, M. A. Cassatella, G. I. Szendrei, et al., “Activation of Microglial cells by β-Amyloid Protein and Interferon-γ,” Nature 374 (1995): 647.

[9] M. F. M. Engel, L. Khemtémourian, C. C. Kleijer, et al., “Membrane Damage by Human Islet Amyloid Polypeptide Through Fibril Growth at the Membrane,” Proceedings of the National Academy of Sciences of the United States of America 105 (2008): 6033.

[10] L. Milanesi, T. Sheynis, W. F. Xue, et al., “Direct Three-Dimensional Visualization of Membrane Disruption by Amyloid Fibrils,” Proceedings of the National Academy of Sciences of the United States of America 109 (2012): 20455.

[11] I. C. Stancu, N. Cremers, H. Vanrusselt, et al., “Aggregated Tau Activates NLRP3-ASC Inflammasome Exacerbating Exogenously Seeded and Non-Exogenously Seeded Tau Pathology In Vivo,” Acta Neuropathologica 137 (2019): 599.

[12] A. W. P. Fitzpatrick, B. Falcon, S. He, et al., “Cryo-EM Structures of Tau Filaments from Alzheimer's Disease,” Nature 547 (2017): 185.

[13] L. Gremer, D. Schölzel, C. Schenk, et al., “Fibril Structure of Amyloid-β(1-42) by Cryo-Electron Microscopy,” Science 358 (2017): 116.

[14] W. Qiang, W. M. Yau, J. X. Lu, J. Collinge, and R. Tycko, “Structural Variation in Amyloid-β Fibrils From Alzheimer's Disease Clinical Subtypes,” Nature 541 (2017): 217.

[15] B. Falcon, W. Zhang, A. G. Murzin, et al., “Structures of Filaments From Pick's Disease Reveal a Novel Tau Protein Fold,” Nature 561 (2018): 137.

[16] D. Li and C. Liu, “Hierarchical Chemical Determination of Amyloid Polymorphs in Neurodegenerative Disease,” Nature Chemical Biology 17 (2021): 237.

[17] Y. Sun, S. Hou, K. Zhao, et al., “Cryo-EM Structure of Full-Length α-Synuclein Amyloid Fibril with Parkinson's Disease Familial A53T Mutation,” Cell Research 30 (2020): 360.

[18] H. Wesseling, W. Mair, M. Kumar, et al., “Tau PTM Profiles Identify Patient Heterogeneity and Stages of Alzheimer's Disease,” Cell 183 (2020): 1699.

[19] K. Zhao, Y. Li, Z. Liu, et al., “Parkinson's Disease Associated Mutation E46K of α-Synuclein Triggers the Formation of a Distinct Fibril Structure,” Nature Communications 11 (2020): 2643.

[20] K. Zhao, Y. J. Lim, Z. Liu, et al., “Parkinson's Disease-Related Phosphorylation at Tyr39 Rearranges α-Synuclein Amyloid Fibril Structure Revealed by Cryo-EM,” Proceedings of the National Academy of Sciences of the United States of America 117 (2020): 20305.

[21] Y. Sun, H. Long, W. Xia, et al., “The Hereditary Mutation G51D Unlocks a Distinct Fibril Strain Transmissible to Wild-Type α-Synuclein,” Nature Communications 12 (2021): 6252.

[22] M. Schweighauser, H. J. Garringer, T. Klingstedt, et al., “Mutation ∆K281 in MAPT Causes Pick's Disease,” Acta Neuropathologica 146 (2023): 211.

[23] S. Zhang, R. Zhu, B. Pan, et al., “Post-Translational Modifications of Soluble α-Synuclein Regulate the Amplification of Pathological α-Synuclein,” Nature Neuroscience 26 (2023): 213.

[24] L. Frey, D. Ghosh, B. M. Qureshi, et al., “On the pH-Dependence of α-Synuclein Amyloid Polymorphism and the Role of Secondary Nucleation in Seed-Based Amyloid Propagation,” Elife 12 (2024): 93562.

[25] J. Hu, W. Xia, S. Zeng, et al., “Phosphorylation and O-GlcNAcylation at the Same α-Synuclein Site Generate Distinct Fibril Structures,” Nature Communications 15 (2024): 2677.

[26] N. Louros, M. Wilkinson, G. Tsaka, et al., “Local Structural Preferences in Shaping Tau Amyloid Polymorphism,” Nature Communications 15 (2024): 1028.

[27] M. H. Polymeropoulos, C. Lavedan, E. Leroy, et al., “Mutation in the α-Synuclein Gene Identified in Families With Parkinson's Disease,” Science 276 (1997): 2045.

[28] G. Tanaka, T. Yamanaka, Y. Furukawa, N. Kajimura, K. Mitsuoka, and N. Nukina, “Biochemical and Morphological Classification of Disease-Associated Alpha-Synuclein Mutants Aggregates,” Biochemical and Biophysical Research Communications 508 (2019): 729.

[29] J. J. Zarranz, J. Alegre, J. C. Gómez-Esteban, et al., “The New Mutation, E46K, of α-Synuclein Causes Parkinson and Lewy Body Dementia,” Annals of Neurology 55 (2004): 164.

[30] K. C. Luk, V. Kehm, J. Carroll, et al., “Pathological α-Synuclein Transmission Initiates Parkinson-Like Neurodegeneration in Nontransgenic Mice,” Science 338 (2012): 949.

[31] F. Chiti and C. M. Dobson, “Protein Misfolding, Functional Amyloid, and Human Disease,” Annual Review of Biochemistry 75 (2006): 333.

[32] A. Makky, L. Bousset, J. Polesel-Maris, and R. Melki, “Nanomechanical Properties of Distinct Fibrillar Polymorphs of the Protein α-Synuclein,” Scientific Reports 6 (2016): 37970.

[33] Q. H. Xu, Y. Y. Ma, Y. P. Sun, D. Li, X. Zhang, and C. Liu, “Protein Amyloid Aggregate: Structure and Function,” Aggregate 4 (2023): e333.

[34] A. L. Mahul-Mellier, F. Vercruysse, B. Maco, et al., “Fibril Growth and Seeding Capacity Play Key Roles in α-Synuclein-Mediated Apoptotic Cell Death,” Cell Death and Differentiation 22 (2015): 2107.

[35] S. Zhang, Y. Q. Liu, C. Jia, et al., “Mechanistic Basis for Receptor-Mediated Pathological α-Synuclein Fibril Cell-to-Cell Transmission in Parkinson's Disease,” Proceedings of the National Academy of Sciences of the United States of America 118 (2021): e2011196118.

[36] J. Adamcik and R. Mezzenga, “Amyloid Polymorphism in the Protein Folding and Aggregation Energy Landscape,” Angewandte Chemie International Edition 57 (2018): 8370.

[37] M. R. Sawaya, M. P. Hughes, J. A. Rodriguez, R. Riek, and D. S. Eisenberg, “The Expanding Amyloid Family: Structure, Stability, Function, and Pathogenesis,” Cell 184 (2021): 4857.

[38] D. Li and C. Liu, “Molecular Rules Governing the Structural Polymorphism of Amyloid Fibrils in Neurodegenerative Diseases,” Structure 31 (2023): 1335.

[39] R. Guerrero-Ferreira, N. M. I. Taylor, D. Mona, et al., “Cryo-EM Structure of Alpha-Synuclein Fibrils,” Elife 7 (2018): e36402.

[40] B. Li, P. Ge, K. A. Murray, et al., “Cryo-EM of Full-Length α-Synuclein Reveals Fibril Polymorphs with a Common Structural Kernel,” Nature Communications 9 (2018): 3609.

[41] M. Schweighauser, Y. Shi, A. Tarutani, et al., “Structures of α-Synuclein Filaments from Multiple System Atrophy,” Nature 585 (2020): 464.

[42] Y. Yang, Y. Shi, M. Schweighauser, et al., “Structures of α-Synuclein Filaments from Human Brains with Lewy Pathology,” Nature 610 (2022): 791.

[43] D. D. Dhavale, A. M. Barclay, C. G. Borcik, et al., “Structure of Alpha-Synuclein Fibrils Derived from Human Lewy Body Dementia Tissue,” Nature Communications 15 (2024): 2750.

[44] Y. Li, C. Zhao, F. Luo, et al., “Amyloid fibril structure of α-synuclein determined by cryo-electron microscopy,” Cell Research 28 (2018): 897.

[45] X. Li, L. Bi, S. Zhang, et al., “Single-Molecule Insight into α-Synuclein Fibril Structure and Mechanics Modulated by Chemical Compounds,” Advanced Science 12 (2025): 2416721.

[46] J. S. Palmer, C. E. Castro, M. Arslan, and M. C. Boyce, “Constitutive Models for the Force-Extension Behavior of Biological Filaments,” Iutam Bookseries 16 (2010): 141.

[47] C. E. Castro, J. Dong, M. C. Boyce, S. Lindquist, and M. J. Lang, “Physical Properties of Polymorphic Yeast Prion Amyloid Fibers,” Biophysical Journal 101 (2011): 439.

[48] S. A. M. Holec and A. L. Woerman, “Evidence of Distinct α-Synuclein Strains Underlying Disease Heterogeneity,” Acta Neuropathologica 142 (2021): 73.

[49] R. W. L. So and J. C. Watts, “α-Synuclein Conformational Strains as Drivers of Phenotypic Heterogeneity in Neurodegenerative Diseases,” Journal of Molecular Biology 435 (2023): 168011.

[50] J. A. Maier, C. Martinez, K. Kasavajhala, L. Wickstrom, K. E. Hauser, and C. Simmerling, “ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB,” Journal of Chemical Theory and Computation 11 (2015): 3696.

[51] J. Huang, S. Rauscher, G. Nawrocki, et al., “CHARMM36m: An Improved Force Field for Folded and Intrinsically Disordered Proteins,” Nature Methods 14 (2017): 71.

[52] H.-N. Wu, F. Jiang, and Y.-D. Wu, “Significantly Improved Protein Folding Thermodynamics Using a Dispersion-Corrected Water Model and a New Residue-Specific Force Field,” Journal of Physical Chemistry Letters 8 (2017): 3199.

[53] W. Wang, “Recent Advances in Atomic Molecular Dynamics Simulation of Intrinsically Disordered Proteins,” Physical Chemistry Chemical Physics 23 (2021): 777.

[54] P. Flagmeier, G. Meisl, M. Vendruscolo, et al., “Mutations Associated With familial Parkinson's Disease Alter the Initiation and Amplification Steps of α-Synuclein Aggregation,” Proceedings of the National Academy of Sciences of the United States of America 113 (2016): 10328.

[55] G. A. P. de Oliveira and J. L. Silva, “Alpha-Synuclein Stepwise Aggregation Reveals Features of an Early Onset Mutation in Parkinson's Disease,” Communications Biology 2 (2019): 374.

[56] J. J. Geldhof, A. M. Malinowska, G. J. L. Wuite, E. J. G. Peterman, and I. Heller, “Temperature Quantification and Temperature Control in Optical Tweezers,” Methods in Molecular Biology 2478 (2022): 123.

[57] L. Loeff, J. W. J. Kerssemakers, C. Joo, and C. Dekker, “AutoStepfinder: A Fast and Automated Step Detection Method for Single-Molecule Analysis,” Patterns 2 (2021): 100256.

[58] M. J. Abraham, T. Murtola, R. Schulz, et al., “GROMACS: High Performance Molecular Simulations through Multi-Level Parallelism from Laptops to Supercomputers,” SoftwareX 1-2 (2015): 19.

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