Parkinson’s disease associated mutation E46K of α-synuclein triggers the formation of a distinct fibril structure

Abstract

Amyloid aggregation of α-synuclein (α-syn) is closely associated with Parkinson's disease (PD) and other synucleinopathies. Several single amino-acid mutations (e.g. E46K) of α-syn have been identified causative to the early onset of familial PD. Here, we report the cryo-EM structure of an α-syn fibril formed by N-terminally acetylated E46K mutant α-syn (Ac-E46K). The fibril structure represents a distinct fold of α-syn, which demonstrates that the E46K mutation breaks the electrostatic interactions in the wild type (WT) α-syn fibril and thus triggers the rearrangement of the overall structure. Furthermore, we show that the Ac-E46K fibril is less resistant to harsh conditions and protease cleavage, and more prone to be fragmented with an enhanced seeding capability than that of the WT fibril. Our work provides a structural view to the severe pathology of the PD familial mutation E46K of α-syn and highlights the importance of electrostatic interactions in defining the fibril polymorphs.

NMR unveils an N-terminal interaction interface on acetylated-α-synuclein monomers for recruitment to fibrils

“…The N-terminal interaction hotspot on the monomer contains several lysine residues, which may be attracted to the highly negatively charged Cterminus of the oligomer. Conversely, αS amyloid fibrils possess both exposed disordered N-and C-termini flanking a rigid Greek-key fibril core, as observed in structural models determined by solid-state NMR and cryo-electron microscopy (18)(19)(20)(21)(22). In this way, αS fibrils also have a "fuzzy-coat" made of disordered C-termini (~residues 97-140) in addition to a "fuzzy-coat" made of disordered N-termini (~residues 1-36), providing the potential for αS monomers to interact with either the N-or C-termini of the fibrils (Figure 7).…”

“…Under the conditions used here, near neutral pH and higher ionic strength, elongation is the predominant mechanism over surface-mediated secondary nucleation (54). The ability to template on the fibril is dependent on an accessible structured core (55), the core dynamics (21,56), and the orientation of the monomer-fibril interaction. Previous work from our group has shown that increased dynamics in the core reduces the seeding capacity of αS fibrils (56), and that N-N, rather than N-C, monomer interactions promote fibril formation (50).…”

“…Structural models derived from solid-state nuclear magnetic resonance (NMR) and cryo-electron microscopy (EM) have revealed that αS fibrils consist of two protofilaments with a rigid "Greek key" core flanked by intrinsically disordered N-and C-termini (~40 residues each) (18)(19)(20)(21)(22) (Figure 1a). These intrinsically disordered regions (IDRs) are a substantial fraction of the fibril structure and make up a "fuzzy coat" that surrounds the rigid core (18)(19)(20).…”

Relevance of Electrostatic Charges in Compactness, Aggregation, and Phase Separation of Intrinsically Disordered Proteins

“…(c) Primary sequence of the wild-type α-Syn fibril core (aa 37-99). Charged residues are shown in colored bold letters and those interacting in the structures of acetylated wild-type (PDB 6A6B; [120]) and E46K (PDB 6L4S; [121]) α-Syn fibrils are connected by black and red solid lines, respectively. Intermolecular interactions are marked by transverse parallel lines.…”

“…Indeed, with respect to the wild type, the E46K variant reshapes the above-mentioned electrostatic network, and forms a smaller fibril core (residues 45-99) and a distinct fold. This is of utmost relevance with regard to the pathogenic mechanism, as E46K fibrils are less resistant to proteases and mechanical stress and, therefore, more prone to propagation [121].…”