Alpha-synuclein oligomers and fibrils originate in two distinct conformer pools: a small angle X-ray scattering and ensemble optimisation modelling study

Abstract

The 140 residue intrinsically disordered protein α-synuclein (α-syn) self-associates to form fibrils that are the major constituent of the Lewy body intracellular protein inclusions, and neurotoxic oligomers. Both of these macromolecular structures are associated with a number of neurodegenerative diseases, including Parkinson's disease and dementia with Lewy bodies. Using ensemble optimisation modelling (EOM) and small angle X-ray scattering (SAXS) on a size-exclusion column equipped beamline, we studied how the distribution of structural conformers in α-syn may be influenced by the presence of the familial early-onset mutations A30P, E45K and A53T, by substituting the four methionine residues with alanines and by reaction with copper (Cu2+) or an anti-fibril organic platinum (Pt) complex. We found that the WT had two major conformer groups, representing ensembles of compact and extended structures. The population of the extended group was increased in the more rapidly fibril-forming E45K and A53T mutants, while the compact group was enlarged in the oligomer-forming A30P mutant. Addition of Cu2+ resulted in the formation of an ensemble of compact conformers, while the anti-fibril agent and alanine substitution substantially reduced the population of extended conformers. Since our observations with the mutants suggest that fibrils may be drawn from the extended conformer ensemble, we propose that the compact and extended ensembles represent the beginning of oligomer and fibril formation pathways respectively, both of which have been reported to lead to a toxic gain of function. Manipulating these pathways and monitoring the results by EOM and SAXS may be useful in the development of anti-Parkinson's disease therapies.

Molecular Determinants of α-Synuclein Mutants’ Oligomerization and Membrane Interactions

“…Further supporting our initial MD results is the fact that the tertiary structures changes reflected in the radii of gyration are consistent with the small-angle X-ray scattering (SAXS) results 50 demonstrated that the gyration radii for wt αsyn and its mutants A30P , E45K , and A53T are between 20 and 42 Å. In our MD simulations for wt asyn and its mutants radii of gyration are in the range from 14 to 40 Å.…”

The Contribution ofα-Synuclein Spreading to Parkinson’s Disease Synaptopathy

“…This may be related to the different conformation of oligomers and fibrils. Consistently, Curtain and coauthors [122], by using small angle X-ray scattering and ensemble optimization modeling studies, were able to demonstrate that α -synuclein oligomers and fibrils originate in two distinct conformer pools, with E53T and E45K mutations enhancing the tendency to form fibrils, while the A30P conferring propensity toward oligomer assembly. This is in line with previous data reporting that A30P mutant α -synuclein forms different fibril structures [123] and that mutations in the KTKE(Q)GV imperfect amino acid repeats in the N-terminal part of the protein can also affect its tendency toward fibril formation [124].…”

Pressure effects on α-synuclein amyloid fibrils: An experimental investigation on their dissociation and reversible nature

“…Results show that high pressures available at the SAXS beamline -until 1.6 kbar -can subtly modify fibrils' sizes and density features, maintaining their overall morphological structure, and induce the fragmentation into soluble disordered aSN oligomers. The possible simultaneous presence of different oligomeric species, as recently detected and described in different experimental conditions by SAXS experiments [61,8,46], can be pointed up from our SAXS curves. Both wild type and familial mutants of aSN amyloid fibrils show that at increasing pressures the fraction of soluble disordered states is increasing.…”

“…It has to be stressed that SAXS curves obtained with the high pressure cell equipped with diamond windows show a signal-to-noise ratio lower than the one usually achieved by standard synchrotron SAXS sample cells, and a simultaneous exact protein concentration measurement by UV detector, as performed in recent SAXS studies on aSN [46], is not possible in our set-up conditions. Hence, the differences between WT and A30P aSN fibrils detected by Nielsen et al [47], suggesting increased β-sheet pairing distances in A30P fibrils and their more compact interfibrillar arrangement, cannot be evidenced in our experimental set-up, neither the use of a single helix form factor newly adopted to analyse lysozyme amyloid aggregates [48] is suitable for our SAXS curves.…”