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

α-Synuclein (α-syn) amyloid fibril, as the major component of Lewy bodies and pathological entity spreading in human brain, 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 determined the cryo-EM structure of a full-length α-syn fibril formed by N-terminal acetylated E46K mutant α-syn (Ac-E46K). The fibril structure represents a new 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 a higher capability of seeding fibril formation 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.

E46K) of α -syn have been identified causative to the early onset of familial PD. Here, we determined the cryo-EM structure of a full-length α -syn fibril formed by N-terminal acetylated E46K mutant α -syn (Ac-E46K). The fibril structure represents a new 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 a higher capability of seeding fibril formation 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

INTRODUCTION
Deposition of α -syn amyloid fibrils in Lewy bodies (LB) and Lewy neuritis (LN) is a common histological hallmark of Parkinson's disease (PD) and synucleinopathies such as dementia with Lewy bodies (DLB) and multiple system atrophy (MSA) 1-3 .
Accumulating evidence show that α -syn amyloid fibrils serve as prion-like seeds for the propagation and cell-to-cell transmission of the pathological entity of α -syn 4,5 . The spread of pathological α -syn fibril is closely associated with the disease progression 6,7 .
Several single point mutations and genomic duplication or triplication of SNCA, the gene encoding α -syn, have been identified to be causative to the familial forms of the diseases with a broad spectrum of distinctive clinical symptoms [8][9][10][11][12] . Moreover, the hereditary mutations exhibit exacerbated pathology in various cellular and PD-like animal models 13,14 . Intriguingly, the cryo-EM structure of full-length wild-type (WT) α -syn fibril demonstrates that in the five mutation sites found in familial PD, four of them, i.e. E46K, A53T/E, G51D and H50Q, are located at the protofilamental interface of the WT fibril 15,16 , which indicates that these mutations may alter the fibril structure and consequently influence α -syn amyloid aggregation and PD pathology.
α -Syn hereditary mutation E46K was originally identified from a Spanish family with autosomal dominant parkinsonism 11 . The clinical phenotype of E46K patient features rapid and severe disease progression with early onset of the parkinsonism of DLB 11 . Previous studies have shown that the E46K mutation can enhance α -syn fibril formation and α -syn pathology in vitro and in cultured cells 13,17,18 . Solid state NMR study has shown that the E46K mutation causes large conformational changes of the fibril structure 19 .
In this work, we determined the cryo-EM structure of the N-terminally acetylated E46K α -syn (Ac-E46K) fibril at an overall resolution of 3.37 Å. The structure reveals a distinct fold of α -syn, in which the electrostatic interactions in the WT fibril are broken and reconfigured due to the E46K mutation. We further show that the Ac-E46K fibril is less stable than the WT fibril under harsh conditions and protease cleavage, while the mutant fibril is more efficient in seeding amyloid fibril formation.
This work provides a structural mechanism for the influence of E46K on the amyloid fibril formation of α -syn, and suggests that electrostatic interactions may serve as one of the driving force for the polymorphic fibril formation of α -syn.  Fig. 2). The Ac-E46K fibril is approximately two times more twisted than the Ac-WT fibril with a pitch of ~ 64 nm in comparison with ~ 120 nm of the Ac-WT. Moreover, the Ac-E46K fibril features a right-handed helical twist ( Supplementary Fig. 2), which is distinct from the left-handed twist commonly found in the WT and other PD-familial mutant α -syn fibrils 15,16,[22][23][24] . The different fibril morphologies suggest different properties of the Ac-E46K and Ac-WT fibrils, which may associate with their different pathologies.

RESULTS
To identify the different properties of the Ac-E46K and Ac-WT fibrils, we first sought to test the stability of the fibrils by cold denaturation 25 . The fibrils were cooled down and incubated at 0 °C for 48 h. Fibril disassociation was assessed by the loss of β structures monitored by circular dichroism (CD) spectroscopy. The result showed that the Ac-E46K fibril denatured significantly faster than that of the Ac-WT fibril ( Fig. 1a). In addition, we found that during the experiment, the Ac-E46K fibril stored at -80 °C underwent apparent denaturation after thawing; in contrast, the Ac-WT fibril were generally stable. To quantitatively measure the stability of the fibrils upon cycles of freeze-thaw, we flash-froze the α -syn fibrils in liquid nitrogen and thawed the fibrils at room temperature in water bath. CD spectra showed that in eight cycles of freeze-thaw, the structure of Ac-WT fibril was well maintained with a consistent content of β structures (Fig. 1b). In contrast, the Ac-E46K fibril gradually lost its β structures as the freeze-thaw cycles increased, indicating the disassembly of amyloid fibrils (Fig. 1b). Thus, these results indicate that the Ac-E46K fibril is less stable than the Ac-WT fibril.
Since α -syn fibrils propagate by seeding the amyloid fibril formation in the transfected neurons, we attempted to compare the seeding property of the Ac-E46K and Ac-WT fibrils. We prepared the fibril seeds by sonication of the preformed fibrils (PFFs). Intriguingly, we noticed that under the same sonication condition, the Ac-E46K fibril broke into significantly shorter fragments than that of the Ac-WT fibril visualized by negative-staining transmission electron microscopy (TEM) and AFM (Fig. 1c, d), which is consistent with the stability tests, and more importantly may reflect an increased pathology of the Ac-E46K fibril since fibril fragmentation has been found as a key factor in nucleation-depended fibrillation 26 . Next, we used the fragmentated PFFs to seed Ac-E46K and Ac-WT, respectively, and monitored the amyloid fibril formation by the ThT fluorescence assay and TEM. The result showed that under the tested condition, there was no obvious fibril formed by the Ac-E46K or Ac-WT α -syn without seeding (Fig. 1e, Supplementary Fig. 3). While, in the presence of fibril seeds, fibril formation was observed, and the Ac-E46K fibril seeds showed markedly higher seeding efficiency than that of the Ac-WT (Fig. 1e, Supplementary   Fig. 3), which suggests that the E46K fibril may have a higher ability of propagation and thus may be more pathological than the WT fibril.
Furthermore, we compared the stability of the fibrils under proteinase K (PK) digestion. The result showed that the Ac-E46K fibril is digested significantly faster than that of the Ac-WT fibril (Fig. 1f). Together, these data indicate that the Ac-E46K fibril is less stable than the Ac-WT fibril in both physical and chemical treatments and more efficient in fragmentation and propagation.

α -syn fibril by cryo-EM
To understand the structural basis underlying the different properties between the Ac-E46K and Ac-WT fibrils, we sought to determine the atomic structure of Ac-E46K fibril by cryo-EM. The Ac-E46K fibril sample was fixed on the carbon grid and frozen in liquid ethane. The cryo-EM micrographs were acquired at 105,000× magnification on the 300 keV Titan Krios microscope equipped with the K2 Summit camera. 13,064 fibrils picked from 754 micrographs were used for the reconstruction of the Ac-E46K fibril ( Table 1). The Ac-E46K fibril sample is morphologically homogeneous with a one dominant species in the 2D classification of the fibrils ( Supplementary Fig. 4). After helical reconstruction of the dominant fibril species by Relion, we obtained a 3D density map of the Ac-E46K fibril to an overall resolution of 3.37 Å ( Supplementary Fig. 5). The density map showed a right-handed helix with a width of ~10 nm and a helical pitch of ~68 nm (Fig. 2), which is consistent with the AFM measurement ( Supplementary Fig. 2). The fibril contains two protofilaments that intertwine along an approximate 2-fold screw axis (Fig. 2). The helical twist between α -syn subunits is -179.37° and the helical rise is 2.38 Å (Fig. 2b, c, Table 1).
The Ac-E46K fibril exhibits a novel fold of α -syn fibril The high quality cryo-EM density map allowed us to unambiguously build an atomic structure model for the Ac-E46K fibril (Fig. 3a). This structure represents the fibril core (FC) of the Ac-E46K fibril consisting of residues 45-99 out of a total of 140 amino acids of α -syn ( Fig. 3a), which is slightly smaller than the Ac-WT FC consisting of residues 37-99 formed under the same condition 15 . While, similar to that of the WT fibril, the N-and C-termini of Ac-E46K α -syn remain flexible and are not visible by cryo-EM.
Compared to the structure of Ac-WT α -syn fibril, the major difference lies in the N-terminal region of the FC (termed as FC-N), which covers residues 37-59 in the Ac-WT fibril or 45-59 in the Ac-E46K fibril. In the Ac-WT structure, the FC-N region stretches and packs around the rest of the α -syn molecule (termed as FC-C) via interactions with T75, A76, A78 and K80 of FC-C (Fig. 3c). In contrast, in the Ac-E46K structure, this segment forms a β hairpin and extends from the FC-C with interactions to the side chains of Q62, T64 and V66 (Fig. 3c).
Aside from the large conformational change of the FC-N, the FC-C regions of the Ac-E46K and Ac-WT fibrils adopt a similar topology with a Greek key-like fold ( Fig. 6). In the Ac-WT fibril, α -syn subunits fold in a flat layer; in contrast, in the Ac-E46K fibril, β6 and β7 of the FC-C swap to the next layer to form inter-molecular side-chain interactions within the same protofilament ( Fig.   3d). Thus, the single mutation of E46K entirely changes the α -syn fibril structure.

E46K mutation rearranges the electrostatic interactions in the α -syn fibril
Since the overall structural rearrangement of α -syn fibril is initiated by one single mutation of E46K, we looked close to residue 46 in the Ac-WT and Ac-E46K fibrils.
In the Ac-WT fibril, E46 forms a salt bridge with K80, which together with the salt bridge formed by E61 and K58, locks the FC-N with FC-C ( Fig. 4a, b). In contrast, in the Ac-E46K fibril, the E46K mutation breaks the salt bridge of E46-K80, which results in the breaking of the other two essential electrostatic interactions (K58-E61 and K45-H50-E57) ( Fig. 4a, b). Alternatively, in the Ac-E46K fibril, K80 forms inter-molecular salt bridge with E61 of the opposing α -syn subunit to involve in the fibril interface, and K45 directly forms salt bridge with E57 to stabilize the β-hairpin conformation of the FC-N (Fig. 4a, b).
The E46K mutation also leads to the disruption of the WT protofilament interface ( Fig. 4c, d). Upon the flipping away of the FC-N from the fibril interface, a new fibril interface is formed. The exposed segments 74-79 of opposing α -syn subunits form typical class I parallel in-register homo-steric zippers to mediate the inter-protofilamental interactions (Fig. 4d). The steric-zipper interface is stabilized by the hydrophobic interactions between the side chains of V74, A76 and A78 and flanked by the inter-molecular salt bridges formed by K80-E61', which further lock the two protofilaments (Fig. 4a).
The release of FC-N from the fibril interface in the Ac-E46K fibril increases the flexibility of this segment, which exhibits higher B-factors and poor density for tracing the N-terminal residues 37-44 that are visible in the Ac-WT FC (Fig. 4c). Thus, the relatively loose packing of the Ac-E46K fibril in comparison with that of the Ac-WT fibril may explain the decreased stability and increased sensitivity of the Ac-E46K fibril to the environment, which may associate with the pathology of this PD familial mutation.

DISCUSSION
Structural polymorphism is a common characteristic of amyloid fibrils formed by different amyloid proteins such as α -syn, tau and Aβ 29-31 . Different fibril polymorphs may represent different pathological entities that are associated with different subsets of neurodegenerative diseases 32-34 . Determination of polymorphic amyloid fibril structures is useful for the mechanistic understanding of amyloid pathology. In this work, we report the cryo-EM structure of an α -syn fibril with N-terminal acetylation and familial mutation E46K found in parkinsonisms. The Ac-E46K α -syn fibril exhibits a novel structure that is distinct from the reported α -syn polymorphs.
Compared to the Ac-WT fibril prepared under the same condition, the E46K mutation breaks an important electrostatic interaction of E46-K80 and thus induces the reconfiguration of the overall structure. In the mutant fibril, the FC-N region that used to form the protofilamental interface in the Ac-WT fibril, flips away from the fibril interface and becomes less stable. In line with the structural information, we find that the Ac-E46K fibril is less stable upon both physical and chemical treatments.  (Fig. 5a). In the Ac-WT fibril, intra-molecular electrostatic interactions of K58-E61 and E46-K80 lock the Greek key-like fold of α -syn, and the inter-molecular interactions of E45-H50-E57' are involved in stabilizing the fibril interface, which pairs α -syn protofilaments to form mature fibrils (Fig. 5a). In contrast, in another WT fibril polymorph 23 , the electrostatic interaction forms between K45 and E57', which mediates the dimerization of the protofilaments (Fig. 5a). Alternatively, the Ac-E46K fibril contains an intra-molecular electrostatic interaction between K45 and E57, which locks the β-hairpin fold of the FC-N region, and an inter-molecular interaction between E61 and K80', which stabilizes the interface of the mutant fibril (Fig. 5a).

Preparation of Ac-WT and Ac-E46K
α -syn. Protein expression and purification of full length α -syn Ac-WT and Ac-E46K mutation follows the same protocol described previously 15 Fig. 1).

Polymorphs of Ac-E46K
α -syn fibril. Two polymorphs at least existed in micrographs. One is twisted filament, and the another one is non-twisted filament which currently cannot be solved since this polymorph has no twist. All filament including twisted and non-twisted filaments in 754 micrographs were count as 18891.
Among them, 13064 was twisted filaments which was used for reconstruction. The percentage of twisted filament is 69.15% and was dominated in the two fibril polymorphs.
Model building and refinement. A homology model based on the NMR structure (PDB entry code 2N0A) was built and modified by COOT 28,44 . The model with 3 adjacent layers (6 promoters) was refined using the real-space refinement program in PHENIX 45 . The subunit dimers in the middle of 3 layers was extracted and used as the final model.