Molecular Dynamics Study to Investigate the Dimeric Structure of the

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Molecular dynamics study to investigate the dimeric structure of the full-length #-synuclein in aqueous solution Tingting Zhang, Yuanxin Tian, Zhonghuang Li, Siming Liu, Xiang Hu, Zichao Yang, Xiaotong Ling, Shu-Wen Liu, and Jiajie Zhang J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.7b00210 • Publication Date (Web): 10 Aug 2017 Downloaded from http://pubs.acs.org on August 12, 2017

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Journal of Chemical Information and Modeling

Molecular Dynamics Study to Investigate the Dimeric Structure of the Full-Length α-Synuclein in Aqueous Solution

Tingting Zhang 1, Yuanxin Tian 1, Zhonghuang Li, Siming Liu, Xiang Hu, Zichao Yang, Xiaotong Ling, Shuwen Liu*, Jiajie Zhang *

Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, 510515, PR China

Table of Contents (TOC)

1

These authors contributed equally to this work. 1

ACS Paragon Plus Environment


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ABSTRACT: The mechanisms of dimerization of α-Synuclein from full-length monomers and the structural features have been investigated through molecular dynamics simulations in this study. The dimerization of α-Syn plays a critical role in the fibrillogenesis mechanism and could initiate and trigger α-Syn to aggregate by conformational transforming. According to the alignment between three regions of α-Syn monomer, 8 diverse starting structures have been constructed. However, only 5 configurations show the dimeric structures and the detailed properties of 3 dimers of them are discussed. During the simulations, both identical α-Syn peptides (P1 and P2) of these three dimers reduce the high contents of α-helix from their native folded structures, while the contents of β-sheet increase. Antiparallel β-hairpin motifs within the α-Syn peptide are formed by intramolecular interactions. The β-hairpin regions are adjacent to the non-amyloid β component (NAC) of α-syn, and these structural features are consistent with the experimental observation. Moreover, intermolecular β-sheets also generate between P1 and P2 through hydrogen bonding interactions. The dimers produce both intramolecular β-hairpin and intermolecular β-sheet characters, the former is presented in monomer and oligomer of α-syn, and the latter occurs in the fibril structure. The simulations also show several other interactions such as hydrophobic interactions and salt-bridges, which would contribute to make the α-Syn dimers more stable with the aforementioned effects. The results may pave the way to design small molecules to inhibit the dimerization in order to block the aggregation of the α-Syn in the future.

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1. Introduction Parkinson’s disease (PD), affected 1-2% of the population over the age of 65,1 is one of the most common neurodegenerative disease currently.2 PD is a progressive brain disorder, the pathological hallmark of which is the aggregation of peptide α-synuclein (α-syn) and assembly to form Lewy bodies.3, 4 This amyloid self-assembly mechanism includes three phases: nucleation, production of critical dimers and oligomers, and elongation and growth to amyloid fibrils.5 Overexpression of α-syn peptides in the animal models clearly led to motor deficits suggestive of PD.6 However, neurodegenerative diseases, including PD and Alzheimer’s disease (AD), are very complex. Many factors could affect the pathological depositions. Reactive oxygen species (ROS) or reactive nitrogen species (RNS) can increase oxidative stress to make damage to the neurons and oxidize metal ions which bind to the α-syn and promote the α-syn to form β-sheets.7-13 Some studies implicated that oxidative stress is partially initiated by Glutamate.7 The role of redox and non-redox metal ions, Fe, Cu, and Zn, has been discussed.8,

14, 15

Therefore, removing ROS to inhibit the

oxidative stress may help to treat the neurodegeneration. Some food or food supplements may work as scavengers to prevent of neurodegeneration through this mechanism.9 Nevertheless, an alternative therapeutic strategy is to design new compounds to prevent neurodegeneration on the level of β amyloid plaques. Nornicotine, a nicotine metabolite that takes part in glycation, was reported to chemically modify amyloid β-peptide (Aβ) resulting in hindering aggregation.16 Although both nornicotine and nicotine have the toxicity and psychoactivity, they 3



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provide valuable information for the treatment of AD and other neurodegenerative disease that forms β amyloid plaques, like PD.16 It’s reported that post-translational chemical modifications which particularly modify Aβ to advanced glycation end product (AGE) could promote the self-assembly of Aβ due to the increase of β-sheet contents.17 In addition, Neelanjana Sengupta et al. demonstrated that glucose environment also play a role on the self-assembly of Aβ.18 As a result, experimental and theoretical work, focusing on the self-association with the formation of β amyloid structures starting from α-syn, plays an important role in the prevention of neurodegeneration. Wild-type (WT) α-syn, an intrinsically disordered protein (IDP), contains 140 amino acids. Under physiological conditions, it shows disordered structure in vitro.19 There are three regions of the primary structure of α-syn (Figure 1).20 The first region (residue 1-60), is termed N-terminal domain (NTD) that tends to form α-helical structure. Following the N-terminal domain, the residue 61-95, identified as the non-amyloid β component (NAC), is involved in the α-syn aggregation and tends to form β-sheet characters.21 The highly acidic C-terminal domain (CTD, residue 96-140), including 14 acidic amino acids, has been found to be critical to hinder rapid α-syn fibrillation.22-32 The NAC region of α-syn undergoes a conformational transition to the extended β-sheet that can stabilize the structure of amyloid-like fibrils.32, 33 This process of dynamics structural change from α-syn monomers to β-sheet assemblies referred to as protein misfolding.

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Figure 1. Structural feature of α-Syn monomer.

α-syn mutants are important in protein assembly and membrane binding.34-36 The role

of α-syn mutants has been reported through extensive parallel tempering molecular dynamics simulations recently.37-39 The structures are found to relate to free energy landscapes. The genetic missense mutants, A30P, E46K, and A53T of α-syn, undergo secondary structure conversions especially the increase of β-sheet contents.37-39 Long-range intramolecular interactions between the three regions of α-syn mutants and stabilities are also different compared with wild-type protein. All the changes above contribute to prompt α-syn mutants to be reactive for aggregation. It is also reported that dimerization of α-syn plays an important role in the aggregation related 5



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to protein misfolding disorders and conformational transition.40-42 For instance, Xavier Roucou et al. demonstrated that dimerization could promote and accelerate α-Syn aggregation which is the central mechanism to aggregate and form amyloid fibrils.43, 44

Later, Yuri L. Lyubchenko et al. found that the dimer structures were much more

stable than that of the monomeric proteins, and dimerization could stabilize the misfolded state of amyloidogenic proteins. The results show that dimers have a higher propensity to aggregate compared with monomers.45 In addition, the accumulated evidence indicates that the soluble dimers and oligomers are the pathogenic species giving rise to neurotoxicity.41, 46-48 As a result, the transformation mechanism from soluble α-Syn monomers to the disease-associated oligomers, especially dimers, has been a hot topic in the recent research. Lyubchenko’s group developed an AFM-based SMFS approach designed to monitor the interaction between monomers tethered to the surface and the AFM tip.45, 49-58 They revealed that α-Syn employs two different types of conformations to form dimeric structure with the lifetimes in the range of 1s.59 However, the characterization of α-Syn oligomers is still an extraordinarily biophysical challenge because of their heterogeneity and propensity to aggregate. As a result, α-Syn oligomers from high-resolution structural characterization by X-ray crystallography are difficult to obtain.60, 61 Several studies of the assembly of small fragments derived from α-Syn have provided insights into oligomeric assemblies of the protein.61-64 Wolfgang Hoyer et al. revealed a β-hairpin of α-Syn monomer comprising residues 37-54 by NMR spectroscopy.65 While the first X-ray crystallographic structure of oligomers formed 6


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by a peptide derived from residue 36-55 of α-Syn was presented by James S. Nowick’s group last year.61 In this study, α-Syn36-55 also folds into a β-hairpin which further assembles into trimers and higher-order oligomers. The authors suggested that trimers may be the unified substructures of amyloid oligomers made of β-hairpin monomers.61 It’s reported that the formation of β-hairpin precedes aggregation of α-Syn.66 This process may play a critical role in oligomerization rather than in

fibrillization.61 Moreover, the structure of the fibrils of α-Syn is different from the oligomers revealed above. The fibril of full-length α-Syn of solid-state NMR structure was released in 2016. In this fibril, the NAC region of the folded monomer interacts with neighboring monomers by hydrogen bonds to form multilayered parallel β-sheets.67 Later, the structures and thermodynamics stabilities of wild-type and

mutated α-Syn fibrils have been determined.68 The binding regions for small molecules have also been provided. Despite some available information, the dimeric structures of the full-length α-Syn remain unknown. The small fragments from α-Syn cannot elucidate the structural properties of the whole chain, since specific amino acid sequence affects the peptide’s conformation. What the most interesting is that what kind of dimeric structure they will form, and which fashion the dimer will follow to assemble itself: through the intermolecular interactions among β-sheets like fibril or the intramolecular interactions of β-hairpin like monomer and oligomer. Therefore, in the present study, we have implemented all-atom molecular dynamics (MD) simulations to study the dimers formed by α-Syn with the full-length peptides. The results will provide useful 7



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information to understand the process of dimerization and help to inhibit the aggregation of α-Syn. 2. Methods 2.1 Initial Models. The initial structure of the full-length α-Syn was downloaded from Protein Data Bank (ID: 2KKW69) and model 3 was selected to construct the starting structures. According to the three regions (NTD, NAC, and CTD) and the secondary structure properties of the monomer, we built the following initial models for dimer comprised by two identical α-Syn peptide (P1 and P2) using the similar method in modeling amyloid β dimers in previous study.70 All the models were shown in Figure 2:

8


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Figure 2. Starting structures for MD simulations.

(1) the N-terminal domain (NTD) of P1 and P2 are parallel (align proximity without intersection) and symmetrical to each other (N−NP); (2) the NTDs of P1 and P2 are antiparallel (in reverse order and symmetrical) (N−NAP); (3) the parallel non-amyloid β component (NAC) of P1 and P2 (A−AP); (4) the antiparallel NAC of P1 and P2

(A−AAP); (5) the NTD of P1 is parallel to the NAC of P2 (N−AP); (6) the NTD of P1 is antiparallel to the NAC of P2 (N−AAP); (7) all the three regions of P1 and P2 arrange shoulder by shoulder (P1 and P2 are parallel, symmetrical and in the same direction) (SP); (8) the three regions of P1 and P2 oppositely align side by side (P1 and P2 are facing each other but one of them is turning 180 degrees in anticlockwise direction) (SAP). At the beginning, the center of mass of P1 and P2 residues in the aligning proximity regions were set to around 20 Å. The details of the initial distances between residues in those aligning proximity regions for all the configurations were shown in Table S1-S9. Based on this design, the bias of intermolecular interactions and the other effects between two monomers such as salt-bridge were ruled out in the initial configurations. To test the model of α-Syn dimer in this work, another starting configuration was created by using reverse approach. S2N dimer was derived from a full-length α-Syn fibril of decamer (PDB ID: 2N0A)67 by deleting eight adjacent monomers.

2.2 Molecular Dynamics Simulations. The MD simulations were implemented with GROMACS 4.5.3.71 The GROMOS 9



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force field 53A6,72 widely utilized to explore the biomolecules including amyloid β peptides in the previous study70, 73-81, was used in all the models. A cubic box was built to immerse the whole structures. The box size should be set 20 Å extended from the border of the structures around all atoms. Periodic boundary conditions (PBC) were utilized to rule out the bias. SPC water models82 were added into the box to solvate the structures. In order to neutralize the systems and keep the physical environment, Na+ and Cl- ions were introduced. Then, the whole systems without any constraints were minimized by 3000 cycles using the steepest descent method. MD simulations were started in the NPT ensemble after energy minimization. Long-range electrostatic interactions were calculated using Particle Mesh Ewald.83,

84

The

temperature was set to 300 K with a coupling coefficient of 0.1 ps and the pressure was 1 atm. The peptides and water molecules were calculated by LINCS algorithm85 and SETTLE algorithm,86 respectively. The former one constrains the bonds, while the latter constrains both the bonds and angles. The time step is 2 fs. In the production phase of MD simulations, the trajectories of all models were analyzed by using the tools of Gromacs and YASARA87, 88. YASARA87, 88 was also applied to visualize and present the structures. The cluster analysis with a 0.3 nm cutoff of root-mean-square deviation was utilized to obtain the most representative structures from the MD simulations. The most representative structures were used for analysis and elucidation in this paper.

2.3 Solvation Energy Calculations. 10


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The solvation energy of peptide1 (P1) and peptide2 (P2) monomers of α-Syn is calculated by using APBS program.89-91 The calculations are carried out under room temperature. The interior dielectric constant of peptides is set to 2.0, whereas use 78.0 for solvent. The ionic radius for solvent surface is 1.5 Å and the van der Waals surface is stated by dielectric boundary. The grid dimensions are set by which separates 20 Å from the peptides to grid boundary. At the beginning, we utilize the PDB2PQR92, 93 online server to convert the dimers from PDB format to PQR format instead. Then the following equations show the computed procedure.

∆ = ∆ + ∆

.

∆ = ∆ _ + ∆ _ . +

= ∑ φ

∆

= γ × SASA

The solvation energy ∆ comprises two terms, ∆ and ∆

,

defined as electrostatic binding energy and non-polar contribution. While the

electrostatic binding energy ∆ consists of components of ∆ _ ,

∆ _ , and . ∆ _ and ∆ _ denote the electrostatic

desolvation free energies of P1 and P2, respectively. These terms are calculated as follow: firstly, compute the electrostatic energy of P1 and solution without P2; then calculate the electrostatic energy of P1 and solution with non partial charge P2 existed; 94

finally, ∆ _ or ∆ _ could be gained by the difference of energies

11



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derived from the former steps. In addition, the electrostatic interaction contribution

was calculated through the third equation of the procedure, in which φi

meaning electrostatic potentials are obtained from the second step computing of electrostatic desolvation free energies, while qi are the atomic charges.

Furthermore, the non-polar binding energy term ∆

is calculated through

the function of solvent accessible surface area (SASA). YASARA program87 was used to computed the SASA for both P1 and P2 monomers and dimers. The surface tension γ is 0.00542 kcal/mol Å2 95, 96 in this equation.

3. Results and Discussion In this paper, we implement 100 ns MD simulations to elucidate the formation of dimeric structures from the wild type α-Syn peptide monomers. In the basis of three regions (NTD, NAC, and CTD) of the two α-Syn monomers (P1 and P2), the individual starting configurations have been created as description in the previous section (Figure 2): N−NP, N−NAP, A−AP, A−AAP, N−AP, N−AAP, SP, and SAP. During the simulations, three structures of the monomers (A−AP, N−AAP, and N−NP) split apart from each other and showed non intermolecular interactions. The snapshots in Figure S18 present the gradual change process of A-AP. The interpeptide center of mass distance between P1 and P2 of A-AP increases dramatically during the simulation. We performed another independent simulation of A-AP configuration and observed the similar increase of distances. The average values of distance are 66.5 and 72.6 Å for the first and second simulations, respectively. None of intermolecular interactions are found between the monomers. The results indicate that the monomers 12


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of A-AP configuration dissociate without forming a dimer. On contrast, the remaining five configurations (N−NAP, A−AAP, N−AP, SP, and SAP) were found to form dimeric structures. The time evolutions of root mean square deviations from Figure S1 indicate that all of the systems have achieved the equilibration after 20 ns. From the data in Table 1 for the study of secondary structure, it shows that all the dimeric structures significantly decrease their helical character which is the dominant conformation in α-Syn monomer. Meanwhile, the β-sheet contents for all the dimers enhance. This conformational transition to the extended β-sheet is regarded as a critical process that can initiate α-Syn aggregation and stabilize the structure of amyloid fibrils.32, 33 In addition, the structural change of the increase in random coil is also related to protein misfolding disorders.40-42 Based on the interactions and energies of the structures, we analyze the characteristics of A−AAP, SP, and SAP of all the five dimers below. Table 1. Secondary Structure Compositions of Initial Structure and Dimers. Structure

Helix

Sheet

Turn

Coil

Initial structure

64.3

--

--

35.7

A−AAP

14.3

12.9

0.7

72.1

SP

32.9

6.4

5.7

55.0

SAP

12.5

8.9

7.9

70.7

N−AP

18.9

8.6

9.3

63.2

N−NAP

22.8

9.6

6.8

60.7

3.1 A-AAP Dimer 13



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The A-AAP dimer is formed by placing the NAC (residue 61-95) region of P1 and P2 antiparallel (align proximity in reverse order and symmetrically) in the initial structure (Figure 2). The NAC part transforms its conformation to β-sheet structure that can stabilize the structure of amyloid-like fibrils.32, 33 This fragment is very important in the α-syn aggregation process and the cytotoxicity.97 It’s reported that removing the residues 71-82 can prevent aggregation of α-syn in vitro, while hinder both the aggregation and neurotoxicity in a Drosophila model of PD.98 Therefore, it is meaningful to investigate the role of NAC region during the dimerization.

Figure 3. The most representative structure of A-AAP obtained from the 100 ns simulation.

The A-AAP dimer is generated by a mass of inter- and intramolecular interactions, including hydrogen bonding, hydrophobic interactions, and salt-bridges. Figure 3 shows the most representative structure came out of the simulations of A-AAP. The most representative structure which has the largest number of neighbors is derived from the cluster analysis that is utilized to group frames with similar structures. From the structure, both P1 and P2 undergo conformational changes which could be 14


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observed from Figure S2 of the definition of secondary structure of protein (DSSP). On the one hand, P1 loses most of the helix structure and retains the only part of Val55-Gly67. On the other hand, the segments of Lys23-Val26, Ala30-Glu35, and Tyr39; Glu45-His50 of P1 form antiparallel intramolecular β-sheet structures, respectively, also termed as β-hairpin motif (Figure 4a). The β-hairpin regions are adjacent to the NAC of α-syn, and these structural features of the A-AAP dimer model are consistent with the experimental observation. Since the segment of residues 37-54 of α-Syn monomer was found to form β-hairpin by NMR spectroscopy,65 while the region 33-58 of α-syn oligomers also folds into β-hairpin characters from X-ray crystallographic structure.61 The snapshots of Glu20-Val55 segments of P1 are shown in Figure 4a. The secondary structures transform during the following process: they have α-helix at the beginning, lose helix structure to form small β-hairpin, β-hairpin increase and elongate. The intramolecular interactions lead to the formation of helix and β-hairpin structures. All these interactions can be revealed from the contact map that shows the distance between pairs of amino acid residues in Figure 5a. For P2, the conformational changes also can be seen from DSSP (Figure S2). The long chain α-helix structures lose many intramolecular interactions to leave only some discrete

helix segments (Tyr39-Gly47, Thr54-Glu61, and Val74-Ala91).

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Figure 4. The truncated snapshots of A-AAP dimer. (a) Segment from P1. (b) Segments from P1 and P2.

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Figure 5. (a) Contact map of P1 and P2. (b) Time-dependent variation of the number of hydrogen bonds between P1 and P2. (c) RMSF of Cα atoms of individual residues from P1 and P2 of A-AAP dimer.

Besides the intramolecular interactions within P1 and P2 monomers, the Lys6-Ser9 region of P1 and the Gly132-Gln134 segment of P2 interact with each other to form β-sheet structure (Figure 3). The snapshots in Figure 4b present the gradual change

process. The amino acid residues between P1 and P2 monomers attach to produce many hydrogen bonds in A-AAP dimer (Figure 5b). All the details of these hydrogen bonding interactions can be seen in Table 2. In particular, the residues of NAC (residue 61-95) region, Ser87, Ser87, Ala89, and Gly93 of P1 and Gly67, Gly73, Asn65, and Lys60 of P2, form hydrogen bonds. The Figure S3 shows the distances of the center of mass between these residues decrease. The interactions between P1 and P2 contribute to the reduction of the distances compared to the initial setup (Table S4). The intermonomer interaction energies in Figure S19 reveal a great decrease during the simulation. It validates the above results that more intermolecular interactions 17



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generate between P1 and P2. The P1 and P2 go through the process from separated monomers, spontaneous dimerization with increased interactions, and stabilized dimer. Furthermore, the side chain of the residues of P1 associate via hydrophobic interactions with Lys32, His50, Glu61, Phe94, Asp115, Val118, Tyr125, Tyr133, and Tyr136 of P2, respectively. The Table 3 shows all the hydrophobic interactions between P1 and P2 in A-AAP. In addition, the negative acidic amino acids and the positive basic amino acids between P1 and P2 have electrostatic attraction termed salt-bridge. The number of salt-bridges between P1 and P2 during the molecular dynamics shows the range of salt-bridges formation in the dimerization process (Figure S4a). Table S10 presents the specific residues that form salt-bridge in dimers, which occurs mostly in the NTD and CTD regions of α-syn since the CTD contains 14 acidic amino acids. These salt-bridges may help to stabilize the dimeric structures and contribute to the electrostatic binding energy for aggregation. The results of hydrogen bonds and salt-bridges shown in Table 2 and S10 validate the experimental data that the CTD forms intermolecular interactions with NTD on account of its great negative charge, and these interactions could increase the aggregation of α-syn.99-106 However, the CTD impedes the process of α-syn from aggregation to fibrillation, since the rate of formation of fibril is increased by removing the CTD.20, 101, 106

Table 2. List of Hydrogen Bonding Interactions between α-Syn Monomers of Dimer Structures. A-AAP P1

SP P2

P1

SAP P2

P1

P2 18


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1

Lys6

Tyr136

Ser9

Ile88

Glu13

Lys43

2

Lys6

Gln134

Glu20

Lys21

Glu28

Asp115

3

Leu8

Gly132

Gln24

Glu28

Lys32

Ala78

4

Leu8

Gly132

Gln24

Glu28

Lys34

Thr92

5

Ser9

Glu130

Tyr39

Leu38

Ser42

Asp119

6

Lys10

Met127

Lys43

Tyr39

Lys43

Ser42

7

Glu13

Gln134

Glu46

Glu46

Glu46

Ser42

8

Val15

Gln134

Glu46

Lys45

His50

Glu126

9

Val15

Asp135

Gly51

Thr33

Gly51

Glu126

10

Thr22

Glu130

Val66

Gln62

Gly51

Met127

11

Glu35

Asn122

Ala69

Thr72

Val55

Ala17

12

Glu35

Asp121

Gln79

Lys80

Ala56

Ala124

13

Gly41

Ala124

Glu83

Glu83

Ala56

Ala124

14

Lys43

Glu123

Glu83

Ala85

Lys58

Asn122

15

Val48

Val118

Ala91

Met1

Lys60

Met127

16

His50

Asp115

Asp98

Asn122

Gly68

Val16

17

Gly51

Asp115

Gln99

Val95

Thr72

Glu35

18

Thr64

Lys97

Leu100

Lys97

Gly73

Ser42

19

Asn65

Val95

Lys102

Lys97

Gln79

Val37

20

Asn65

Val95

Asn103

Glu105

Gln79

Gln24

21

Ser87

Gly73

Asn103

Glu105

Lys80

Leu38

22

Ser87

Gly67

Glu104

Gln99

Gln99

Thr54

23

Ala89

Asn65

Ala107

Glu105

24

Gly93

Lys60

Gln109

Glu105

25

Glu105

Val71

Asp121

Pro117

26

Glu105

Val70

Asn122

Glu126

27

Glu105

Ala69

Glu123

Ala124

28

Gly106

Thr59

Glu123

Glu126

29

Gln109

Val48

Gly132

Tyr136 19



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30

Glu110

Ser42

Tyr133

Gln134

31

Pro117

Ala53

Tyr133

Tyr133

32

Asp119

Gly51

Gln134

Asp135

33

Asp121

His50

Asp135

Asp135

34

Met127

Lys23

Tyr136

Asp135

35

Glu139

Lys23

Ala140

Ser129

Ala140

Glu130

36

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Table 3. List of CH-π, and NH-π Interactions between α-Syn Monomers of A−AAP Dimer Structure. P1(residue)

P2(residue)

interaction

P1(residue)

P2(residue)

interaction

Val3

Tyr136

CH-π

His50

Asp115

CH-π

Lys6

Tyr133

CH-π

Asn65

Phe94

CH-π

Tyr136

NH-π

Val66

Phe94

CH-π

Gly7

Tyr133

CH-π

Phe94

Glu61

CH-π

Val15

Tyr133

CH-π

Val118

His50

CH-π

Val16

Tyr133

CH-π

Asp119

His50

CH-π

Gly36

Tyr125

CH-π

Pro120

His50

CH-π

Tyr39

Val118

CH-π

Asp121

His50

CH-π

Val40

Tyr125

CH-π

Tyr125

Lys32

CH-π

Gly41

Tyr125

CH-π

All the interactions aforementioned are shown to agree with the previous studies that dimer structure of α-syn is more stable.45 The electrostatic binding energies of the dimers could be found in Table S11 to reveal the binding affinity. The binding free energy for A−AAP dimer is -46.0 kcal/mol. However, the energies of SP and SAP are less favorable, shown as -40.7 and -3.9 respectively. On this basis the non-polar 20


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contribution lowers all the energies but retains three dimers the same order. It further enhances the relative binding affinity of A−AAP (−91.4 kcal/mol) and is followed by SP (−67.1 kcal/mol) and SAP (−28.6 kcal/mol). The root mean square deviation (RMSD) values of α-carbon atoms from the backbone vs time of P1 and P2 from A−AAP are shown in Figure S5. The RMSD of the NTD and CTD regions of P1 and the CTD of P2 reveal that they change their conformation substantially. The phenomenon is due to the conversion from helix and random coil structure of NTD and CTD respectively to the β-hairpin motif. The root mean square fluctuation (RMSF) could assess the fluctuations of atomic position for each residue’s α-carbon atoms. The RMSF plot also validates the results above in Figure 5c. It

indicates that the NTD and CTD regions of P1 are rigid because of the formation of β-sheet structures. The P2 monomer is more flexible than P1 in the A-AAP dimer.

To test the dependence of the trajectories, another independent 100 ns simulation of A-AAP configuration has been performed. Figure S20a shows the time evolutions of root mean square deviations for two trajectories. The new trajectory have similar change tendency with the previous one. The interpeptide center of mass distances between P1 and P2 in Figure S20b show a great decrease for both trajectories. Both P1 and P2 lose most of the helix structure. The segments of Lys23-Val26, Ala30-Glu35, and Tyr39 of P1 form β-hairpin character, which are the same regions of the previous model (Figure 3 & S20c). The results suggest that the A-AAP dimeric structures from two independent simulations have high structural similarity.

21



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3.2 SP Dimer In the initial model, all the three regions (NTD, NAC, and CTD) of P1 and P2, facing each other in the same direction, are parallel and symmetrical (Figure 2). The formation of numerous hydrogen bonding and hydrophobic interactions prompts the progress of dimerization for this configuration (Table 2 & 4). The secondary structure compositions of both monomers change and the contents of helix character decrease compared with initial structure (Table 1 and Figure S6). Figure 6 shows the most representative structure for SP dimer. The NTD and NAC regions of P1 lose their long chain helix structure and retain some pieces instead (Met1-Val15, Glu20-Gly25, Lys43-His50, Glu57-Val63, and Val70-Val82), and these intramolecular interactions are observed from the contact map in Figure S7a. The change of structure conformation of P2 is quite similar to P1. For instance, the NTD and NAC regions of P2 also contain a lower α-helix character. The contact map of the simulation shows the contacts between Phe4-Gly36, Thr75-Lys80, and Val82-Ala91, which form helix structures through intramolecular hydrogen bonds. Similar to A-AAP dimer, the segment of Thr54-Thr59 of P2 also forms antiparallel intra β-sheet structures (β-hairpin motif) in particular (Figure 6). In addition, the principal component analysis has been implemented to analyze the structures of simulations. The horizontal coordinate and vertical coordinate denote the first and the second principal components, respectively, in Figure S8. Each point is related to a specific structure, and the close points on the plot represent the similarity of their structures. Several separate basins are generated observed from Figure S8. The most representative 22


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structure is located in the darkest region which is the most stable. The truncated snapshots shown in Figure 7 are derived from the structures which are located in other basins.

Figure 6. The most representative structure of SP obtained from the 100 ns simulation.

In Figure 7b, the CTD regions start from random coil to β-sheet character gradually during the simulation time. These regions of P1 and P2 are stabilized by intermolecular

interactions.

The

Leu100-Asn103,

Glu105-Pro108,

and

Asn122-Glu123 segments of P1 and Lys97, Glu105, and Ala124-Glu126 regions of P2, respectively, form the parallel β-sheet structures (Figure 6). The distances of the center of mass between Glu46, Glu83, Tyr133, and Asp135 from P1 and Glu46, Glu83, Tyr133, and Asp135 from P2 shorten compared with the original ones in the starting configuration (Figure S9 & Table S7). The intermonomer interaction energies in Figure S19b also decrease during the simulation. Seen in Figure S7b, the amino 23



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Page 24 of 41

acid residues between P1 and P2 possess attractions to form hydrogen bonds with the number of ~36. All the details of these hydrogen bonding interactions can be seen in Table 2. The residue side chains of P1 associate via CH-π, NH-π, and π-π interactions with Thr33, Val37, Tyr39, Phe94, Val95, Lys96, Tyr125, Tyr133, and Tyr136 of P2, respectively, besides the intra- and intermolecular hydrogen bonds, The Table 4 reveals all the hydrophobic interactions between P1 and P2. Moreover, the salt-bridges that derived from electrostatic attraction among monomers are presented in Table S10. The Figure S4b shows the number of salt-bridges that generate in SP of the simulation, whereas, the amount is decreased compared with that in A-AAP dimer. During the simulation, the NAC and CTD regions of P1 are quite flexible and the RMSD values are relatively larger than the ones for NTD, which are shown in Figure S10. Since both the NTD and NAC fragments only lose some helix structure, the CTD appears to be the most flexible among these regions. It adopts random coil conformation at the initial state and then transforms into the structure that possesses some β-sheet character. In P2, this region has similar performance and therefore reveals larger RMSD values than others. As can be seen from Figure S7c, the RMSF plot of P1 almost overlaps with that of P2. This is consistent with the RMSD variations and the structural transformations. It’s observed that the CTD of all the three regions has the maximum fluctuations in P1 and P2.

24


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Figure 7. The truncated snapshots of SP dimer. (a) Segment from P2. (b) Segments from P1 and P2. Table 4. List of π-π, CH-π, and NH-π Interactions between α-Syn Monomers of SP Dimer Structure. P1(residue)

P2(residue)

interaction

P1(residue)

P2(residue)

interaction

Phe4

Lys96

NH-π

Gln99

Phe94

CH-π

Met5

Phe94

CH-π

Leu100

Phe94

CH-π 25



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Leu8

Phe94

CH-π

Asn122

Tyr125

CH-π

Tyr39

Val37

CH-π

Glu123

Tyr125

CH-π

Gly41

Tyr39

CH-π

Ala124

Tyr125

CH-π

Lys43

Tyr39

CH-π

Tyr125

Tyr125

CH-π

Gly47

Tyr39

CH-π

Met127

Tyr125

CH-π

His50

Tyr39

CH-π

Gly132

Tyr136

CH-π

Thr33

CH-π

Tyr133

Tyr133

CH-π

Gly86

Phe94

CH-π

Tyr136

CH-π

Ala89

Phe94

CH-π

Gln134

Tyr136

CH-π

Ala90

Phe94

CH-π

Tyr136

Tyr133

π-π

Phe94

Val95

CH-π

Otherwise, to test the force field parameters of the simulation, the same SP configuration was selected to run MD simulation with CHARMM 27 force field that is usually utilized in the simulation of IDP.68, 107-109 The time evolution of root mean square deviations and radius of gyration from Figure S11a & b indicate that the system reach equilibrium within 100 ns simulations. As a result, the last 50 ns period of trajectory was selected for analysis. The most representative structure which was obtained from this equilibration phase by utilizing cluster analysis represented the most population of the conformations. The radius of gyration of SP undergo an obvious decrease, indicating that the P1 and P2 monomers generate interactions to make SP form a more compact dimer. In contrast, the values of SP(CHARMM) increase (Figure S11b). The segments of Met5-Ala29 and Gly41-Gly93 of P1 and Ser9-Ala30 and Glu46-Thr92 of P2 still remain long-chain α-helix character (Figure S11c). The secondary structure compositions of SP(CHARMM) are different with 26


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those of SP structure. The content of helix character is 51.4%, higher than that of SP, while the content of β-sheet character is 3.9%, lower compared with SP (Table 1). Similar with SP, some small β-sheet structures are formed from the CTD regions of P1 and P2 (Figure S11). The observations suggest that the compactness, conformations, and secondary structure contents are sensitive to the selected force field.

3.3 SAP Dimer The SAP dimer is originated from the three regions of P1 and P2 facing each other but one of them is turning 180 degrees in anticlockwise direction (Figure 2). As seen from Figure 8 & S12, the dominant helix structures in the NTD and NAC regions of P1 shorten to Lys6-Glu20 and Val77-Ala90. An antiparallel intra β-sheet conformation is produced from Lys96-Asp98 and Gly101-Asn103 segments linking through a turn. The same manner is observed between Asp121 and Tyr125. In P2, the contact map shows strong contacts between Val52-Gln62 that is the α-helix structure region (Figure 8 & S13a). Whereas, the segments of Gly25-Glu28, Ala30-Gly36, and Tyr39; Lys97-Asp98 and Ile112-Glu114 from P2 also form the β-hairpin motif, respectively. Figure 9a shows the changes of those segments: the loss of helix structures and the growth of β-hairpin. These structure conversions are similar to those of SP and A-AAP dimers.

27



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Page 28 of 41

Figure 8. The most representative structure of SAP obtained from the 100 ns simulation.

Between the P1 and P2 monomers, a small antiparallel β-sheet structure is formed by Ala56-Glu57 region of P1 and Ala124-Tyr125 region of P2. Different kind of interactions including hydrogen bonding, π-π stacking, and hydrophobic interactions between P1 and P2 contribute to the generation of the SAP dimer. Throughout the simulation time, 19~22 hydrogen bonds occur as a result of the interactions between amino acid residues of P1 and P2 (Figure S13b). Table 2 shows the details of the hydrogen bonding interactions. Figure S14 shows the distances of the center of mass between P1 and P2 residues. Obvious decrease of the distances during the simulation is observed between the center of mass of these residues to form hydrogen bonds. The interaction strengths between P1 and P2 in Figure S19c demonstrate more and more strong intermolecular interactions formed. In addition, some residues from the NTD 28


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and CTD regions of P1 and P2 form CH-π and π-π interactions. However, both the hydrogen bonding and hydrophobic interactions between P1 and P2 monomers decrease in SAP, compared with SP and A-AAP dimer (Table 2 & 5). The same situation happens in the formation of salt-bridges. SAP only possess ~21 salt-bridges (Figure S4 & Table S10), less than those of A-AAP and SP. The CTD and NTD segments of both P1 and P2 change their conformation significantly, revealed from the RMSD (Figure S15). This may be due to that the CTD of both P1 and P2 monomers fold their random coil structures to form some β-hairpin motif and the NTD regions convert to turn, bend, and coil structures. The RMSD values of the NAC regions are relatively smaller. The aforementioned results are in line with the values of the RMSF shown in Figure S13c. The RMSF plot indicates that the NTD and CTD regions of both P1 and P2 are more flexible compared with the NAC segments.

29



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Page 30 of 41

Figure 9. The truncated snapshots of SAP dimer. (a) Segment from P2. (b) Segments from P1 and P2. 30


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Table 5. List of π-π, CH-π, and NH-π Interactions between α-Syn Monomers of SAP Dimer Structure. P1(residue)

P2(residue)

interaction

P1(residue)

P2(residue)

interaction

Tyr39

Pro117

CH-π

Ala53

Tyr136

CH-π

His50

Tyr125

π-π

Phe4

CH-π

Gly51

Tyr136

CH-π

Val74

Tyr125

CH-π

Val52

Phe4

CH-π

Thr75

Tyr125

CH-π

Tyr125

CH-π

To test the model of α-Syn dimer constructed and discussed above in this work, we have created another starting configuration by using reverse approach. A full-length α-Syn fibril of decamer (PDB ID: 2N0A)67 has been downloaded and pretreated to

obtain S2N dimer (Figure S16a). In the initial state, only intermolecular β-sheet structure exists. At 30 ns of the simulation, the Ser9-Gly14 and Val16-Glu20 segments of P1 produce an intramolecular β-hairpin motif, continually increase and elongate it (Figure S16b). This result verifies the structural features of our dimer models aforementioned.

4. Structural Features of Dimers The radius of gyration plotted against time for five dimers have been calculated and showed in Figure S21. Obvious decreases are observed for all of the configurations during the simulations. It reveals that the compactness of the configurations is enhanced to form more compact dimers. Those changes mostly due to the intermolecular interactions that generate between the P1 and P2 monomers of the 31



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Page 32 of 41

configurations. During the simulations, both P1 and P2 of the dimers A−AAP, SP, SAP, N−AP, and N−NAP reduce their dominant long chain helix structures of NTD and NAC regions from 64.3% of initial structure to 14.3, 32.9, 12.5, 18.9, and 22.8%, respectively. In contrast, they transform the structures to intramolecular β-sheet fragments also termed as β-hairpin. The details of structures are presented in Figure S22. In A−AAP, the segments of Lys23-Val26, Ala30-Glu35, and Tyr39; Glu45-His50 form β-hairpin. Similar to A−AAP, the segment of Thr54-Thr59 of SP; Gly25-Glu28, Ala30-Gly36, and Tyr39 of SAP; Lys34-Gly36 and Tyr39, Lys45-Gly47 and His50-Val52 of N−NAP; Val49-Val52 and Gly67-Val71 of N−AP also form intra β-hairpin motifs, respectively. These β-hairpin regions are all adjacent to the NAC of α-syn. The structural features of the dimers are consistent with the experimental observation.61, 65 The formation of β-hairpin that usually exists in monomer or oligomer could accelerate the aggregation of α-Syn.66 However, the intramolecular β-hairpins will disappear during the fibrillization process replaced by intermolecular β-sheets between monomers. However, in our α-Syn dimer models, intermolecular β-sheets also generate through multiple hydrogen bonding interactions. Therefore, the dimer is a key intermediate that transform the conformation from monomer to fibril. In addition, some other intermolecular interactions, including hydrophobic interactions and salt-bridges, also participate in the process of dimerization. All of the effects above suggest contributing to make the α-Syn dimers more stable.

32


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5. Summary and Conclusions The dimerization of α-Syn plays a critical role in the fibrillogenesis mechanism, which could initiate and trigger the α-Syn to aggregate by conformational transforming. Therefore, it is crucial to reveal the mechanism and specific structural details from the molecular level of α-Syn dimer in order to have a deeper understanding of Parkinson’s disease. In this paper, the dimerization of α-Syn and the roles of specific segments (NTD, NAC, and CTD) have been explored by implementing MD simulations. According to the alignment between three regions of the two α-Syn monomers (P1 and P2), we develop different starting models as follow (N−NP, N−NAP, A−AP, A−AAP, N−AP, N−AAP, SP, and SAP). During the simulations, only five configurations (N−NAP, A−AAP, N−AP, SP, and SAP) are found to form dimeric structures. Furthermore, the characteristics of A−AAP, SP, and SAP of the five dimers are discussed based on the interactions and energies of the dimer structures. α-Syn dimerization triggers the formation of not only the neurotoxic oligomers, but

also amyloid fibrils.44 As we know, both of these species are related to PD pathology.44 Therefore, a strategy to hinder the aggregation is to reduce the dimerization rate or even prevent it. Small molecules designed for this process may be a prospective solution. Our results show that the β-hairpin regions of dimers are adjacent to the non-amyloid β component (NAC) of α-syn. Since the formation of β-hairpin can accelerate the aggregation of α-Syn, small molecules that could bind to

these regions and inhibit the process of β-hairpin forming may play an important role 33



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Page 34 of 41

in α-Syn aggregation. These regions could be a promising binding target for designing molecules that interact with them. The results of this study provide detailed information regarding the dimerization mechanism and structural details of α-Syn. It may provide helpful information to prevent and treat PD in future.

ASSOCIATED CONTENT Support information (a) Figures S1-S22. The supplementary details of the simulations: RMSD, RMSF, DSSP, timelines of the number of salt-bridges & hydrogen bonds, timelines of distances, intermonomer interaction energies, and radius of gyration, contact map, and snapshots. (b) Table S1-S11. Initial distances between residues, residue-specific salt-bridges between α-Syn monomers and the solvation free energies of dimers.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]

Prof. Jiajie Zhang, Phone: 86-20-61648548.

* E-mail: [email protected]

Prof. Shuwen Liu, Phone: 86-20-61648538

Present Address School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, 510515, P.R China.

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Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by Medical Scientific Research Foundation of Guangdong Province, China (No. A2016378), the National Natural Science Foundation of China (No. 21603095), Scientific Research Foundation of Southern Medical University (No. LX2016N005), and Special Program for Applied Research on Super Computation of the NSFC-Guangdong Joint Fund (the second phase). We thank Dr. Jingwei Zhou for many helpful discussions and the assistance in the preparation of the manuscript.

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Molecular Dynamics Study to Investigate the Dimeric Structure of the

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