Aggregation and Fibrillar
Structure of α-synuclein
Literature Seminar
2020/12/17
B4 Keiichi Kawabata

Contents• Introduction• α-synuclein• Aggregation• Fibril• Familial PD• Oxidation of α-synuclein• SummaryParkinson’s Disease• Symptom• tremor, rigidity, stiffness, postural instability,
constipation• Number of patients• 6.9 million (world, 2015)• 160,000 (Japan, 2017)• Cause• Aggregation and accumulation of misfolded α-synuclein→Formation of Lewy body
→Death of dopamine producing neurons in substantianigra→Decrease of dopamine

Other synucleinopathies• Dementia with Lewy bodies• Symptom: memory problems, Parkinsonism,
hallucinations• Cause: Lewy body

• Multiple System Atrophy (MSA)• Symptom: Shy-Drager syndrome, striatonigral
degeneration, olivopontocerebellar atrophy (OPCA)• Cause: glial cytoplasmic inclusions (GCI) in
oligodendroglia

α-synuclein• 140-residue protein in the brain• In neurons, located at the presynaptic termini• In the cytosol, monomeric and no persistent
structure at physiological conditions

HM D V F M K G L S K

A K E G V V A A A EK T K Q G V A E A A

G K T K E G V L Y V

G S K T K E G V V H

G V A T V A E K T K

E Q V T N V G G A V

V T G V T A V A Q K

T V E G A G S I A A

A T G F V K K D Q L

G K N E E G A P Q E

G I L E D M P V D P

D N E A Y E M P S E

E G Y Q D Y E P E A

OHCremades, N.; Chen, S. W.; Dobson, C. M., International Review ofCell and Molecular Biology, 2017, 329, 79-143.

α-synuclein• Three regions• N-terminal region (residues 1–60)• NAC (nonamyloid-β component) region (residues 61–95)-- hydrophobic→fibril formation• C-terminal region (residues 96–140)--highly acidic

Cremades, N.; Chen, S. W.; Dobson, C. M., International Review ofCell and Molecular Biology, 2017, 329, 79-143.

α-synuclein• N-terminal is generally acetylated as a posttranslational modification• In solution, α-synuclein adopts an α-helicalconformation in the presence of membranes withacidic phospholipid headgroups or high curvature• Loss of all three synucleins does not produce
parkinsonism or any typical form ofneurodegeneration.
Cremades, N.; Chen, S. W.; Dobson, C. M., International Review ofCell and Molecular Biology, 2017, 329, 79-143.
D. Sulzer and R. H. Edwards, J. Neurochem., 2019, 150, 475-486

Possible role of α-synuclein• Regulating synaptic trafficking, homeostasis,
neurotransmitter release and so on• Exocytosis• acts at membrane fusion↑synuclein (α-and β-) inhibits regulated exocytosis• synuclein normally serves to promote dilation of thefusion pore
↑Loss of synuclein delays the release of peptide cargo fromdense core vesicles, and increases the likelihood that the fusion
pore will close

D. Sulzer and R. H. Edwards, J. Neurochem., 2019, 150, 475-486

Contents• Introduction• α-synuclein• Aggregation• Fibril• Familial PD• Oxidation of α-synuclein• SummaryMonomer• Monomeric α-synuclein is dynamic and populates
an ensemble of conformational states• Each conformation has a life span that is dependent
on intramolecular interactions• Intramolecular interactions are stabilized byhydrogen bonds, electrostatic and hydrophobicinteractions (depend on surrounding conditions)• All the conformations α-synuclein adopt are in
equilibrium
Alam, P., et al, J. Neurochem., 2019, 150, 522-534

Tetramer of α-synuclein• α-synuclein could exist in large part (up to 70%) asa folded tetramer with α-helical structure• This tetrameric structure resists aggregation→ Destabilization of the tetramer precedes αsynuclein misfolding in vivo

Cremades, N.; Chen, S. W.; Dobson, C. M., International Review ofCell and Molecular Biology, 2017, 329, 79-143.

Stability of tetramer• Helical tetramer has the most balanced structurewith the lowest activation energy• Familial mutations destabilize α-helical tetramers
and induce neurotoxicity and inclusions.

Calculation by moleculardynamics computer simulations

Xu, L., Bhattacharya, S. and Thompson, D.,
Phys. Chem. Chem. Phys., 2019, 21, 12036-12043

Toxicity of oligomer and fibril

Alam, P., et al, J. Neurochem., 2019, 150, 522-534

Pathological fibrils of α-synclein

Negative-staining TEM image of therecombinant α-syn fibrils
Incubated in a buffer containing 50mM Tris, pH 7.5,150mM KCl and pre-formed fibril seeds, 37 ℃, 3 daysCytotoxicity of the fibrils to HEK 293T cells
Assessed by the MTT assay.
Cells were treated with indicated concentrationof α-syn fibrils for 24 hY. Li et al., Cell Research, 2018, 28, 897–903

Structural characteristics of fibril• Fibril polymorphs vary in the numberand disposition of the protofilaments• Amyloid cross-β structure• Core structure generally include residues
30-110• Monomers adopt an antiparallel inregister β-sandwich fold
• C- and N-terminal could be involved in Obtained by the combination of cryo-EMinteractions between protofilaments imaging with solid-state NMR analysis
Cremades, N.; Chen, S. W.; Dobson, C. M., International Review ofCell and Molecular Biology, 2017, 329, 79-143.

Structure of different fibrillar
polymorphs

Red: NAC region(60-95)
Green: E46
Blue: A53

• The structures of fibrillar α-synuclein obtained by solid-state
NMR [pdb ID# 2n0a] or Cryo-Electron Microscopy [PDB id#6cu7, 6h6b, 6flt, 6a6b, and 6cu8] Alam, P., et al, J. Neurochem., 2019, 150, 522-534

Models for acquisition of amyloid structure• Nucleation–polymerization model• The structural conversion
from random coil to β-sheet
structure take place at themonomeric level

Cremades, N.; Chen, S. W.; Dobson, C. M., International Review ofCell and Molecular Biology, 2017, 329, 79-143.

Models for acquisition of amyloid structure• Nucleation–conversion–
polymerization model• The structural conversion
occurs at the oligomericlevel.

Cremades, N.; Chen, S. W.; Dobson, C. M., International Review ofCell and Molecular Biology, 2017, 329, 79-143.

Familial early onset PD• Point mutations (A30P, E46K, H50Q, G51D, A53T)• Increase the number of possible conformations→Increase the life span of assembly-competentconformers→Aggregation• Duplication and triplication of SNCA gene
• Increase the concentration of assembly-competentconformers→Aggregation

Alam, P., et al, J. Neurochem., 2019, 150, 522-534

Structure of Familial PD mutationsites• Four mutations (H50Q, G51D, A53T/E) change thedimer interface• G51D, A53T/E mutations→introduce hydrophilic or charged residues into thehydrophobic steric-zipper interface→ disrupt the dimer interface

Y. Li et al., Cell Research, 2018, 28, 897–903Structure of Familial PD mutationsites• Four mutations (H50Q, G51D, A53T/E) change thedimer interface• H50Q mutation→break electrostatic interactions among H50, K45, E57’

Y. Li et al., Cell Research, 2018, 28, 897–903

Structure of Familial PD mutation
sites• K58-E61 and E46-K80 form intramolecular salt
bridges, important for the folding of the Greek-key
topology• E46K mutation break the salt bridge between E46
and K80

Y. Li et al., Cell Research, 2018, 28, 897–903

Structures of mutant fibrils• Mutations (except for A30P) lead to polymorphic
fibril structures with distinct features• Fibrils formed by A30P mutant showed nodifference from that of WT(A30 is not involved in the formation of fibril core)

Measured by AFM

Y. Li et al., Cell Research, 2018, 28, 897–903

Contents• Introduction• α-synuclein• Aggregation• Fibril• Familial PD• Oxidation of α-synuclein• SummaryOxidation of methionine• Methionine side-chains are oxidation-prone andreact with physiological oxidants• H2O2 oxidizes all four methionines• Lewy body contains oxidative modifications, suchas nitrated tyrosines and oxidized methionines• Methionine oxidation triggers the formation ofintermediate oligomer species

Binolfi, A. et al. Nat. Commun. 2016
7:10251 doi: 10.1038/ncomms10251

Oxidation of methionine

2D NMR spectra• Structural alterations by methionineoxidation is greater on N-terminus
than C-terminus• methionine oxidation diminished theincrease in residual α-Syn helicity that
occurs in response to physiological Nterminal acetylation

Chemical shiftdifference (Δδ) mapping

secondary structure
propensity (SSP) score

Binolfi, A. et al. Nat. Commun. 2016
7:10251 doi: 10.1038/ncomms10251

Stepwise repair of N-terminal
methionine sulfoxides• Sulfoxide reduction
occurred at Met5
before Met1
• Met1 was not reduced
as long as oxidized
Met5 was present• R/S diastereoisomers
were repaired equallywell• Met116 and Met127
sulfoxides stably
persisted
Binolfi, A. et al. Nat. Commun. 2016
7:10251 doi: 10.1038/ncomms10251

time-resolved NMR (A2780 lysate)

C-terminal methionine sulfoxides
impair phosphorylation• Tyr125 phosphorylation ofoxidized α-synuclein wasimpaired• Oxidized C-terminal
methionines did notcompromise phosphorylationof Ser129 by Plk3
• An age- and diseasedependent decline of Tyr125
phosphorylation is reported
Binolfi, A. et al. Nat. Commun. 2016
7:10251 doi: 10.1038/ncomms10251

Contents• Introduction• α-synuclein• Aggregation• Fibril• Familial PD• Oxidation of α-synuclein• SummarySummary• Aggregation of α-synuclein is explained by twomodels• Familial PD mutations break local interactions andchange fibril structure• C-terminal α-synclein sulfoxides are stablypreserved