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Letter

Two distinct polymorphic folding states of self-assembly of the non-amyloid # component differ in the arrangement of the residues Maya Pollock-Gagolashvili, and Yifat Miller ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00334 • Publication Date (Web): 15 Sep 2017 Downloaded from http://pubs.acs.org on September 17, 2017

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ACS Chemical Neuroscience

Two distinct polymorphic folding states of selfassembly of the non-amyloid β component differ in the arrangement of the residues

Maya Pollock-Gagolashvili1,2 and Yifat Miller1,2

1

Department of Chemistry and 2Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel

* Author to whom correspondence should be addressed: [email protected] Yifat Miller, Tel: 972-86428705; Fax: 972-86428709

ACS Paragon Plus Environment


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Abstract Parkinson’s disease is a degenerative disorder of the central nerves system. It is characterized by presence of Lew bodies (LBs), in which the main components of the LBs are α-synuclein (AS) aggregates. The central domain of AS, known as the “non-amyloid β component” (NAC) is responsible for the aggregation properties of AS. It is proposed that AS fibrillar structure is a well-packed cross-β structure of the NAC domains, while the N- and C-termini are disordered. Therefore, the study of the self-assembly of NAC domains are crucial in order to understand the molecular mechanisms of AS aggregation. This is a first study that illustrates two distinct polymorphic folding states of NAC that differ in the arrangement of the residues along the sequence. One of the polymorphic folding states reveals a conformational change that is similar to the other polymorphic folding state in the backbone shape, but differ in the arrangement of the residues along the backbone. This work provides insight into the molecular mechanisms through which AS can self-assembled in two different pathways yielding to a conformational change between the two polymorphic folding states.

Keywords: α-synuclein, non-amyloid β component, Parkinson’s disease, polymorphism, neurodegenerative diseases, amyloids


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Misfolded protein diseases are well-known phenomena of many brain disorder diseases.14

These proteins are amyloidogenic proteins that are self-assembled to form oligomers and

fibrillary aggregates.5 Among the large group of amyloidogenic proteins, α-synuclein (AS) is an amyloid protein that is related to Parkinson’s disease (PD). PD is the second most common age-related degenerative neurological disorder after Alzheimer’s disease (AD).6 The progression of PD is caused by the reduction of dopamine in neurons in the substania nigra, which are located in the midbrain.7 Cytosolic filamentous inclusions named as Lewy bodies (LB) are presented in these nigral dopaminergic neurons. The main component in these LB is the pre-synaptic protein AS, which is involved in synaptic maintenance and regulating mobilization of vesicles in the presynaptic neuronal terminal.8-10 Therefore, abnormal oligomers and fibrils which are the results of AS aggregation are the hallmark lesion of PD. AS protein consists of 140 amino acids and is composed of an amphipathic lysine-rich Nterminus and an acidic disordered C-terminus. The central domain of AS, residues 61-95, which is known as a non-amyloid-β component (NAC), is highly hydrophobic motif that is indispensable for AS aggregation.11,

12

The structure of AS monomer has been

previously determined by solution nuclear magnetic resonance (NMR).13-15 The AS monomers has an α-helix conformation in the unfolded form. The monomers of AS can self-assembled to form dimers, trimers and larger oligomers, which ultimately generate β-sheet-rich oligomers. These β-sheet-rich oligomers eventually yielding amyloid fibrils. The oligomers are highly toxic and known as species that induce neurodegeneration. Previous solid-state NMR (ssNMR) studies, presented the location of the β-strands along the sequence of AS in the fibrillary form,16-18 however these studies did not solve the three-dimensional structure of the self-assembled AS. Recently, the three-dimensional structure of the self-assembly of AS has been solved by ssNMR19 and has been proposed by computational molecular modeling study at the atomic resolution.20 The self-assembly of the central NAC domain of AS was recently proposed by two computational studies.21, 22 These extensive studies demonstrate that selfassembled AS and self-assembled NAC demonstrates polymorphic states, similarly as have shown in Aβ,23-26 Tau,23, 27 and amylin.28-30 Furthermore, it has been shown that differentiation in fibril structures of prions, i.e. various deposits of prions leads to different pathological toxic activities.31 Therefore, it is crucial to investigate the polymorphic states of amyloids.



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The common self-assembled structure of amyloids is the formation of a cross-β structure along the fibril axis, with hydrogen bonds that are perpendicular to the fibrillar axis. Yet, the fibrils of amyloids can self-assembled into a wide range of folding states. For example, Aβ fibrils can form two polymorphic states of parallel in register β-sheet arrangement in different condition,15, 29 and sometimes in similar conditions.26 Aβ can also self-assembled into triangular structures,32,

33

tubular structures34,

35

and double-horse-shoe-like

structures.36 Previously, we investigated the two polymorphic states of the AS fibrils.20 We have shown that similarly to Aβ, the two AS fibrils form cross-β structure, but the backbone of these two folding states are differ. Amylin peptides that are related to type 2 diabetes, also are self-assembled to form polymorphic states of cross-β fibrillar structures. However, oppositely to Aβ an AS, the polymorphic states of amylin illustrate similar backbone folding states, but different arrangements of the residues along the sequence of amylin.28-30 Yet, the polymorphism of self-assembled NAC has not been investigated. Herein, we investigated for the first time two polymorphic states of self-assembled NAC octamers at the atomic resolution: models M1 and M2 (Figure 1). The two polymorphic states have different backbone fibrillar folding states. The first polymorphic state model M1, which has been previously proposed by computational modeling tools has a β-arch structure.21 The second model M2 has an orthogonal Greek-key topology, and was extracted from a recent ssNMR of the three-dimensional structure of the self-assembled full-length AS.15 The construction procedures of these two polymorphic states are detailed in the Supporting Information text. These two models illustrate similar secondary structure, consisting of three main β-strands that are connected by two turn domains. However, these two models demonstrate differences in the arrangements of the residues along the sequence of NAC (Figure 1). To examine the stability of these two models we applied all-atom explicit molecular dynamics (MD) simulations (Supporting Information). The models were simulated for 80 ns. In order to examine whether 80 ns is a sufficient timescale for these models, we computed the root-mean-square deviation (RMSD) analysis. The RMSD analysis has shown that the two simulated models were converged at 10 ns, which is at relatively early time scale of the simulations (Figure S1). Therefore, the RMSD analysis indicating that 80 ns is definitely a satisfactory timescale. The early convergence of the simulated model M1 can be explained due to the use of the converged β-arch structure from our previous


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study. Since the simulated model M2 is an experimental-based structure of an orthogonal Greek-key topology which is the core of the fibrillar AS structure, the convergence occurs at an early stage of the simulations. This Greek-key topology is the core domain of the AS fibrils, thus it is characterized with absence of fluctuations according to the ssNMR study. The simulated models are seen in Figure 2. Interestingly, simulations of the orthogonal Greek-key topology model M2 led to a conformational change of the backbone folding state to form a β-arch-like structure. This new shape of β-arch-like structure is similar to the shape of the backbone of model M1, but differ in the arrangement of the residues along the sequence of NAC. Previously, Wineman-Fisher et al have shown that fibrillar amylin have similar backbone folding states, but differ in the arrangement of the residues along the sequence, indicate on polymorphism.28 Our study illustrates that this phenomenon also occurs in the fibrillary NAC. We first computed the conformational energies of each simulated model, using the generalized Born model based on molecular volume (GBMV) method37, 38 (Supporting Information). Interestingly, the averaged values of the conformational energies are similar (Figure 3a, Table S1) as well as the distribution of the conformers of each model. Moreover, population analysis demonstrated that both models have similar populations (Figure 3b). M1 occupies 52% of the population while M2 occupies 48% of the population, indicating on two polymorphic folding states of the self-assembled NAC. This is a first study that shows the phenomenon that two polymorphic folding states that differ in the arrangement of the residues along the sequence in amyloids have similar conformational energies and populations. While these two polymorphic folding states show differences in the arrangement of all residues along the sequence, there is a great interest to compare their structural properties. We first, examined the structural stability of the two polymorphic states along the MD simulations. The two polymorphic states showed a fast convergence of the structures (10 ns) according to the RMSD analysis (Figure S1). Interestingly, also other structural analyses of these two models have shown a convergence after 10 ns of the simulations. One can see, that the percentage of the hydrogen bonds that retained during the simulation is ~93%. This relatively high percentage appears at 10 ns and is retained throughout the MD simulations for both models (Figure S2). The inter-sheet distance values of both models also show a convergence at 10 ns (Figure S3). The averaged inter-sheet distance values for models M1 and M2 are 6.2 ± 0.2 Å and 7.0 ± 0.3 Å, respectively, indicating that these models are well-packed β-cross structures. We further estimated the fibrillar



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diameter values of the two models (Figure S4). Similarly, to the RMSD results, hydrogen bond values and the inter-sheet distance measurements, also the fibrillar dimeter values demonstrated a fast convergence at 10 ns of the simulations. The averaged estimated fibril diameter value for model M1 is 4.2 ± 0.02 nm and for model M2, it is 4.00 ± 0.05 nm. These values are in correlation with the experimental fibril diameter value (3.8 ± 0.6 nm).39 The experimental fibrillar diameter value was measured for the full-length AS(1−140) and for AS(30−110). Our result provides a support for the premise that the NAC central domain of AS plays a role in fibrillization. While the N-termini and the Ctermini are fluctuated with absence of well-packed fibrillar structure, the well-packed organized fibrillar structure is the central NAC domain. Therefore, it is reasonable to compare the fibrillar diameter values of the two simulated models with the experimental measured fibrillar diameter. It is known that polymorphic states of amyloids, such distinct morphologies of Aβ fibrils share a common secondary structure.40 To examine whether the two polymorphic folding states M1 and M2 share a common secondary structure, we estimated the secondary structure using two different methods. The first method is the database of secondary structure of protein (DSSP) method (Figure 4a), which assigning secondary structure using hydrogen bond algorithm. The second method estimates the secondary structure according to the values of the backbone dihedral angles ψ and φ of each residue along the sequence of the self-assembled NAC (Figure 4b). Both methods illustrated similar locations of β-strands along the sequence of the self-assembled NAC. The secondary structure that were estimated in these two methods is consistent with the experimental results.15, 19 Finally, we examined the structural stability of the two fibrillar models by investigating the backbone solvation (Figure 4c). The NAC domain in AS is known as a domain with relatively a large number of hydrophobic residues. The folding states of the two models have well-packed organized fibrillar cross-β structures, due to the relatively large hydrophobic interactions that produce the hydrophobic cores. Indeed, the residues that are located along the β-strands are less solvated, while in the two turn domains (Gly67 and K80-G86) the residues are relatively more solvated. Moreover, the property of flexibility of residues along the fibrillary structure can indicate the level of the solvation of these residues. Residues with relatively large flexibility/fluctuations are more prone to solvate with the water molecules, e.g. Glycine residues. Indeed, the root-mean square fluctuations


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(RMSF) analysis have shown that these two turn domains that are more solvated, particularly the turn domain that is located in residues K80-G86 are more fluctuates (Figure S5). The study of polymorphism in amyloids is a fundamental challenge issue. The polymorphism can illustrate a wide range of potential conformations under various conditions or under similar conditions. It is crucial to investigate the various molecular mechanisms pathways of the formation of the wide range of the amyloids’ conformations. This study will open up future studies on polymorphism of NAC and AS fibrils. It is expected that further polymorphic states of fibrillary NAC will be solved experimentally and will be predicted by computational studies. Obviously, one cannot slightly change one fibrillary structure and run all-atom explicit MD simulations to produce a different structure, due to the high barriers that allow to conformational change. Therefore, the backbone and the three-dimensional structure of the fibrils will determine the polymorph fibrillary structure. Determination of the wide range of conformational ensembles may assist to pave the way to find a cocktail of target drugs for inhibition of the wide range of the self-assembled amyloidogenic proteins. In our previous study, the focus was to find for the first time the fibrillary structure of NAC, while examining relatively large pools of structural candidates in order to find the stable fibrillary structure, this study illustrates a first research on the polymorphism of NAC fibrils. This is a first study that shows two distinct polymorphic folding states of NAC fibrils that differ both in the backbone shape and the arrangements of the residues along the sequence of NAC: orthogonal Greek-key topology and β-arch structure. Interestingly, we found a conformational change of the orthogonal Greek-key topology to a new backbone folding state of a β-arch-like structure. This new β-arch-like structure shows similar conformational energies, similar populations and similar structural properties. The two polymorphic folding states demonstrate similar scenario that was found in polymorphic fibrillar amylin:28-30 The polymorphic states differ by the arrangements of the residues along the sequence. The novelty of this work proposes the three-dimensional structure of two co-existing structures of the self-assembled fibril-like structures of the NAC that during the folding process the arrangements of the residues led to two different molecular mechanisms pathways. This is a unique phenomenon, that may affect the continuation of the folding state of AS. The initial stage of the seeding of selfassembly of NAC or AS is based on the initial interactions between residues. Due to the different arrangement of the amino acid residues along the backbone, there are different



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interactions between different residues and thus leading to different molecular mechanisms pathways.

Supporting information The Supporting Information is available free of charge on the ACS Publications website at DOI: Constructions of models; MD simulations protocol; GBMV method; structural analyses details; Secondary structure analyses; Table of conformational energies of models; RMSD, the fraction of number of hydrogen bonds between all β-strands, inter-sheet distances, calculations of diameters of fibrils and RMSF.

Author information Corresponding author Email: [email protected] Phone: 972-86428705 Fax: 973-86428709

ORCID Yifat Miller: 0000-0002-1163-9745

Author Contributions M.P.G and Y.M. design the study. M.P.G. carried out the simulations and analyses. M.P.G and Y.M. wrote the paper.

Funding The study is supported by the Israel Science Foundation (grant no. 532/15).

Notes The authors declare no competing financial interest.


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Acknowledgement This research was supported by the Israel Science Foundation (grant no. 532/15). All simulations were performed in the high-performance computational facilities of the Miller Lab in the BGU HPC computational center. The support of the BGU HPC computational center staff is greatly appreciated.



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Figure 1: Initial NAC fibrillary structural models. Model M1 is constructed from our previous proposed model.21 Model M2 is constructed from the experimental ssNMR of the full-length α-synuclein.19 The top structures illustrate one monomer from the octamer fibrillary structural models (bottom).


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Figure 2: Simulated NAC fibrillary structural models. The top structures illustrate one monomer from the octamer fibrillary structural models (bottom).



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Figure 3: Distributions of the conformational energy values (a) and populations analysis (b) for models M1 and M2.


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Figure 4: The secondary structures of the simulated fibrillary NAC models (a) according to the DSSP analysis and (b) according to dihedral angles’ calculations. The arrows illustrate the β-strand structure. (c) Average number of water molecules around each sidechain of Cβ carbon (within 4 Å) for models M1 and M2.



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For Table of Contents Use Only: Title: Two distinct polymorphic folding states of self-assembly of the non-amyloid β component differ in the arrangement of the residues Authors: Maya Pollock-Gagolashvili and Yifat Miller


Two Distinct Polymorphic Folding States of Self ... - ACS Publications

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