Familial Mutations May Switch Conformational ... - ACS Publications

Subscriber access provided by University of Newcastle, Australia

Article

Familial Mutations May Switch Conformational Preferences in #-Synuclein Fibrils Liang Xu, Buyong Ma, Ruth Nussinov, and Damien Thompson ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00406 • Publication Date (Web): 11 Jan 2017 Downloaded from http://pubs.acs.org on January 14, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Chemical Neuroscience is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Neuroscience

Familial Mutations May Switch Conformational Preferences in α-Synuclein Fibrils Liang Xu*†, Buyong Ma§, Ruth Nussinov‡§, and Damien Thompson*† †

Department of Physics, Bernal Institute, University of Limerick, Limerick, Ireland.

‡

Sackler Inst. of Molecular Medicine Department of Human Genetics and Molecular Medicine Sackler

School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel. §

Basic Science Program, Leidos Biomedical Research, Inc. Cancer and Inflammation Program,

National Cancer Institute, Frederick, MD 21702, USA.

1

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT The pathogenesis of Parkinson’s disease is closely associated with the aggregation of the α-synuclein protein. Several familial mutants have been identified and shown to affect the aggregation kinetics of α-synuclein through distinct molecular mechanisms. Quantitative evaluation of the relative stabilities of the wild type and mutant fibrils is crucial for understanding the aggregation process and identifying the key component steps. In this work, we examined two topologically different α-synuclein fibril structures that are either determined by solid-state NMR method or modelled based on solid-state NMR data, and characterized their conformational properties and thermodynamic stabilities using molecular dynamics simulations. We show that the two fibril morphologies have comparable size, solvent exposure, secondary structures, and similar molecule/peptide binding modes; but different stabilities. Familial mutations do not significantly alter the overall fibril structures but shift their relative stabilities. Distinct mutations display altered fibril conformational behavior, suggesting different propagation preferences, reminiscent of cross-seeding among prion strains and tau deletion mutants. The simulations quantify the hydrophobic and electrostatic interactions, as well as N-terminal dynamics, that may contribute to the divergent aggregation kinetics that has been observed experimentally. Our results indicate that small molecule and peptide inhibitors may share the same binding region, providing molecular recognition that is independent of fibril conformation. Keywords: α-synuclein; mutation; Parkinson’s disease; amyloid aggregation; molecular dynamics simulations

2


Page 2 of 39

Page 3 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION Presence of Lewy bodies and Lewy neurites in the patient’s brain are prominent neuropathological hallmarks of Parkinson’s disease (PD).1-4 Their major constituent is α-synuclein (α-Syn) fibrils, the final stage of aggregation by α-Syn protein.5-7 α-Syn has been implicated in synaptic plasticity, vesicle dynamics, dopamine metabolism, suppression of apoptosis, regulation of glucose activity, modulation of calmodulin activity, and ATP synthase function.8-13 Increasing evidence has shown that α-Syn aggregates, especially oligomers, may play a key role in triggering neurotoxicity,14 possibly by interacting with and disrupting membranes,15 and impairing protein degradation and function of several organelles like the mitochondria and endoplasmic reticulum.14-18 α-Syn oligomers can spread between interconnected brain regions, and thereby act as seeds for prion-like pathology.19-21 The identification of a series of rare familial mutations, A30P,22 E46K,23 H50Q,24, 25 G51D,26, 27 A53E,28 and A53T,29 in the SNCA gene that encodes the α-Syn protein further establishes it as causative for the pathogenesis of related neurodegenerative disorders such as PD and dementia with Lewy bodies.30 The effects of these mutations on α-Syn aggregation kinetics and membrane binding have been investigated in in vitro experiments.31-47 However, aggregation is sensitive to experimental conditions such as solution pH, temperature, protein concentration, and concentration of metal ions, and so it can be difficult to obtain consistent results for specific mutants. Here we overview the conclusions relating to the effects of point mutations on the kinetics of aggregation of α-Syn into oligomers (on- or off-pathway) and fibrils. Compared to wild type α-Syn, the A30P variant accelerates nonfibrillar aggregate formation (oligomers or protofibrils) but significantly reduces the formation of 3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

mature fibrils.31, 33, 38, 44 However, recently A30P was also shown to retard the formation of both oligomers and fibrils.45 The E46K mutant increases membrane affinity,48 and accelerates α-Syn aggregation and fibrillation.41,

42

H50Q mutation dramatically accelerates α-Syn aggregation into

fibrils and strongly stabilizes fibrils.36 The G51D mutation significantly slows down the rate of α-Syn aggregation into oligomers and fibrils, but forms amorphous aggregates.27, 49 The A53E mutation significantly reduces aggregation in vitro and at cell membranes, but can still form very thin amyloid fibrils with similar morphology as wild type.40, 43 The A53T mutant considerably accelerates α-Syn aggregation, and fibrillation is faster than wild type.31, 46, 50 It has been suggested that these mutations could alter the initiation and proliferation steps.37 These studies provide insights into the effects of familial mutations on the kinetics of aggregation, but little is known about the influence of each mutant on the relative thermodynamic stability of α-Syn fibrils. α-Syn monomer predominantly displays α-helical conformations in the presence of biological micelles or micelles of detergent sodium lauroylsarcosinate,51, 52 but it is an intrinsically disordered protein (IDP) under normal physiological conditions, adopting a random coil conformation.53, 54 The core of α-Syn fibril is organized in a parallel in-register structure rich in cross β-sheet conformation,55, 56

a characteristic of fibrils such as Alzheimer’s disease-associated amyloid β(Aβ).57-59 The full

sequence of α-Syn contains 140 residues, and can be divided into three distinct domains: the amphipathic N-terminal region (residues 1–60), which is believed to be responsible for membrane binding;60,

61

the highly hydrophobic non-amyloid-β component (NAC) domain (residues 61–95),

which is assumed to be essential for α-Syn aggregation;62 and the acidic (negatively charged) C-terminal domain (residues 96–140), which is critical for the chaperone-like activity of α-Syn.63, 64 In 4


Page 4 of 39

Page 5 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


addition to interactions with membranes, the N-terminal also plays an important role in the formation of α-Syn oligomers,45 and all familial variants occur in this domain, indicating the critical role of this region in modulating the aggregation tendency of α-Syn. Also note that E46 is in one highly conserved hexameric repeat of K43T44K45E46G47V48. This N-terminal repeat domain (XKTKEGVXXXX) has been shown to mediate the aggregation kinetics of α-Syn.65, 66 However, the underlying mechanism of how the N-terminus tunes the aggregation kinetics is not well understood. More importantly, α-Syn aggregates, oligomers or fibrils, are potential targets for therapeutic intervention. Recently, the human monoclonal antibody, aducanumab was shown to selectively interact with Aβ aggregates and reduce Aβ plaques in the brains of Alzheimer’s patients (AD).67 As such, elucidation of the effects of familial mutations on the conformation and relative stability of α-Syn fibrils should provide important information on modulating α-Syn aggregate formation. Amyloid polymorphism can affect seed and fibril propagation in neurodegenerative diseases.68 Single mutations can modulate the distribution of different conformations, thereby influencing fibril growth, as in the case of α-Syn,69 Tau protein amyloid formation,70 and prion species.71 On the basis of solid-state NMR (ssNMR) experimental results,72, 73 there are at least two polymorphic variants of α-Syn fibrils (Fig. 1 and Fig. 2). A pathogenic fibril of full-length human α-Syn was recently determined by ssNMR method, in which the core residues are arranged in parallel, in-register β-sheets with a Greek key topology (G-Key)73 (Fig. 1). Based on the Riek’s model,63 the structure of NAC was predicted by Miller’s group;74 and a different fibril structure of α-Syn(30–110) was also predicted,75 which has many residue contacts (for example Val37-Ala53, and His50-Asn65) that are not possible in the G-Key structure. Both ssNMR results63 and computational75 studies indicated that the α-Syn(30–110) 5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

may have five β-strands packed in the conventional β-strand-loop-β-strand fold (5-Fold) (Fig. 2). Therefore, it is important to compare the stabilities of the two human α-synuclein fibril structures and investigate the possible effects of known mutations on their relative stabilities. In this study, we characterized the conformational dynamics of the two α-Syn fibril structures using extensive molecular dynamics (MD) simulations in aqueous solution. The effects of each point mutation (A30P, E46K, H50Q, G51D, A53E, and A53T) on fibril structure and stability were compared. The data from our systematic simulation study reveals that these two fibril structures, although exhibiting quite different folds, are similar in their overall structural properties and interaction with small molecules/peptides. The point mutations do not significantly alter the general folds of the two fibril structures, but there are structural changes in regions far away from the mutation sites. We find that α-Syn fibril is more stable in the G-Key fold than in the 5-Fold. However, the mutants display different conformational preferences. Computed fibril stabilities do not correlate with experientally observed aggregation kinetics, indicating that the divergent aggregation propensity of these mutants is more likely due to the early aggregation stage that is modulated by hydrophobic and electrostatic interactions, as well as the N-terminal dynamics of α-Syn proteins. RESULTS Two morphologies of α-Syn fibrils have comparable structural properties. We first compare the structural similarity and difference of the two α-Syn fibril structures. The folds are distinctly different (Fig. 1 and Fig. 2), yet the computed molecular dynamics structures reveal that they are comparable in overall fibril size, solvent-exposed area (Fig. 3), and secondary structure (Fig. 4). The average radii of gyration for G-Key and 5-Fold fibrils are 27.2±0.1 Å and 27.7±0.1 Å, respectively (Fig. S1). 6


Page 6 of 39

Page 7 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Moreover, both fibrils have a core dimension of ~45 Å. The calculated values of solvent-exposed surface area (SASA) are 25522±28 Å2 and 25857±175 Å2 for G-Key and 5-Fold fibrils, respectively. The values of SASA for the component monomers in G-Key and 5-Fold fibrils are ~4034±10 Å2 and ~4206±17 Å2, respectively. Such results seem to indicate that 5-Fold fibril is slightly more exposed to solvent. However, the relative differences in the SASA of fibril and monomer are only 1% and 4%, respectively, illustrating how rapidly the difference in SASA of the two fibril morphologies would decrease with increasing number of monomers. Both fibril cores are rich in β structures (Fig. 4) but the distribution of these β structures differs from each other (Fig. 5 and Fig. 6). Notably, the NAC region (residues 61–95) in both fibril models has a high tendency to form β structures. The average percentages of β structure in G-Key and 5-Fold are 28% and 33%, respectively, implying comparable contents of β structure in the two fibrils with different morphologies. The high degree of similarity in populations of other secondary structures (random coil, bend, helix, and turn) can also be seen in Fig. 4, with the largest difference less than 5% in turn structures. Both fibril structures display helical conformations (predominantly α-helix) within the N-terminal region containing residues 20–40, with Val26–Ala30 showing high propensity to form helix conformations (Fig. 5 and Fig. 6). The relative stability of these two fibril structures was estimated by their calculated conformational energies, as shown in Fig. 7. The α-Syn fibril structure in G-Key fold has a signifcantly lower energy (-10021 ± 17 kcal/mol) than that in 5-Fold (-9684 ± 22 kcal/mol). Torsional energies contribute the most to this difference (Fig. S4), suggesting that β-sheet-turn-β-sheet (U-turn) structures in the 5-Fold fibril may induce greater backbone strain. This strain could be compensated for, and the 5-fold structure made stable, under specific experimental conditions, or with additional optimization of its internal 7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

interactions (for example, mutation-triggered shifts, as discussed later). The different folds make the interactions between NT and NAC regions, and between CT and NAC regions, more favorable in the G-Key model than in the 5-Fold model (Fig. 8), which may contribute to the stability of α-Syn fibril structure in G-Key fold. In addition, no direct interactions were observed between NT and CT regions in both fibril structures. We also compared the ligand binding propensity of the α-Syn fibril structure in the different folds by performing molecular docking. Small molecule epigallocatechin-3-gallate (EGCG) and short peptides (WWP, cyclo-WWP), which could prevent the formation of α-Syn fibril,76-78 were blindly docked to the α-Syn fibril structure using the method outlined in our previous study.75 The docking results are summaried in Table S3. EGCG predominantly binds to the NAC domain and the N-terminal of α-Syn fibril structure, independent of the type of fold. The short β-peptides preferentially bind to the NAC domain, and the cyclo-peptide interacts with the α-Syn C-terminal. The above docking results indicate that small molecule/peptide inhibitors could potentially bind to the same region of α-Syn fibrils in both morphologies. Familial mutations do not significantly alter WT α-Syn fibril structures. Having characterized the WT α-Syn fibril structures, we now examine the influence of each N-terminal mutation on the fibril structures. We find no significant alterations in the overall structural properties, but subtle mutation-specific changes are found. Fig. 3 and Fig. S1 show that the radius of gyration of all mutants varies from 26.3 Å to 28.8 Å, and from 27.7 Å to 28.5 Å for α-Syn fibrils in G-Key and 5-Fold, respectively, comparable to WT fibrils. Computed SASA ranges for WT and mutants were also very similar, for fibril and monomer structures in the two different folds. These findings suggest that the 8


Page 8 of 39

Page 9 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


single-site mutations do not give a notable difference in the overall size and solvent exposure of the α-Syn fibrils. The changes in the contents of secondary structures vary by mutant (Fig. 4), and all changes are within the same order of magnitude relative to that of WT fibril structures. The same mutants in the 5-Fold structure possess comparable or slightly higher contents of β-strand compared to those in the G-key motif. The same mutant in different folds might have different effects on the secondary structures. For example, slight (2%) decrease and increase in β content was observed for G51D in G-Key and 5-Fold, respectively. In addition, no significant mutation-triggered alterations in the overall helical conformations were observed. The residue-specific distributions of helix and β-strand for mutants in G-Key and 5-Fold are shown in Fig. 5 and Fig. 6, respectively. For all but A30P, mutants in the G-Key model increase the helical structures differently in the same N-terminal region compared to the WT fibril. Lower helix content was also observed in the C-terminal in variants E46K and H50Q (Fig. 5). Similarly, most mutants (E46K, H50Q, A53E, and A53T) in the 5-Fold structure promote the formation of helical conformation in the same N-terminal region as WT (Fig. 6). All mutants in the G-Key fold increase the propensity for β structure around Val37 and Leu38, and decrease the β-structure tendency of regions Lys45–Val55 and Lys60–Val66 to varying extents (Fig. 5). Note that both these two regions run into highly conserved hexameric repeats K43T44K45E46G47V48 and K58T59K60E61Q62V63, respectively. Here, we show that charge swap of -1 → +1 in this region (E46K) or charge alteration of 0 → -1 (G51D and A53E) in regions close to the K58T59K60E61Q62V63 motif disrupt the secondary structures of these repeats. Moreover, discrete regions involving residues 78–98 that are far away from the mutation sites are also affected, but such perturbations are generally small except that residues 9



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Lys80 and Thr81 in A53E display increased β structure by 73% and 55% compared to WT, respectively (Fig. 5). In all cases of α-Syn mutants in 5-Fold (Fig. 6), the mutation sites are in the flexible N-terminal (A30), the first β strand (E46K), or the second β strand (H50, G51, and A53). As expected, point mutations not only affect the propensity of β structure of their neighboring residues, but could influence residues of other β strands through triggered interactions in adjacent β strands. In addition to direct propagation by intramolecular interactions, long-range effects including electrostatic interactions and allosteric effects could also contribute to the divergent alterations of secondary structures caused by point mutations.79 A30P illustrates such long-range effects. The changes in β structures are on the same order of magnitude as for the other mutants even though this mutation site is in the dynamic N-terminal. Taken together, our simulation results suggest that residue-specific changes allosterically spread through the fibril, even though point mutations do not significantly alter the overall structures of α-Syn fibrils. Conformational preference of mutant α-Syn fibril structures. The conformational energy for each mutant was calculated and summarized in Fig. 7. Note that the difference in conformational energy is about 337 kcal/mol, indicating that WT α-Syn fibril may predominantly assume the G-Key conformation. However, the conformational energy difference for the mutants decreases to about 44 kcal/mol, 11 kcal/mol, 101 kcal/mol, 70 kcal/mol, 61 kcal/mol, and 104 kcal/mol for A30P, E46K, H50Q, G51D, A53E, and A53T, respectively. The computed relatively small difference in the conformational energy of E46K (11 kcal/mol) suggests that the E46K fibril is similarly stable in both folds. We note that the E46K mutant behaves differently than the other mutants. In the G-Key 10


Page 10 of 39

Page 11 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


morphology, E46 originally establishes a salt-bridge with K80, and replacement of E with K results in unfavorable electrostatic repulsion between K46 and K80 (Fig. 1), destabilizing the E46K fibril structure in G-Key fold. However, in the 5-Fold topology, we observed that one of the C-terminal residues E110 interacts with K46 by forming a salt-bridge (Fig. 2), which seems to stabilize the E46K mutant. (The possible conformation switch between G-Key and 5-Fold structure is provided below.) The largest difference in the conformational energy (104 kcal/mol) indicates that A53T fibril strongly prefers G-Key conformation. Similarly, we found that A53E prefers G-Key fold, while A30P, H50Q and G51D are less strongly penalised in the 5-Fold conformation. This finding suggests that single point mutation in α-Syn fibril leads to different conformational preference, and thereby different populations under specific conditions, which is consistent with the known polymorphism of α-Syn fibrils.56, 80 E46K mutant in 5-Fold morphology prefers extended conformation. In order to further examine the contribution of the interactions between K46 and E110 to the stability of E46K mutant in 5-Fold, we constructed another E46K mutant (henceforth referred to as E46K_II) where all K46 residues interact with corresponding E110 residues within the same monomers (Fig. 9), and performed a 100-ns MD simulation without constraints after minimization and equilibration with harmonic constraints between K46 and E110 residues. Only three E110 residues persistently interact with five K46 residues, and the other two E110 residues move far away from K46 in aqueous solution, indicating that the E46K_II mutant might not predominate. The E46K_II fibril structure shows a reduced radius of gyration of (~25.4 Å) compared to the extended E46K (~27.9 Å) (Fig. 3). No significant differences in SASAs of both fibril and monomer are observed between the compact 11



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

E46K_II and extended E46K mutant (Fig. 3). As expected, the interactions between NAC and C-terminal regions increase due to the formation of salt-bridges (Fig. 8), and E46K has a slightly better time-averaged conformational energy in the extended conformation (-9863 ± 8 kcal/mol for E46K, and -9789 ± 42 kcal/mol for E46K_II), suggesting that the compact E46K morphology is less favorable than the extended morphology (Fig. 9). Further analyses of the contribution of each component to the total energy suggest that compact E46K_II has improved van der Waals contacts (by 1475 kcal/mol) with an associated loss in solvation of 2097 kcal/mol, indicating that a delicate balance between hydrophobic interactions and solvation energy could determine whether E46K will adopt an extended or compact morphology. The relatively small difference in electrostatic interactions (3128 ± 4 kcal/mol and 3096 ± 5 kcal/mol for E46K and E46K_II, respectively) implies that the contribution of salt-bridges beween K46 and E110 to the stability of fibril structures are not so significant compared to the other components like van der Waals and solvation energies caused by the alteration of the organization of the C-terminal. Note that the formation of different morphology of α-Syn fibrils is sensitive to solution conditions.81,

82

The fibril structure in G-Key was grown in 50 mM sodium phosphate buffer,73

whereas the fibril structure of 5-Fold was grown in 0.01% sodium azide.72 In most of our simulations we simply added Cl- ions to neutralize the net protein charge in each system (8 mM Cl- and 10 mM Clfor the G-Key and 5-Fold structures, respectively). To further probe the effect of ion concentrations on the weak salt-bridge that formed in the E46K system, we performed another 100ns-MD simulation of E46K in physiological strength 150 mM NaCl, and found that the average number of salt-bridges decreases from 3.3 to 0.8 due to electrostatic screening by the salt ions. However, no significant 12


Page 12 of 39

Page 13 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


changes in the general structure properties and conformational energies were observed (Table S2). DISCUSSION Familial mutations contribute to the polymorphism of α-Syn fibrils. The polymorphism of amyloid fibrils has been well established.56,

80

For example, Aβ42 fibrils, which are one of the

hallmarks of AD, have four NMR structures available in the Protein Data Bank (PDB ID: 2BEG,57 2MXU,58 5KK3,59 and 2NAO83), showing distinctive morphologies (U-turn containing two β-strands, S-shape containing three β-strands, and S-shape containing four β-strands) under different experimental conditions. Solid-state NMR experiments suggested that the landscape of α-Syn fibrils may be more complex.7,

56

Previous studies have characterized two α-Syn strains with different

structures, levels of toxicity, seeding and propagation properties84, 85 but to date only one ssNMR structure has been reported (PDB ID: 2N0A)73 for α-Syn fibril (G-Key). The 5-Fold morphology of α-Syn fibril was suggested in earlier ssNMR experimental studies,55,

72

but the atomic molecular

structure was only recently proposed from computational models.75 Based on the atomic structures of G-Key and 5-Fold, we characterized the structural properties of both WT and six familial mutants. It is interesting to find that the two morphologies have comparable size, solvent exposure, and secondary structures, implying that the morphological changes in α-Syn fibrils may originate from different folds (tertiary and/or quaternary, Fig. S5), consistent with recent experimental observations.86 Moreover, no significant alterations in the overall size, solvent exposure, and secondary structures were observed between WT and mutants in the two polymorphs, suggesting that mutants could exhibit the same structure as either of the WT forms. However, fibrils could consist of various number of monomers in in vitro experiments, and so caution must be used in comparing the fibril size estimated here using the 13



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 39

radius of gyration with experimental data.87 Experimentally, A30P has been observed to show a conserved β-sheet core and adopt the WT fibril structure.33 However, A30P could also form a fibril structure different from WT.38 H50Q, A53E, and A53T have been suggested to have minor secondary structure perturbations.39,

40, 43, 46, 88

By

contrast, E46K could have significant effects, and may change the overall structural arrangement of the fibrils.46 E46K has also been reported to enhance the interactions of the C-terminal with either the NAC or the N-terminal, resulting in a more compact structure compared to WT fibrils.35, 41, 45 In line with these experimental observations, we found that E46K has the highest tendency to shift between 5-Fold and G-Key fold morphologies due to electrostatic repulsion between proximate K46 and K80 sites. In the proposed structural model E46K_II, we observed interactions of the N-terminal residues K46 with the C-terminal residues E110, which could contribute to enhanced interactions between the N- and the C-terminal regions. Thus, the E46K mutant is highly polymorphic, and could induce new morphologies by rearranging its C-terminal region. Recently, the Arctic E22G mutant of Aβ40 has been identified to yield a highly polymorphic Aβ40 fibril landscape.89 Thus, introduction of an additional charge, or electrostatic interactions in general, can have profound effects on amyloid polymorphs. Cross-seeding experiments suggest that A30P monomers cannot bind to A53T fibril seeds, and A53T monomers cannot bind to A30P fibril seeds either.69 Consistent with this finding, our results show that A30P fibril has a preference for 5-Fold morphology, but A53T fibril prefers G-Key fold. In addition, formation of helical structures in the N-terminal (residues 20–30) of all fibril models is consistent with experimental observations of α-helices at residues 20–34.90 This helical segment may 14


Page 15 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


play a role in binding to cell membranes.91 However, direct correlation of the membrane binding affinity to the contents of helical conformations is not feasible because the first 20 N-terminal residues, which display high tendency to form helices, are not included (by necessity, see Methods) in the present fibril structures. Familial mutations alter the conformational preferences of α-Syn fibrils. The effects of PD-related familial mutations of α-Syn on the aggregation thermodynamics, kinetics, and the underlying molecular mechanism has been intensively studied. Differential scanning calorimetry experiments suggested a positive enthalpy change (heat absorption) in α-Syn fibril formation.92 Recent studies revealed that point mutations dramatically affect the rate of lipid-induced fibril formation and secondary nucleation.37 The presence of nonfibrillar α-Syn oligomers has been suggested in the aggregation process of amyloid.47, 93 These oligomers may hinder elongation of α-Syn fibrils and slow down the kinetics of amyloid formation in a concentration-dependent manner.94 On the other hand, certain α-Syn oligomers that are kinetically trapped during the formation of fibrils have been characterized and found to have a much lower tendency to elongate due to the difference in the arrangement of β-strands in the oligomers (antiparallel) and fibrils (parallel).95 The rearrangement of β-strands involved in the formation of fibrils in different folds from those oligomers seems rather slow. These minor populations of relatively unstable species prevent amyloid formation by increasing the energy barrier for amyloid assembly.96 We thus speculate that the mutations could alter the relative population of oligomeric states that determine the aggregation kinetics of α-Syn. The coexistence of polymorphs of the same mutants or redistribution of the ensemble of conformers of α-Syn fibrils has been suggested in NMR studies of A30P and A53T mutations.97 Based 15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

on the calculated conformational energies, we further show that WT α-Syn fibril preferentially adopts G-Key conformation. But different mutants shift the relative stability (and relative population) of the same α-Syn fibril in both morphologies. Direct correlation of our findings with experimental aggregation kinetics is not straightforward as we found there is no clear relationship between the computed conformational preference of α-Syn fibrils and their known aggregation propensity. A30P has been reported to form fibrils more slowly than WT,31, 33, 38, 44, 45 and our results indicate that the A30P mutant becomes more stable when adopting 5-Fold morphology. Fibrillation in H50Q and A53T is more rapid,31, 36, 46, 50 and their fibrils are more prone to form 5-Fold and G-Key morphology, respectively. Both G51D and A53E have excess negative charge, and have been reported to form fibrils significantly slower than WT,27, 40, 43, 49 and their fibrils are respectively found to be more stable in 5-Fold and G-Key. E46K accelerates fibrillation41, 42 and is comparably stable in both morphologies. From this comparison, it appears that subtle changes in α-Syn sequence have more profound effects on the early stage of aggregation of oligomers rather than on the final fibril conformation. Previous experimental studies suggested that long-range intramolecular interactions within the α-Syn monomer could stabilize those conformations that inhibit aggregation.98 Familial mutations are likely to enhance or disrupt such tertiary interactions, leading to conformations with opposite aggregation tendencies. In this respect, the introduction of the more hydrophobic residue Q/T may accelerate the initial assembly of α-Syn monomers. The introduction of a negatively charged residue D/E may slow α-Syn aggregation due to intermolecular electrostatic repulsion, resulting in a high energy barrier that would need to be surmounted to form amyloids. The switch of charge from E to K in the E46K mutant has an opposite effect, which may contribute to its fast aggregation. The Pro residue is known to 16


Page 16 of 39

Page 17 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


preferentially display β-turn rather than α-helix or β-sheet in soluble proteins.99 The conformational constraints induced by A30P in the N-terminal may retard formation of oligomers or conversion of oligomers to fibrils. In summary, here we provide molecular details of the effect of familial mutations on the structure and stability of α-Syn fibrils. Two distinct morphologies of WT α-Syn fibrils display comparable size, solvent exposure, secondary structure contents, and ligand-binding mode, but different stabilities. Familial mutants shift the relative stabilities (and relative populations) of these fibrils without significantly altering their overall structures. All mutations are in the N-terminal region, but their influence on fibril structures is intrinsically long-ranged. In particular, A30P, H50Q and G51D fibrils exhibit a calculate preference for the 5-Fold conformation, whereas WT, A53E and A53T prefer G-Key and E46K fibrils show comparable stability in both conformations. Formation of oligomers appears more important in modulating aggregation kinetics of α-Syn mutants, resulting in divergent effects on the conformational preference of the fibrils. Nevertheless recognition of the fibril by small molecule/peptide

drugs

may

share

a

common

binding

region

and

appears

to

be

conformation-independent. METHODS Based on available ssNMR data,72, 73 we constructed two α-Syn fibril structures with each fibril structure consisting of five monomers. The full-length α-Syn fibril that has been determined by ssNMR structure was used to build the G-Key model.73 The five β-strand model was derived from ssNMR data in our previous study.75 Approximately 20 N-terminal residues and 30 C-terminal residues of α-Syn fibril are highly unstructured, and the core region of α-Syn fibril comprises ~70 17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

residues,72, 88, 100-102 and so our fibril models are composed of residues 20 to 110. The six familial mutants (A30P, E46K, H50Q, G51D, A53E, and A53T) were constructed by replacement of A30, E46, H50, G51, and A53 with Pro, Glu, Gln, Asp, and Glu/Thr, respectively. MD simulations were performed at constant pressure-temperature ensemble (NPT) using the NAMD2 program.103 Each fibril structure was represented by the CHARMM27 force field with CMAP corrections.104, 105 TIP3P water molecules were used to solvate each fibril structure in a cubic water box.106 The minimum distance between protein and the edge of water box was at least 12 Å. Each system was neutralized by adding counter-ions. The Langevin piston method and Langevin dynamics were applied to control the pressure at 1 atm and the temperature at 310 K.107 The long-range electrostatics were treated using the Particle Mesh Ewald (PME) method,108 and the van der Waals interactions were calculated using a switching function with a twin cutoff of 10 Å and 12 Å. The SHAKE algorithm was applied to constrain the hydrogen bonds.109 An integration time step of 2 fs was used and structures were saved every 5000 steps (10 ps). Two WT fibril structures (G-Key and 5-Fold) were first energy minimized for 10000 steps, and then heated to 310 K gradually. The two structures were equilibrated for 2 ns before running production dynamics at 1 atm and 310 K. MD simulations of structures in G-Key and 5-Fold were carried out for 50 ns and 100 ns, respectively. The conformations of WT at 50 or 100 ns were taken as the starting conformations for MD simulations of mutants. The production runs of MD simulations of WT and mutants in either G-Key or 5-Fold were performed for 200 ns each. The time evolution of the radius of gyration was monitored and shown in Fig. S1. Extending MD simulations of wild types to 400 ns gives comparable results (Fig. S2 and Table S1), indicating that both systems seem to reach an equilibrium. The cumulative average of the 18


Page 18 of 39

Page 19 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


percentage of β-strand was also calculated and shown in Fig. S3. The changes of the radius of gyration and the content of β-strand suggest that each system could reach an equilibrium within 200-ns MD simulations. Consequently, the last 50-ns trajectory of each system was used for analysis. The relative stability of each fibril model was evaluated in terms of the conformational energy, which includes molecular mechanics energy (MM, sum of bonded energy terms), non-bonded energy terms (electrostatics and van der Waals), and solvation energy calculated by the generalized Born model using the molecular volume (GBMV) method implemented in CHARMM program (c40b2).104, 110

To obtain the bonded and non-bonded energy contribution, each fibril structure was first minimized

for 200 steps using the steepest decedent method. The standard parameters in the GBMV II algorithm were applied to obtain the solvent free energy of the fibril structures after energy minimization. The average values and standard deviations in all data analysis were obtained using the block average method, i.e., the last 50-ns trajectory was divided into two parts and an average value was calculated for each part; the final average value and standard deviation were obtained from the average values of two parts. It should be noted that the current GBMV method uses implicit continuum solvation models to calculate protein-solvent interactions. Emerging results suggest that H-bonds are extraordinarily stable in amyloid fibrils dues to hyperpolarization and hypercooperativity effects,111 and future models should include more explicit treatment of water-mediated H-bonds for more precise calculation of conformational stabilities. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Radius of gyration of wild type α-Syn fibrils and mutants during MD simulations (Fig. S1 and S2); Cumulative average of the percentage of β-strand (Fig. S3); Energy components of conformational energy (Fig. S4); Conformational change of NAC quaternary structures (Fig. S5); Characterization of wild type α-Syn fibril structures on the basis of the last 50-ns trajectories of total 400-ns MD simulations (Table S1); Comparison of 5-Fold E46K fibrils simulated in aqueous solution with different NaCl concentrations (Table S2); and summary of molecular docking result (Table S3). The calculated structures are available upon request from the authors. AUTHOR INFORMATION Corresponding Author *Email: [email protected]; [email protected]. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS We acknowledge Science Foundation Ireland (SFI) for financial support under Grant Number 11/SIRG/B2111 and computing resources at the SFI/Higher Education Authority Irish Center for High-End Computing (ICHEC). We acknowledge financial support from NCI, NIH, under contract number HHSN261200800001E. This research was supported (in part) by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. 20


Page 20 of 39

Page 21 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


REFERENCES 1.

2. 3.

4. 5. 6. 7. 8. 9. 10.

11. 12.

13. 14.

15.

16.

Spillantini, M. G., Crowther, R. A., Jakes, R., Hasegawa, M., and Goedert, M. (1998) α-Synuclein in filamentous inclusions of Lewy bodies from Parkinson's disease and dementia with Lewy bodies. Proc. Nat. Acad. Sci. U. S. A. 95, 6469–6473. Recchia, A. (2004) α-Synuclein and Parkinson's disease. FASEB J. 18, 617–626. Volpicelli-Daley, Laura A., Luk, Kelvin C., Patel, Tapan P., Tanik, Selcuk A., Riddle, Dawn M., Stieber, A., Meaney, David F., Trojanowski, John Q., and Lee, Virginia M. Y. (2011) Exogenous α-synuclein fibrils induce Lewy body pathology leading to synaptic dysfunction and neuron death. Neuron 72, 57–71. Dauer, W., and Przedborski, S. (2003) Parkinson's disease: Mechanisms and models. Neuron 39, 889–909. Recasens, A., and Dehay, B. (2014) Alpha-synuclein spreading in Parkinson's disease. Front. Neuroanat. 8, 159. Breydo, L., Wu, J. W., and Uversky, V. N. (2012) α-Synuclein misfolding and Parkinson's disease. Biochim. Biophys. Acta (BBA) - Mol. Basis Dis. 1822, 261–285. Mor, D. E., Ugras, S. E., Daniels, M. J., and Ischiropoulos, H. (2016) Dynamic structural flexibility of α-synuclein. Neurobiol. Dis. 88, 66–74. Burré, J. (2015) The Synaptic function of α-synuclein. J. Parkinson Dis. 5, 699–713. Dettmer, U., Selkoe, D., and Bartels, T. (2016) New insights into cellular α-synuclein homeostasis in health and disease. Curr. Opin. Neurobiol. 36, 15–22. Uchihara, T., and Giasson, B. I. (2015) Propagation of alpha-synuclein pathology: Hypotheses, discoveries, and yet unresolved questions from experimental and human brain studies. Acta Neuropathol. 131, 49–73. Fink, A. L. (2006) The aggregation and fibrillation of α-synuclein. Acc. Chem. Res. 39, 628– 634. Ludtmann, M. H. R., Angelova, P. R., Ninkina, N. N., Gandhi, S., Buchman, V. L., and Abramov, A. Y. (2016) Monomeric alpha-synuclein exerts a physiological role on brain ATP synthase. J. Neurosci. 36, 10510–10521. Emamzadeh, F. (2016) Alpha-synuclein structure, functions, and interactions. J. Res. Med. Sci. 21, 29. Roberts, R. F., Wade-Martins, R., and Alegre-Abarrategui, J. (2015) Direct visualization of alpha-synuclein oligomers reveals previously undetected pathology in Parkinson’s disease brain. Brain 138, 1642–1657. Stefanovic, A. N. D., Stöckl, M. T., Claessens, M. M. A. E., and Subramaniam, V. (2014) α-Synuclein oligomers distinctively permeabilize complex model membranes. FEBS J. 281, 2838–2850. Winner, B., Jappelli, R., Maji, S. K., Desplats, P. A., Boyer, L., Aigner, S., Hetzer, C., Loher, T., Vilar, M., Campioni, S., Tzitzilonis, C., Soragni, A., Jessberger, S., Mira, H., Consiglio, A., Pham, E., Masliah, E., Gage, F. H., and Riek, R. (2011) In vivo demonstration that α-synuclein oligomers are toxic. Proc. Nat. Acad. Sci. U. S. A. 108, 4194–4199. 21



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

17.

18.

19.

20.

21. 22.

23.

24.

25.

26.

27.

28.

29.

Guardia-Laguarta, C., Area-Gomez, E., Rub, C., Liu, Y., Magrane, J., Becker, D., Voos, W., Schon, E. A., and Przedborski, S. (2013) α-Synuclein is localized to mitochondria-associated ER membranes. J. Neurosci. 34, 249–259. Khalife, M., Morshedi, D., Aliakbari, F., Tayaranian Marvian, A., Mohammad Beigi, H., Azimzadeh Jamalkandi, S., and Pan-Montojo, F. (2015) Alpha-synuclein fibrils interact with dopamine reducing its cytotoxicity on PC12 cells. Protein J. 34, 291–303. Masuda-Suzukake, M., Nonaka, T., Hosokawa, M., Oikawa, T., Arai, T., Akiyama, H., Mann, D. M. A., and Hasegawa, M. (2013) Prion-like spreading of pathological α-synuclein in brain. Brain 136, 1128–1138. Aulić, S., Le, T. T., Moda, F., Abounit, S., Corvaglia, S., Casalis, L., Gustincich, S., Zurzolo, C., Tagliavini, F., and Legname, G. (2014) Defined α-synuclein prion-like molecular assemblies spreading in cell culture. BMC Neurosci. 15, 69. Olanow, C. W., and Brundin, P. (2013) Parkinson's disease and alpha synuclein: Is Parkinson's disease a prion-like disorder? Mov. Disord. 28, 31–40. Krüger, R., Kuhn, W., Müller, T., Woitalla, D., Graeber, M., Kösel, S., Przuntek, H., Epplen, J. T., Schols, L., and Riess, O. (1998) Ala30Pro mutation in the gene encoding α-synuclein in Parkinson's disease. Nat. Genet. 18, 106–108. Zarranz, J. J., Alegre, J., Gómez-Esteban, J. C., Lezcano, E., Ros, R., Ampuero, I., Vidal, L., Hoenicka, J., Rodriguez, O., Atarés, B., Llorens, V., Tortosa, E. G., del Ser, T., Muñoz, D. G., and de Yebenes, J. G. (2004) The new mutation, E46K, of α-synuclein causes parkinson and Lewy body dementia. Ann. Neurol. 55, 164–173. Appel-Cresswell, S., Vilarino-Guell, C., Encarnacion, M., Sherman, H., Yu, I., Shah, B., Weir, D., Thompson, C., Szu-Tu, C., Trinh, J., Aasly, J. O., Rajput, A., Rajput, A. H., Jon Stoessl, A., and Farrer, M. J. (2013) Alpha-synuclein p.H50Q, a novel pathogenic mutation for Parkinson's disease. Mov. Disord. 28, 811–813. Proukakis, C., Dudzik, C. G., Brier, T., MacKay, D. S., Cooper, J. M., Millhauser, G. L., Houlden, H., and Schapira, A. H. (2013) A novel α-synuclein missense mutation in Parkinson disease. Neurology 80, 1062–1064. Kiely, A. P., Asi, Y. T., Kara, E., Limousin, P., Ling, H., Lewis, P., Proukakis, C., Quinn, N., Lees, A. J., Hardy, J., Revesz, T., Houlden, H., and Holton, J. L. (2013) α-Synucleinopathy associated with G51D SNCA mutation: A link between Parkinson’s disease and multiple system atrophy? Acta Neuropathol. 125, 753–769. Lesage, S., Anheim, M., Letournel, F., Bousset, L., Honoré, A., Rozas, N., Pieri, L., Madiona, K., Dürr, A., Melki, R., Verny, C., and Brice, A. (2013) G51D α-synuclein mutation causes a novel Parkinsonian-pyramidal syndrome. Ann. Neurol. 73, 459–471. Pasanen, P., Myllykangas, L., Siitonen, M., Raunio, A., Kaakkola, S., Lyytinen, J., Tienari, P. J., Pöyhönen, M., and Paetau, A. (2014) A novel α-synuclein mutation A53E associated with atypical multiple system atrophy and Parkinson's disease-type pathology. Neurobiol. Aging 35, 2180.e2181–2180.e2185. Polymeropoulos, M. H., Lavedan, C., Leroy, E., Ide, S. E., Dehejia, A., Dutra, A., Pike, B., Root, H., Rubenstein, J., Boyer, R., Stenroos, E. S., Chandrasekharappa, S., Athanassiadou, 22


Page 22 of 39

Page 23 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

A., Papapetropoulos, T., Johnson, W. G., Lazzarini, A. M., Duvoisin, R. C., Di Iorio, G., Golbe, L. I., and Nussbaum, R. L. (1997) Mutation in the alpha-synuclein gene identified in families with Parkinson's disease. Science 276, 2045–2047. Petrucci, S., Ginevrino, M., and Valente, E. M. (2016) Phenotypic spectrum of alpha-synuclein mutations: New insights from patients and cellular models. Parkinsonism Relat. Disord. 22, S16–S20. Li, J., Uversky, V. N., and Fink, A. L. (2001) Effect of familial Parkinson's disease point mutations A30P and A53T on the structural properties, aggregation, and fibrillation of human α-synuclein. Biochemistry 40, 11604–11613. Fares, M. B., Ait-Bouziad, N., Dikiy, I., Mbefo, M. K., Jovi i , A., Kiely, A., Holton, J. L., Lee, S. J., Gitler, A. D., Eliezer, D., and Lashuel, H. A. (2014) The novel Parkinson's disease linked mutation G51D attenuates in vitro aggregation and membrane binding of α-synuclein, and enhances its secretion and nuclear localization in cells. Hum. Mol. Genet. 23, 4491–4509. Lemkau, L. R., Comellas, G., Kloepper, K. D., Woods, W. S., George, J. M., and Rienstra, C. M. (2012) Mutant protein A30P α-synuclein adopts wild-type fibril structure, despite slower fibrillation kinetics. J. Biol. Chem. 287, 11526–11532. Narhi, L., Wood, S. J., Steavenson, S., Jiang, Y., Wu, G. M., Anafi, D., Kaufman, S. A., Martin, F., Sitney, K., Denis, P., Louis, J. C., Wypych, J., Biere, A. L., and Citron, M. (1999) Both familial Parkinson's disease mutations accelerate α-synuclein aggregation. J. Biol. Chem. 274, 9843–9846. Rospigliosi, C. C., McClendon, S., Schmid, A. W., Ramlall, T. F., Barré, P., Lashuel, H. A., and Eliezer, D. (2009) E46K Parkinson’s-linked mutation enhances C-terminal-to-N-terminal contacts in α-synuclein. J. Mol. Biol. 388, 1022–1032. Khalaf, O., Fauvet, B., Oueslati, A., Dikiy, I., Mahul-Mellier, A. L., Ruggeri, F. S., Mbefo, M. K., Vercruysse, F., Dietler, G., Lee, S. J., Eliezer, D., and Lashuel, H. A. (2014) The H50Q mutation enhances α-synuclein aggregation, secretion, and toxicity. J. Biol. Chem. 289, 21856–21876. Flagmeier, P., Meisl, G., Vendruscolo, M., Knowles, T. P. J., Dobson, C. M., Buell, A. K., and Galvagnion, C. (2016) Mutations associated with familial Parkinson’s disease alter the initiation and amplification steps of α-synuclein aggregation. Proc. Nat. Acad. Sci. U. S. A. 113, 10328–10333. Kahle, P. J., Nielsen, S. B., Macchi, F., Raccosta, S., Langkilde, A. E., Giehm, L., Kyrsting, A., Svane, A. S. P., Manno, M., Christiansen, G., Nielsen, N. C., Oddershede, L., Vestergaard, B., and Otzen, D. E. (2013) Wildtype and A30P mutant alpha-synuclein form different fibril structures. PLoS ONE 8, e67713. Ghosh, D., Mondal, M., Mohite, G. M., Singh, P. K., Ranjan, P., Anoop, A., Ghosh, S., Jha, N. N., Kumar, A., and Maji, S. K. (2013) The Parkinson’s disease-associated H50Q mutation accelerates α-synuclein aggregationin vitro. Biochemistry 52, 6925–6927. Ghosh, D., Sahay, S., Ranjan, P., Salot, S., Mohite, G. M., Singh, P. K., Dwivedi, S., Carvalho, E., Banerjee, R., Kumar, A., and Maji, S. K. (2014) The newly discovered Parkinson’s disease associated finnish mutation (A53E) attenuates α-synuclein aggregation and membrane 23



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

41.

42. 43.

44.

45.

46.

47.

48.

49.

50. 51. 52.

53.

54.

binding. Biochemistry 53, 6419–6421. Fredenburg, R. A., Rospigliosi, C., Meray, R. K., Kessler, J. C., Lashuel, H. A., Eliezer, D., and Lansbury, P. T. (2007) The impact of the E46K mutation on the properties of α-synuclein in its monomeric and oligomeric states. Biochemistry 46, 7107–7118. Pandey, N., Schmidt, R. E., and Galvin, J. E. (2006) The alpha-synuclein mutation E46K promotes aggregation in cultured cells. Exp. Neurol. 197, 515–520. Rutherford, N. J., and Giasson, B. I. (2015) The A53E α-synuclein pathological mutation demonstrates reduced aggregation propensity in vitro and in cell culture. Neurosci. Lett. 597, 43–48. Li, J., Uversky, V. N., and Fink, A. L. (2002) Conformational behavior of human alpha-synuclein is modulated by familial Parkinson's disease point mutations A30P and A53T. Neurotoxicol. 23, 553–567. Tosatto, L., Horrocks, M. H., Dear, A. J., Knowles, T. P. J., Dalla Serra, M., Cremades, N., Dobson, C. M., and Klenerman, D. (2015) Single-molecule FRET studies on alpha-synuclein oligomerization of Parkinson’s disease genetically related mutants. Sci. Rep. 5, 16696. Greggio, E., Lemkau, L. R., Comellas, G., Lee, S. W., Rikardsen, L. K., Woods, W. S., George, J. M., and Rienstra, C. M. (2013) Site-specific perturbations of alpha-synuclein fibril structure by the Parkinson's disease associated mutations A53T and E46K. PLoS ONE 8, e49750. Kaganovich, D., Lázaro, D. F., Rodrigues, E. F., Langohr, R., Shahpasandzadeh, H., Ribeiro, T., Guerreiro, P., Gerhardt, E., Kröhnert, K., Klucken, J., Pereira, M. D., Popova, B., Kruse, N., Mollenhauer, B., Rizzoli, S. O., Braus, G. H., Danzer, K. M., and Outeiro, T. F. (2014) Systematic comparison of the effects of alpha-synuclein mutations on its oligomerization and aggregation. PLoS Genet. 10, e1004741. Giannakis, E., Pacífico, J., Smith, D. P., Hung, L. W., Masters, C. L., Cappai, R., Wade, J. D., and Barnham, K. J. (2008) Dimeric structures of α-synuclein bind preferentially to lipid membranes. Biochim. Biophys. Acta (BBA) - Biomem. 1778, 1112–1119. Rutherford, N. J., Moore, B. D., Golde, T. E., and Giasson, B. I. (2014) Divergent effects of the H50Q and G51D SNCA mutations on the aggregation of α-synuclein. J. Neurochem. 131, 859–867. Conway, K. A., Harper, J. D., and Lansbury, P. T. (1998) Accelerated in vitro fibril formation by a mutant α-synuclein linked to early-onset Parkinson disease. Nat. Med. 4, 1318–1320. Ulmer, T. S., Bax, A., Cole, N. B., and Nussbaum, R. L. (2004) Structure and dynamics of micelle-bound human α-synuclein. J. Biol. Chem. 280, 9595–9603. Rao, J. N., Jao, C. C., Hegde, B. G., Langen, R., and Ulmer, T. S. (2010) A combinatorial NMR and EPR approach for evaluating the structural ensemble of partially folded proteins, J. Am. Chem. Soc. 132, 8657–8668. Wu, K.-P., Kim, S., Fela, D. A., and Baum, J. (2008) Characterization of conformational and dynamic properties of natively unfolded human and mouse α-synuclein ensembles by NMR: Implication for aggregation, J. Mol. Biol. 378, 1104–1115. Berrocal, R., Vasquez, V., Krs, S. R., Gadad, B. S., and Ks, R. (2014) α-Synuclein misfolding 24


Page 24 of 39

Page 25 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


55. 56.

57.

58.

59.

60.

61. 62. 63. 64.

65. 66.

67.

68.

versus aggregation relevance to Parkinson’s disease: Critical assessment and modeling. Mol. Neurobiol. 51, 1417–1431. Der-Sarkissian, A., Jao, C. C., Chen, J., and Langen, R. (2003) Structural organization of α-synuclein fibrils studied by site-directed spin labeling. J. Biol. Chem. 278, 37530–37535. Heise, H., Hoyer, W., Becker, S., Andronesi, O. C., Riedel, D., and Baldus, M. (2005) Molecular-level secondary structure, polymorphism, and dynamics of full-length α-synuclein fibrils studied by solid-state NMR. Proc. Nat. Acad. Sci. U. S. A. 102, 15871–15876. Luhrs, T., Ritter, C., Adrian, M., Riek-Loher, D., Bohrmann, B., Dobeli, H., Schubert, D., and Riek, R. (2005) 3D structure of Alzheimer's amyloid-β (1-42) fibrils. Proc. Nat. Acad. Sci. U. S. A. 102, 17342–17347. Xiao, Y., Ma, B., McElheny, D., Parthasarathy, S., Long, F., Hoshi, M., Nussinov, R., and Ishii, Y. (2015) Aβ(1–42) fibril structure illuminates self-recognition and replication of amyloid in Alzheimer's disease. Nat. Struct. Mol. Biol. 22, 499–505. Colvin, M. T., Silvers, R., Ni, Q. Z., Can, T. V., Sergeyev, I., Rosay, M., Donovan, K. J., Michael, B., Wall, J., Linse, S., and Griffin, R. G. (2016) Atomic resolution structure of monomorphic Aβ42 amyloid fibrils. J. Am. Chem. Soc. 138, 9663–9674. Lorenzen, N., Lemminger, L., Pedersen, J. N., Nielsen, S. B., and Otzen, D. E. (2014) The N-terminus of α-synuclein is essential for both monomeric and oligomeric interactions with membranes. FEBS Lett. 588, 497–502. Eliezer, D., Kutluay, E., Bussell, R., and Browne, G. (2001) Conformational properties of α-synuclein in its free and lipid-associated states. J. Mol. Biol. 307, 1061–1073. Hashimoto, M., Takenouchi, T., Mallory, M., Masliah, E., and Takeda, A. (2000) The role of NAC in amyloidogenesis in Alzheimer's disease. Am. J. Pathol. 156, 734–736. Kim, T. D., Paik, S. R., and Yang, C.-H. (2002) Structural and functional implications of C-terminal regions of α-synuclein. Biochemistry 41, 13782–13790. Hong, D.-P., Xiong, W., Chang, J.-Y., and Jiang, C. (2011) The role of the C-terminus of human α-synuclein: Intra-disulfide bonds between the C-terminus and other regions stabilize non-fibrillar monomeric isomers. FEBS Lett. 585, 561–566. Kessler, J. C., Rochet, J.-C., and Lansbury, P. T. (2003) The N-terminal repeat domain of α-synuclein inhibits β-sheet and amyloid fibril formation. Biochemistry 42, 672–678. Harada, R., Kobayashi, N., Kim, J., Nakamura, C., Han, S.-W., Ikebukuro, K., and Sode, K. (2009) The effect of amino acid substitution in the imperfect repeat sequences of α-synuclein on fibrillation. Biochim. Biophys. Acta (BBA) - Mol. Basis Dis. 1792, 998–1003. Sevigny, J., Chiao, P., Bussière, T., Weinreb, P. H., Williams, L., Maier, M., Dunstan, R., Salloway, S., Chen, T., Ling, Y., O’Gorman, J., Qian, F., Arastu, M., Li, M., Chollate, S., Brennan, M. S., Quintero-Monzon, O., Scannevin, R. H., Arnold, H. M., Engber, T., Rhodes, K., Ferrero, J., Hang, Y., Mikulskis, A., Grimm, J., Hock, C., Nitsch, R. M., and Sandrock, A. (2016) The antibody aducanumab reduces Aβ plaques in Alzheimer’s disease. Nature 537, 50–56. Ma, B., and Nussinov, R. (2012) Selective molecular recognition in amyloid growth and transmission and cross-species barriers. J. Mol. Biol. 421, 172–184. 25



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

69. 70.

71.

72.

73.

74.

75.

76.

77. 78.

79. 80.

81.

82. 83.

Sidhu, A., Segers-Nolten, I., and Subramaniam, V. (2016) Conformational compatibility is essential for heterologous aggregation of α-synuclein. ACS Chem. Neurosci. 7, 719–727. Meyer, V., Dinkel, P. D., Luo, Y., Yu, X., Wei, G., Zheng, J., Eaton, G. R., Ma, B., Nussinov, R., Eaton, S. S., and Margittai, M. (2014) Single mutations in tau modulate the populations of fibril conformers through seed selection. Angew. Chem. Int. Ed. 53, 1590–1593. Zhou, S., Shi, D., Liu, X., Liu, H., and Yao, X. (2016) Protective V127 prion variant prevents prion disease by interrupting the formation of dimer and fibril from molecular dynamics simulations. Sci. Rep. 6, 21804. Vilar, M., Chou, H. T., Luhrs, T., Maji, S. K., Riek-Loher, D., Verel, R., Manning, G., Stahlberg, H., and Riek, R. (2008) The fold of α-synuclein fibrils. Proc. Nat. Acad. Sci. U. S. A. 105, 8637–8642. Tuttle, M. D., Comellas, G., Nieuwkoop, A. J., Covell, D. J., Berthold, D. A., Kloepper, K. D., Courtney, J. M., Kim, J. K., Barclay, A. M., Kendall, A., Wan, W., Stubbs, G., Schwieters, C. D., Lee, V. M. Y., George, J. M., and Rienstra, C. M. (2016) Solid-state NMR structure of a pathogenic fibril of full-length human α-synuclein. Nat. Struct. Mol. Biol. 23, 409–415. Atsmon-Raz, Y., and Miller, Y. (2015) A proposed atomic structure of the self-assembly of the non-amyloid-β component of human α-synuclein as derived by computational tools. J. Phys. Chem. B 119, 10005–10015. Xu, L., Nussinov, R., and Ma, B. (2016) Coupling of the non-amyloid-component (NAC) domain and the KTK(E/Q)GV repeats stabilize the α-synuclein fibrils. Eur. J. Med. Chem. 121, 841–850. Eidenschink, L., Kier, B. L., Huggins, K. N. L., and Andersen, N. H. (2009) Very short peptides with stable folds: Building on the interrelationship of Trp/Trp, Trp/cation, and Trp/backbone-amide interaction geometries. Proteins 75, 308–322. Sivanesam, K., Byrne, A., Bisaglia, M., Bubacco, L., and Andersen, N. (2015) Binding interactions of agents that alter α-synuclein aggregation. RSC Adv. 5, 11577–11590. Huggins, K. N. L., Bisaglia, M., Bubacco, L., Tatarek-Nossol, M., Kapurniotu, A., and Andersen, N. H. (2011) Designed hairpin peptides interfere with amyloidogenesis pathways: Fibril formation and cytotoxicity inhibition, interception of the preamyloid state. Biochemistry 50, 8202–8212. Xu, L., Nussinov, R., and Ma, B. (2016) Allosteric stabilization of the amyloid-β peptide hairpin by the fluctuating N-terminal. Chem. Commun. 52, 1733–1736. Miller, Y., Ma, B., and Nussinov, R. (2010) Polymorphism in Alzheimer Aβ amyloid organization reflects conformational selection in a rugged energy landscape. Chem. Rev. 110, 4820–4838. Hoyer, W., Antony, T., Cherny, D., Heim, G., Jovin, T. M., and Subramaniam, V. (2002) Dependence of α-synuclein aggregate morphology on solution conditions. J. Mol. Biol. 322, 383–393. Makky, A., Bousset, L., Polesel-Maris, J., and Melki, R. (2016) Nanomechanical properties of distinct fibrillar polymorphs of the protein α-synuclein. Sci. Rep. 6, 37970. Wälti, M. A., Ravotti, F., Arai, H., Glabe, C. G., Wall, J. S., Böckmann, A., Güntert, P., Meier, 26


Page 26 of 39

Page 27 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


84.

85.

86.

87.

88.

89.

90.

91.

92. 93.

94.

95.

B. H., and Riek, R. (2016) Atomic-resolution structure of a disease-relevant Aβ(1–42) amyloid fibril. Proc. Nat. Acad. Sci. U. S. A. 113, E4976–E4984. Bousset, L., Pieri, L., Ruiz-Arlandis, G., Gath, J., Jensen, P. H., Habenstein, B., Madiona, K., Olieric, V., Böckmann, A., Meier, B. H., and Melki, R. (2013) Structural and functional characterization of two alpha-synuclein strains. Nat. Commun. 4, 2575. Guo, Jing L., Covell, Dustin J., Daniels, Joshua P., Iba, M., Stieber, A., Zhang, B., Riddle, Dawn M., Kwong, Linda K., Xu, Y., Trojanowski, John Q., and Lee, Virginia M. Y. (2013) Distinct α-synuclein strains differentially promote tau inclusions in neurons. Cell 154, 103– 117. Sidhu, A., Nolten, I. S., Raussens, V., Claessens, M. M. A. E., and Subramaniam, V. (2016) Distinct mechanisms determine α-synuclein fibril morphology during growth and maturation. ACS Chem. Neurosci. doi: 10.1021/acschemneuro.6b00287. Curtain, C. C., Kirby, N. M., Mertens, H. D. T., Barnham, K. J., Knott, R. B., Masters, C. L., Cappai, R., Rekas, A., Kenche, V. B., and Ryan, T. (2015) Alpha-synuclein oligomers and fibrils originate in two distinct conformer pools: a small angle X-ray scattering and ensemble optimisation modelling study. Mol. BioSyst. 11, 190–196. Comellas, G., Lemkau, L. R., Nieuwkoop, A. J., Kloepper, K. D., Ladror, D. T., Ebisu, R., Woods, W. S., Lipton, A. S., George, J. M., and Rienstra, C. M. (2011) Structured regions of α-synuclein fibrils include the early-onset Parkinson's disease mutation sites. J. Mol. Biol. 411, 881–895. Elkins, M. R., Wang, T., Nick, M., Jo, H., Lemmin, T., Prusiner, S. B., DeGrado, W. F., Stöhr, J., and Hong, M. (2016) Structural polymorphism of Alzheimer’s β-amyloid fibrils as controlled by an E22 switch: A solid-state NMR study, J. Am. Chem. Soc. 138, 9840–9852. Zhao, M., Cascio, D., Sawaya, M. R., and Eisenberg, D. (2011) Structures of segments of α-synuclein fused to maltose-binding protein suggest intermediate states during amyloid formation. Protein Sci. 20, 996–1004. Fusco, G., De Simone, A., Gopinath, T., Vostrikov, V., Vendruscolo, M., Dobson, C. M., and Veglia, G. (2014) Direct observation of the three regions in α-synuclein that determine its membrane-bound behaviour. Nat. Commun. 5, 3827. Ikenoue, T., Lee, Y.-H., Kardos, J., Saiki, M., Yagi, H., Kawata, Y., and Goto, Y. (2014) Cold denaturation of α-synuclein amyloid fibrils. Angew. Chem. Int. Ed. 53, 7799–7804. Conway, K. A., Lee, S. J., Rochet, J. C., Ding, T. T., Williamson, R. E., and Lansbury, P. T. (2000) Acceleration of oligomerization, not fibrillization, is a shared property of both alpha -synuclein mutations linked to early-onset Parkinson's disease: Implications for pathogenesis and therapy. Proc. Nat. Acad. Sci. U. S. A. 97, 571–576. Lorenzen, N., Nielsen, S. B., Buell, A. K., Kaspersen, J. D., Arosio, P., Vad, B. S., Paslawski, W., Christiansen, G., Valnickova-Hansen, Z., Andreasen, M., Enghild, J. J., Pedersen, J. S., Dobson, C. M., Knowles, T. P. J., and Otzen, D. E. (2014) The role of stable α-synuclein oligomers in the molecular events underlying amyloid formation. J. Am. Chem. Soc. 136, 3859–3868. Chen, S. W., Drakulic, S., Deas, E., Ouberai, M., Aprile, F. A., Arranz, R., Ness, S., 27



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

96.

97.

98.

99. 100. 101.

102. 103.

104.

105.

106. 107. 108. 109.

Roodveldt, C., Guilliams, T., De-Genst, E. J., Klenerman, D., Wood, N. W., Knowles, T. P. J., Alfonso, C., Rivas, G., Abramov, A. Y., Valpuesta, J. M., Dobson, C. M., and Cremades, N. (2015) Structural characterization of toxic oligomers that are kinetically trapped during α-synuclein fibril formation. Proc. Nat. Acad. Sci. U. S. A. 112, E1994–E2003. Karamanos, T. K., Pashley, C. L., Kalverda, A. P., Thompson, G. S., Mayzel, M., Orekhov, V. Y., and Radford, S. E. (2016) A population shift between sparsely populated folding intermediates determines amyloidogenicity. J. Am. Chem. Soc. 138, 6271–6280. Bertoncini, C. W., Fernandez, C. O., Griesinger, C., Jovin, T. M., and Zweckstetter, M. (2005) Familial mutants of α-synuclein with increased neurotoxicity have a destabilized conformation. J. Biol. Chem. 280, 30649–30652. Bertoncini, C. W., Jung, Y. S., Fernandez, C. O., Hoyer, W., Griesinger, C., Jovin, T. M., and Zweckstetter, M. (2005) Release of long-range tertiary interactions potentiates aggregation of natively unstructured α-synuclein, Proc. Nat. Acad. Sci. U. S. A. 102, 1430–1435. Chou, P. Y., and Fasman, G. D. (1978) Empirical predictions of protein conformation. Ann. Rev. Biochem. 47, 251–276. Miake, H. (2002) Biochemical Characterization of the core structure of alpha-synuclein filaments. J. Biol. Chem. 277, 19213–19219. Giasson, B. I., Murray, I. V. J., Trojanowski, J. Q., and Lee, V. M. Y. (2000) A hydrophobic stretch of 12 amino acid residues in the middle of α-synuclein is essential for filament assembly. J. Biol. Chem. 276, 2380–2386. Chen, M., Margittai, M., Chen, J., and Langen, R. (2007) Investigation of α-synuclein fibril structure by site-directed spin labeling. J. Biol. Chem. 282, 24970–24979. Phillips, J. C., Braun, R., Wang, W., Gumbart, J., Tajkhorshid, E., Villa, E., Chipot, C., Skeel, R. D., Kalé, L., and Schulten, K. (2005) Scalable molecular dynamics with NAMD. J. Comput. Chem. 26, 1781–1802. Brooks, B. R., Bruccoleri, R. E., Olafson, B. D., States, D. J., Swaminathan, S., and Karplus, M. (1983) CHARMM: A program for macromolecular energy, minimization, and dynamics calculations. J. Comput. Chem. 4, 187–217. MacKerell, A. D., Bashford, D., Bellott, M., Dunbrack, R. L., Evanseck, J. D., Field, M. J., Fischer, S., Gao, J., Guo, H., Ha, S., Joseph-McCarthy, D., Kuchnir, L., Kuczera, K., Lau, F. T. K., Mattos, C., Michnick, S., Ngo, T., Nguyen, D. T., Prodhom, B., Reiher, W. E., Roux, B., Schlenkrich, M., Smith, J. C., Stote, R., Straub, J., Watanabe, M., Wiórkiewicz-Kuczera, J., Yin, D., and Karplus, M. (1998) All-atom empirical potential for molecular modeling and dynamics studies of proteins. J. Phys. Chem. B 102, 3586–3616. Jorgensen, W. L., Chandrasekhar, J., Madura, J. D., Impey, R. W., and Klein, M. L. (1983) Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 79, 926. Feller, S. E., Zhang, Y., Pastor, R. W., and Brooks, B. R. (1995) Constant pressure molecular dynamics simulation: The Langevin piston method. J. Chem. Phys. 103, 4613. Darden, T., York, D., and Pedersen, L. (1993) Particle mesh Ewald: An N⋅log(N) method for Ewald sums in large systems. J. Chem. Phys. 98, 10089. Ryckaert, J.-P., Ciccotti, G., and Berendsen, H. J. C. (1977) Numerical integration of the 28


Page 28 of 39

Page 29 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


110.

111.

cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes. J. Comput. Phys. 23, 327–341. Lee, M. S., Feig, M., Salsbury, F. R., and Brooks, C. L. (2003) New analytic approximation to the standard molecular volume definition and its application to generalized Born calculations. J. Comput. Chem. 24, 1348–1356. Mompeán, M., Nogales, A., Ezquerra, T. A., and Laurents, D. V. (2016) Complex system assembly underlies a two-tiered model of highly delocalized electrons. J. Phys. Chem. Lett. 7, 1859–1864.

29



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig. 1. Representative conformations of α-Syn fibrils in the Greek key fold (G-Key). The starting (t=0 ns) WT structure and structures after 200 ns are shown for WT and all mutants. The mutation sites are shown in CPK representation, as well as transparent surface areas colored according to the residue types: red for acidic residue; blue for basic residue; green for polar residue; and white for nonpolar residue. For the sake of clarity, G51 is not highlighted in the WT structures. Fig. 1 57x20mm (300 x 300 DPI)


Page 30 of 39

Page 31 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig. 2. Representative conformations of α-Syn fibrils in the 5-Fold. The representation and color are the same as shown in Fig. 1. Fig. 2 111x77mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig. 3. (A) The values of radius of gyration (Rg), and solvent accessible surface area (SASA) for (B) fibril and (C) monomer. The SASA of (buried) monomer was averaged over the SASA of the three inner monomers. Fig. 3 172x369mm (300 x 300 DPI)


Page 32 of 39

Page 33 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig. 4. The overall percentage of secondary structures for WT and mutants of α-Syn fibrils. The helix structures includes α-helix, π-helix and 310-helix, but α-helix is the major contribution. Fig. 4 58x43mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig. 5. (A) The residue-specific population of helical conformation. (B) The residue-specific β-strand conformation of WT α-Syn fibrils in G-Key fold. (C-H) The difference in residue-specific β-strand conformation of α-Syn familial mutants relative to the WT. Fig. 5 220x304mm (300 x 300 DPI)


Page 34 of 39

Page 35 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig. 6. (A) The residue-specific population of helical conformation. (B) The residue-specific β-strand conformation of WT α-Syn fibrils in 5-fold. (C-H) The difference in residue-specific β-strand conformation of α-Syn familial mutants relative to the WT. Fig. 6 221x307mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig. 7. (A)The conformational energy (enthalpy) of WT and familial mutants of α-Syn fibrils. (B) Conformational energy difference of WT and familial mutants of α-Syn fibrils (G-Key fold relative to 5-Fold). Fig. 7 106x142mm (300 x 300 DPI)


Page 36 of 39

Page 37 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig. 8. The interaction energy between different regions (N-terminal, NT; C-terminal, CT; and NAC) of WT and familial mutants of α-Syn fibrils. Fig. 8 55x39mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig. 9. (A) Conformations of E46K_II at the starting (t=0 ns) and 100 ns of MD simulations. (B) The conformational energy of two polymorphs of E46K mutant, as well as different contributions to the total energy. Fig. 9 90x103mm (300 x 300 DPI)


Page 38 of 39

Page 39 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


TOC Table of Content 61x46mm (300 x 300 DPI)


Familial Mutations May Switch Conformational ... - ACS Publications

Recommend Documents