Structure based aggregation studies reveal the presence of helix-rich intermediate during α-Synuclein aggregation

Abstract

Mechanistic understanding of nucleation dependent polymerization by α-synuclein (α-Syn) into toxic oligomers and amyloids is important for the drug development against Parkinson's disease. However the structural and morphological characterization during nucleation and subsequent fibrillation process of α-Syn is not clearly understood. Using a variety of complementary biophysical techniques monitoring entire pathway of nine different synucleins, we found that transition of unstructured conformation into β-sheet rich fibril formation involves helix-rich intermediates. These intermediates are common for all aggregating synucleins, contain high solvent-exposed hydrophobic surfaces, are cytotoxic to SHSY-5Y cells and accelerate α-Syn aggregation efficiently. A multidimensional NMR study characterizing the intermediate accompanied with site-specific fluorescence study suggests that the N-terminal and central portions mainly participate in the helix-rich intermediate formation while the C-terminus remained in an extended conformation. However, significant conformational transitions occur at the middle and at the C-terminus during helix to β-sheet transition as evident from Trp fluorescence study. Since partial helix-rich intermediates were also observed for other amyloidogenic proteins such as Aβ and IAPP, we hypothesize that this class of intermediates may be one of the important intermediates for amyloid formation pathway by many natively unstructured protein/peptides and represent a potential target for drug development against amyloid diseases.

Structural, morphological, and functional diversity of amyloid oligomers

“…In some cases (e.g. a-synuclein), a-helical intermediates may also be involved [80,81,107]. A number of stable, yet highly disordered oligomers have been observed as well [32,67,108].…”

“…Under some conditions, the oligomers formed at the early stages of a-synuclein aggregation were also found to be primarily a-helical [80,81]. a-Helix rich oligomers and protofibrils were formed from the primarily disordered ones during incubation at neutral pH (20 mM glycine, pH 7.5) and converted to fibrils upon further incubation [81].…”

“…a-Helix rich oligomers and protofibrils were formed from the primarily disordered ones during incubation at neutral pH (20 mM glycine, pH 7.5) and converted to fibrils upon further incubation [81]. These oligomers were intermediate in stability between fibrils and monomers, and had higher hydrophobic surface exposure than either fibrils or monomers [81].…”

α-synuclein dimer structures found from computational simulations

“…Finally, the C-terminal region, residues 96-140, is highly acidic and negatively charged. This region is believed to stay in extended conformation during polymerization [28].…”

α-Synuclein and Lewy pathology in Parkinson's disease

“…a-Synuclein has the capability of forming multiple soluble oligomeric species, ranging from dimers to more than 100mers [4]. Larger conformations of a-synuclein can also develop and, via intermediate helix-rich conformations [5], can fold into b-pleated sheets. Insoluble amyloid-like fibrils can form from multiple b-pleated sheets.…”