Structure
Volume 31, Issue 4, 6 April 2023, Pages 411-423.e6
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Article
Structural and dynamic insights into α-synuclein dimer conformations

https://doi.org/10.1016/j.str.2023.01.011Get rights and content

Highlights

  • α-Synuclein exists mostly as monomers or dimers at few micromolars at most

  • Experimentally driven computational prediction of α-synuclein dimer structures

  • The stable and frequent conformation of the α-synuclein dimer is compact

  • An α-synuclein dimer conformation exhibits proximity between tyrosine 39 residues

Summary

Parkinson disease is associated with the aggregation of the protein α-synuclein. While α-synuclein can exist in multiple oligomeric states, the dimer has been a subject of extensive debates. Here, using an array of biophysical approaches, we demonstrate that α-synuclein in vitro exhibits primarily a monomer-dimer equilibrium in nanomolar concentrations and up to a few micromolars. We then use spatial information from hetero-isotopic cross-linking mass spectrometry experiments as restrains in discrete molecular dynamics simulations to obtain the ensemble structure of dimeric species. Out of eight structural sub-populations of dimers, we identify one that is compact, stable, abundant, and exhibits partially exposed β-sheet structures. This compact dimer is the only one where the hydroxyls of tyrosine 39 are in proximity that may promote dityrosine covalent linkage upon hydroxyl radicalization, which is implicated in α-synuclein amyloid fibrils. We propose that this α-synuclein dimer features etiological relevance to Parkinson disease.

Introduction

α-Synuclein (αSyn) is a small soluble protein localized mainly in nuclei and in presynaptic terminals of dopaminergic neurons in the substantia nigra pars compacta.1 In the brain, αSyn is involved in several stages of neurotransmitter trafficking, including the stabilization of dopamine vesicles and supporting fast and efficient formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex.2 However, αSyn is involved in multiple disorders termed synucleinopathies, such as Parkinson disease (PD). One of the hallmarks of PD is the accumulation of αSyn amyloid-like fibrils in Lewy bodies.1

The protein is 140 amino acids long and can be divided into three peptide segments: (1) the N-terminal segment or domain (NTD; residues1–60), an amphipathic and lysine-rich region that plays critical roles in phospholipid attachment;3 (2) the non-amyloid-β component (NAC) segment (residues 61–95), a hydrophobic region that is involved in aggregation and fibrilization;4,5 and (3) the C-terminal segment or domain (CTD; residues 96–140), an acidic tail that contains multiple prolines and acidic amino acids that disrupt secondary structure formation.6

αSyn attaches to different types of negatively charged membrane surfaces, including the ones that form dopamine vesicles in neurons.7,8 In addition, αSyn can spontaneously self-associate into oligomers. αSyn amyloid-like fibrils and certain oligomeric species are biochemical features of PD. Other oligomeric species have been documented, such as the α-helical stable tetramer.9,10,11 These different oligomeric species may support different biological functions.

In aqueous solutions, αSyn is intrinsically disordered. Therefore, αSyn exhibits features of misfolded proteins, such as high structural heterogeneity and conformational plasticity. Yet, oftentimes when αSyn binds to a biomolecular target, such as the outer leaflet of phospholipid membranes, or undergoes self-association, it tends to co-fold in a given pathway.12

αSyn aggregation kinetics include a long (tens of hours) delay, which is thought of as the time it takes for αSyn to form a fibrillization nucleus.13 Since exposure of the hydrophobic residues of the NAC segment to the solvent is one factor that can help nucleate oligomerization in the fibril formation pathway, delay of the fibrillization nucleation can be attained via αSyn forms that sterically occlude the NAC from exposure to the solvent. In the free-form monomer, the CTD and NTD sterically interact to shield the NAC from being exposed to the solvent, and by that protect the NAC segment from aggregation-prone interactions with the NAC segment of other αSyn molecules.14 However, there may be other αSyn forms, including small oligomers, which may act to sterically occlude the NAC.

αSyn self-associates and some of its self-associated species lead to pathophysiological-promoting conditions, such as amyloid-like fibrils or toxic oligomers. Therefore, it is of utmost importance to study the mechanism by which αSyn self-associates, and specifically the earliest stages of oligomerization. Logically, the earliest stage of self-association would be the result of dimerization. It is noteworthy that αSyn dimers and trimers have previously been implicated with neurotoxicity.15,16 In addition, the αSyn dimer would serve as the smallest and most elementary self-associated species. The monomer-dimer interaction could be considered controversial, as several studies had demonstrated that αSyn exists as a monomer in solution,17,18,19 or as a tetramer.9

Previously, there have been computational and experimental works providing evidence for the existence of αSyn dimers using ion-mobility mass spectrometry (MS), paramagnetic resonance enhancement (PRE) nuclear magnetic resonance (NMR), and spin-label NMR, as well as potential models of the αSyn dimer from molecular dynamics (MD) simulations.20,21,22,23 Nevertheless, structure models of the αSyn dimer that are based on experimental and computational works integratively combined have not yet been introduced.

Therefore, studying the structural features of an αSyn dimer is important for understanding how self-association starts in αSyn at the first place. In addition, it is hypothesized that specific inter-molecular interactions within αSyn oligomers can act as seeds of aggregation, especially irreversible interactions, such as covalent bonds between pairs of amino acid side chains of two αSyn subunits.24 One such covalent linkage that is related to the stabilization of the fibrillization pathway is the dityrosine covalent linkages.24,25,26,27 Dityrosine linkages can form only after hydroxyl radicalization of tyrosines, which can occur in specific stress conditions, implicated in PD.28 Therefore, upon elucidation of structure models of the αSyn dimer, inter-molecular proximal tyrosines may serve as a signature of the potential for this covalent linkage to occur under such stress conditions.

Several experimental and computational studies have previously focused on the αSyn dimer.24,29,30,31,32,33,34,35 Using high-speed atomic force imaging, Zhang et al.33 have revealed the αSyn dimer exists in several different configurations, at low spatial resolution: (1) with one compact globular subunit and another expanded and dynamic, (2) with both subunits being compact and globular, and (3) with both subunits being expanded and flexible.

Here, we perform an array of experiments that show fresh αSyn molecules are predominantly monomers and dimers in vitro at concentrations of a few micromolars (μM) at most, which is below the known concentrations at which αSyn exhibits efficient aggregation that leads to fibril formation.13 Using single-molecule photo-isomerization-related fluorescence enhancement (smPIFE; commonly known as protein-induced fluorescence enhancement),36,37,38,39 we find that dimer formation is accompanied by local structural changes in the vicinity of specific residues, at the subunit level, pointing toward local structural differences between the monomer and dimer αSyn.

Encouraged by the results, we performed hybrid structural modeling of the αSyn dimer, using hetero-isotopic cross-linking MS (CL-MS)40,41,42 as experimentally derived restraints for discrete MD (DMD) simulations, to elucidate structure models of the αSyn dimer. We report the features of the structure models of different conformations of the αSyn dimer.

Section snippets

MALS: αSyn monomer-dimer mixture

We perform analytical anion exchange (AIEX) chromatography coupled to multi-angle light scattering (MALS), yielding a direct readout of the molecular mass of the different species in solution with high detection sensitivity and minimal chemical modifications or perturbations.43 AIEX-MALS of wt-αSyn yields a single elution peak with an average molecular mass of 21.6 ± 0.8 kDa, which is larger than that of a monomer (14.4 kDa) and smaller than that of a dimer (28.8 kDa; Figure 1A). wt-αSyn eluted

Discussion

We performed AIEX-MALS and SEC-MALS, WB, inter-molecular FRET assays, and smPIFE measurements to demonstrate a monomer-dimer mixture, and potentially a monomer-dimer equilibrium exists in αSyn at the concentration range of a few micromolars. Therefore, when probing a solution of freshly thawed recombinant αSyn with a nominal concentration of a few micromolars, a significant number of dimers are formed (Figures 1 and 2). To gain insight into dimer structures, we perform hetero-isotopic CL-MS,

Key resources table

REAGENT or RESOURCESOURCEIDENTIFIER
Bacterial and virus strains
BL21(DE3) Singles™ Competent Cells - NovagenSigma-AldrichCat#70235-M
MAX Efficiency™ DH5α Competent CellsThermoFisherCat#18258012
Chemicals, peptides, and recombinant proteins
Tris-HClSigma-AldrichCat#93363
Isopropyl β-d-1-thiogalactopyranoside (IPTG)Sigma-AldrichCat#I5502
Ethylenediaminetetraacetic acid (EDTA)Sigma-AldrichCat#E5134
Dithiothreitol (DTT)Sigma-AldrichCat#43815
Magnesium chlorideSigma-AldrichCat#63068
Streptomycin sulfate

Acknowledgments

We thank the following: (1) Drs. Asaf Grupi, Dan Amir, and Elisha Haas from the Mina & Everard Goodman Faculty of Life Sciences in Bar Ilan University for sharing the plasmids of αSyn bearing single cysteine mutations; (2) Dr. Yuval Garini for inviting us to perform the bulk inter-molecular time-resolved- and FCS-FRET measurements of the mixtures of donor- and acceptor-labeled residues 39 αSyn on his experimental setup; (3) Dr. William Breuer from the Proteomics and Mass Spectrometry Unit in

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