Conformational switch in the alpha-synuclein C-terminus domain directs its fibril polymorphs

Abstract

α-Synuclein inclusion bodies are a pathological hallmark of several neurodegenerative diseases. Although it has been hypothesized a relationship between fibril polymorphism and different pathologies, the molecular origins of polymorphism are not understood. Here, employing biophysical approaches, we demonstrate that the conformational state of the monomeric αSyn is responsible for fibril polymorphism: αSyn can exist as a compact monomer that produces rod fibrils, and as extended monomers that generate twisted fibrils. Using NMR, we found that the compaction relies on a polar interaction between the initial part of the NAC region and a wide section of the C-terminus domain. The compaction can be commonly affected by changes in the chemical environment, like NaCl, the presence of Ca2+ or cellular components, like endotoxins, that alter the interaction NAC/C-terminus domain. Our compaction model also provides mechanistic insights that explain how the behavior of the C-terminus domain imparts the polymorphism during the fibril formation.

Ultrastiff Amyloid-Fibril Network of α-Synuclein Formed by Surface Seeding Reaction Confirmed by Multichannel Electrodeless Quartz-Crystal-Microbalance Biosensor

“…The rod seeds predominantly have two parallel protofilaments, while the twist seeds comprise two intertwined protofilaments. These morphologies are consistent with those reported. ,, Both types of seeds have an equivalent average length of around 50 nm. From Figure B, we can clearly see the formation of the fibril network composed of dense fibrils on the QCM surfaces, on which these seeds were immobilized by flowing the monomer solution.…”

“…To further investigate the relationship between the fibril morphology and the network stiffness, we monitored the α-syn aggregation reaction using the wireless QCM at a higher NaCl concentration of 500 mM. At this salt concentration, elongated fibrils show different morphologies due to the conformation change of α-syn monomer as has been reported in our recent study, which is also confirmed in bulk solutions as shown in Figure S2: In the early stage of the seeding reaction, there is no remarkable difference in the seeding reaction between with rod and twist seeds at the low NaCl concentration (Figure S2A), but the seeding reaction with rod seeds proceeds faster than that with twist seeds at the high NaCl concentration (Figure S2B).…”

“…Depending on solution conditions as well as the seed, α-syn fibrils formed from the same monomers have different morphologies. ,, We prepared α-syn fibrils with two distinct morphologies (rod and twist types) and broke them into fibril fragments by sonication Figure A shows their TEM images.…”

“…To prepare the seeds from the patients, the protein-misfolding-cyclic-amplification (PMCA) method was adopted as previously described. , In brief, the amygdala brain homogenates from patients of PD or MSA were added to 0.5 mg·mL –1 α-syn monomer solution in Tris-HCl buffer (50 mM, pH7.4, 150 mM NaCl). The mixture was placed in a 96-well plate with 200 μL solution in each well containing 10 μM ThT and was then subjected to cyclic sonication (0.3 s irradiation followed by 0.5 s pause) at 37 °C.…”

Generating Ensembles of Dynamic Misfolding Proteins

“…Instead of performing clustering analysis using global features as shown in Figure 3 , we can extend these ideas to include local features. Due to the complex nature of the problem, in many cases information about which residues/regions of the protein are important/irrelevant for misfolding/aggregation is sparse ( Aguirre et al, 2022 ; Seetaloo et al, 2022 ). Perhaps the most informative results come from mutational studies that assess the effect of mutations on misfolding/aggregation rate in a rigorous way.…”