α-Synuclein (α-syn) is a cellular acidic protein of 140 amino acid residues that release neurotransmitters in the presynaptic nerve terminals [1-4] and whose abnormal aggregation results at the onset of Parkinson’s disease (PD) [5-9]. Accumulation of α-syn fibrils in neuronal inclusions is the major pathological process involved in PD. PD is a neuro-degenerative disorder, sporadically affecting nearly 10 million people around the world and associated with common ageing of a person [8,10-14]. It is considered to be a progressive and movement disorder which results in slow movement and develops cardinal symptoms such as tremor, bradykinesia, rigidity and postural instability [15]. There are also various other motor symptoms associated with PD such as freezing of gait, altered gait pattern, and motor co-ordination deficits [16,17]. Apart from that, some non-motor symptoms are also observed to be associated with PD which is anxiety and depression [18,19]. Hence in general, PD shows a direct link with mobility and motor control. Aggregation of α-syn as Lewy Neurites (LN) or Lewy Bodies (LB) is a pathological demonstration for dementia in both PD and DLB (Dementia in Lewy Bodies) in cortical areas [20-22]. The mechanism underlying protein aggregation and corresponding neurochemical correlations are still not properly understood. α-Syn protein consists of three distinct structural and dynamical properties which includes a membrane binding N-terminal helical segment (1-60); an acidic C-terminal region, weakly associated with the membrane (95-140) and a hydrophobic central fibril core region (NAC: non-amyloid β component) (61-95) which acts as a sensor of lipid properties, determining the affinity of α-syn membrane binding [23-25]. The central region that includes the NAC fragment is involved in the mechanism of α-syn aggregation, and is reported to play a key role in modulating the affinity of α-syn for cellular membranes [26-32]. Studies reveal that NAC domain is partially protected by the charges present in the positive N-terminal and negative C-terminal regions [33]. However, α-syn possesses dynamic conformations that are stabilized by long-range interactions providing a substantial degree of compactness [33]. These interactions occur between NAC region and C-terminal region and also between N and C-terminal region due to the effect of electrostatic and hydrophobic contacts which might prevent aggregation [33]. It has also been reported that the NAC domain, that plays a significant role in α-syn aggregation propensity is likely to be included in the membrane-sensor region and also to have functional relevance [30,34,35]. The NAC domain has the capability for defining the affinity of α-syn with lipid membranes and also in modulating partition between the bound and free states of membrane in the synaptic terminal [36-40]. Indeed, it is evident from earlier reports that α-syn exists in a state of equilibrium in vivo between the membrane-bound and cytosolic states, with membrane partition tightly being regulated [23]. During the process of aggregation, these membrane interactions highlight the importance of the interaction between various functional states of α-syn and its aggregation mechanism leading to PD.

Though the function of native α-syn is not clear, but it has been shown that α-syn can regulate the transport of neurotransmitter-filled vesicle to the release zone and assembly of complexes in presynaptic protein [41,42]. α-syn form aggregates which later forms insoluble

intracellular filamentous inclusions called as LB or LN [20] that are the significant histo-pathological symptoms of PD. Though the mechanism leading to the fibrillation process of α-syn are not known properly, but alteration and truncation of the C-terminal region of α-syn was shown to induce the aggregation mechanism in several models. Among the various mechanisms that can promote the aggregation propensity of α- syn, C-terminal truncation in α-syn have been recognized as a promoter/enhancer for α-syn fibrillization and oligomerization [43-46]. It has been reported earlier that the C-terminal truncations decreases the reductive capabilities and can potentially results in permanent oxidation of α-syn [47]. Hence it was shown that Cterminal region of α-syn is required for protein stability as the truncation mutants present in α-syn might have a higher tendency to fibrillate [48-50]. Also the C-terminal truncated forms have been observed to aggregate faster than the full length protein of α-syn [43,45]. The C-terminal region of α-syn plays a major part in the interaction of α-syn with other proteins and also with some small molecules [42,51-56]. The binding of important metals such as iron, copper and various other metals have been shown to influence the aggregation propensity of α-syn [14]. Consequently, inhibiting the α- syn C-terminal truncation might modify the period of disease in multiple system atrophy (MSA) and various other related neuropathogenesis [57] by reducing the α-syn aggregation and oligomerization process. Studies have shown that the carboxyl terminal truncation present in human α-syn was also observed in human DLB brain, and these truncations can increase α-syn propensity to aggregate in vitro [49,58]. Several evidences have been seen from literature which states that the missense mutations linked with PD can increase the proliferation of C-terminally truncated α-syn species. This type of truncations present in α-syn might occur in healthy brains too. Remarkably it has been observed that various truncated species might accelerate the fibrillation propensity of the full-length protein of α-syn both in vivo [59] and in vitro [46], and hence may contribute or initiate the aggregation process. The C-terminal region of α-syn is however less conserved than the N-terminal region which shows that interaction between these two regions helps to stabilize the conformations and protect it from various truncations and aggregation. The modifications done in C-terminal region may protect the various cleavage sites and also stabilize α-syn protein against proteolytically accessible conformations. As a result, the C-terminal truncation can generate production of toxic aggregates and hence promotes the neuro-degeneration process [43-45,60-70].

In order to understand the mechanism by which the C-terminal truncations affect the aggregation propensity of α-syn, the intermolecular interactions between them might play a vital role. In this work, we demonstrate the interactions between the WT α-syn and its three important C-terminal truncations 95, 120 and 123, using Molecular Dynamics (MD) Simulation and Potential of Mean Force study (PMF) [70]. The starting structure of the individual hetero-dimer containing WT α-syn and one of its three important C-terminal truncations 95, 120 and 123 for PMF study were obtained from the PatchDock [71] online docking server and the structures were then refined using FireDock [72] server. The interacting residues between the monomeric units of the hetero-dimer of α-syn and C-terminal truncations (α-syn 95, 120 and 123) were analyzed using PDBSum online server [73]. Using this server, the various bonding and nonbonding interactions was studied. We also noticed number of hydrogen bonds to be larger, high geometrical shape complementarity score, larger number of interface residues and interface area for the hetero-dimer (WT α-syn - α-syn 120) than other hetero-dimers (WT α-syn - α-syn 95/123). From the PMF study, we observed C-terminal truncation 120 (α-syn 120) to bind strongly to WT α-syn than the other truncated forms of α-syn. Thus, we can infer that among α-syn 95, α-syn 120 and α-syn 123, α-syn 120 show more association with WT α-syn and can easily form a hetero-dimer and aggregate at a faster rate than the other C-terminal truncated form of α-syn. Our findings suggest that more the truncation on the C-terminal region of α-syn more will be its impact on the aggregation.

Building the initial structures of the C-terminal truncated counter-parts of α-syn

The model structure of the three-truncated counter-parts of α-syn: C-terminal truncation 95 (α-syn 95), C-terminal truncation 120 (α-syn 120) and C-terminal truncation 123 (α-syn 123) (Figure 1) were prepared from the WT structure of α-syn with PDB ID: 1XQ8 taken from Protein Data Bank [74] by truncating the various residues present in C-terminal region using Pymol visualizing software (PyMOL Molecular Graphics System).

Figure 1: 3D structures of WT and the three truncated counterparts of α-Synuclein (Syn-95/120/123) obtained using Chimera softwares.

Construction of dimer

Using the PatchDock [71] online docking server, we constructed the hetero-dimers (WT α-syn - α-syn 95, WT α-syn - α-syn 120, WT α-syn - α-syn 123) by docking the two corresponding monomeric units. PatchDock is a rigid docking server that is based on geometry docking algorithm [71] which finds the optimum candidate conformers and uses Root Mean Square Deviation (RMSD) clustering to eradicate the redundant solutions. Based on the atomic desolvation energy [75] as well as geometric fit, a particular score was given to the candidate solution. In our study, the RMSD value was taken as 4 Å as default value for clustering the solutions. For refining the conformers obtained from the PatchDock docking server, we used the FireDock server [72]. This server targets the flexibility problem and solutions scoring that are formed by fast rigid-body docking algorithms. It refines based on an energy function by spending 3.5 seconds per candidate solution from a total of 1000 potential docking candidates. Based on an energy function, the candidate model conformers were refined and scored in the server. The individual candidate solutions are refined by optimizing the side-chain conformations and also by rigid body orientation. This energy score consists of atomic contact energy (ACE), electrostatics, hydrogen bonding, soften van der Waals interactions and estimation of binding free enegy.

Further to obtain the details about the various protein-protein interactions between the monomeric units of dimer of WT α-syn and the C-terminal truncated α-syn (α-syn 95/120/123) conformers, we used PDBSum online server. This server gives a brief illustration about the varying interacting residues, interfaces between the chains, overview of which chains interact with the other chain and interactions across any selected interface of the dimer complex of WT α-syn and Cterminal truncations.

The dimer conformer having the best geometric shape complementarity score, surface area, atomic contact energy (ACE) obtained from PatchDock (Figure 2) was submitted onto the PDBSum online server for inter-molecular interactions study (Figure 3). A detailed summary of the various bonded, non-bonded contacts and the interacting residues involved between the monomeric units of the dimer of WT α-syn and C-terminal truncations (α-syn 95/120/123) were obtained from this server.

Figure 2: Dimer conformers of the three complex αS and Cterminal truncated 95 (Syn 95), aS and C-terminal truncated 120 (Syn 120) and aS and C-terminal truncated 123 (Syn 123) obtained from PatchDock server.

Figure 3: Residue-residue interactions of the hetero-dimer of αS and C-terminal truncated (Syn 95/120/123) showing the various bonded and non-bonded interactions like salt-bridge (red colour), disulphide bonds (yellow colour), hydrogen bonds (blue colour) and non-bonded contacts (orange colour) as predicted by PDBsum server.

Potential of mean force study

The conformer having best atomic contact energy (ACE) value, interface area and geometric complementary score obtained from PatchDock was subjected to energy minimization, heating and equilibration dynamics and then proceeded for PMF study. Using Weighted Histogram Analysis Method (WHAM) [76-78], we calculated the PMF of WT α-syn and C-terminal truncation (α-syn 95/120/123) conformers using the equilibrium umbrella sampling simulations. We used the umbrella sampling method [79] to calculate the free energy profiles of the corresponding dimers. In umbrella sampling, the exploration of phase space relies on MD simulations over a series of regions (windows) and they are distributed along a predefined reaction path. Around the selected regions of phase space, biasing potentials are added to Hamiltonian to confine the molecular system. The biasing potential is a harmonic potential [79] to keep the system near a specified value in the reaction path. This process is done for a number of windows along the reaction path. For every window, equilibrium simulations are carried out and biased probability distributions (histogram) are obtained. The WHAM is used to determine the optimal free energy constants for the combined simulations. To study the degree of association of monomeric units of WT α-syn and C-terminal truncation (α-syn 95/120/123) conformers, we carried out PMF computation by varying the distance between center of masses (COMs) of the two monomeric units between them in two opposite directions (increasing and decreasing). The PMF has been computed as a function of inter-chain distances between the two monomeric units of the dimer complexes. The “inter-chain distance” is the distance between COM of the two monomers. Thereby, umbrella samplings were performed with reaction coordinate as the distance between the monomeric units for WT α-syn and C-terminal truncation (α-syn 95/120/123) conformers. By performing COM distance with constrained MD simulations, initial configurations were generated for the series of umbrella sampling MD simulation in WT α- syn and C-terminal truncation (α-syn 95/120/123) conformers. Distance between the COMs of the monomeric units of the dimer present in WT α-syn and C-terminal truncation (α-syn 95/120/123) was changed with time from 4 to 40 Å spanning different configurations. Initial configurations were extracted as a grid of spacing 15 Ǻ for WT α-syn - α-syn 120, 24 Å for WT α-syn - α-syn 95 and 30 Å for WT α-syn - α-syn 123.

Secondary structure analysis for the C-terminal truncated conformers

The secondary structure analysis for the C-terminal truncated conformers were carried out using online server Self-Optimized Prediction from Multiple Alignment (SOPMA). This server uses the homologue method by taking the short homologous sequence of amino acids that tend to form similar secondary structures. This database merely consists of 126 chains of non-homologous proteins. From the analysis of secondary structure contents we noticed α-syn 95 to have higher number of extended strand and beta turns (Table 1). From Table 1, we observed that the helical and strand contents are more in α-syn 95 whereas the coil content is more in α-syn 120 and α- syn 123. Hence we can infer that C-terminally truncated counterparts might play an important role in the aggregation propensity as it contains the necessary secondary structural contents for fibrillation of α-syn.

Table 1: Secondary Structure details of the truncated conformers of αS95, αS120 and αS123 monomers showing the percentage of secondary contents, graph and similarity threshold as predicted by SOPMA (Self-Optimized Prediction method With Alignment).

Dimer study

To study the docking conformations for the corresponding heterodimer of WT α-syn and C-terminal truncation (α-syn 95/120/123) of synuclein, we built the model hetero-dimer complex structures using the PatchDock online server. From the PatchDock server, the complex structure of hetero-dimer of WT α-syn and C-terminal truncation (α- syn 95/120/123) conformers having the best ACE value, interface area and geometrical shape complementarity score was obtained (Figure 2). From our results, we noticed that WT α-syn interacts with α-syn 95 with a geometric shape complementarity score of 12154, approximate interface area of 3054.60 Ǻ2 and atomic contact energy (ACE) of -563.83 kcal/mol. In the same manner, we observed that WT α-syn interacts with α-syn 120 with a geometric shape complementarity score of 20950, approximate interface area of 4043.30 Ǻ2 and atomic contact energy (ACE) of -840.07 kcal/mol whereas WT α-syn interacts with α- syn 123 with a geometric score of 16995, approximate interface area of 3883.00 Ǻ2 and atomic contact energy (ACE) of -460.85 kcal/mol respectively. The complex dimer structures obtained from PatchDock were refined using FireDock online server that are ranked based on Global energy, ACE value, Attractive van der Waals, Repulsive van der Waals and Hydrogen Bond.

We analyzed the interacting residues and the interface area for the complex model structure of WT α-syn and C-terminal truncation (α- syn 95/120/123) hetero-dimer having maximum interface area using the PDBSum online server. From this server, the interface and possible interacting residues across the interface were obtained. The results for the PDBSum were summarized in Table 2. From Table 2, we noticed the total number of interface residues in WT α-syn and α-syn 95 complex was found to be 95 and the interface area for each monomeric unit involved in the interaction was observed to be more than 2238 Å2. While in the case of WT α-syn and α-syn 120 complex we observed the total number of interface residues to be 122 and the interface area to be more than 2880 Å2. Similarly, for WT α-syn and α-syn 123 complex, we noticed the total number of interface residues to be 110 and the interface area to be more than 2543 Å2.

Table 2: Interface plot statistics of αS (1-140)-αS (1-95), αS (1-1s40)-αS (1-120) and αS (1-140)-αS (1-123) showing the total number of interface residues, interface area, number of salt bridge and total non-bonded contacts.

We observed that all the three-complex hetero-dimer structures WT α-syn and C-terminal truncation (α-syn 95/120/123) were stabilized by molecular interactions like hydrogen bonding, salt-bridges and nonbonded contacts (Figure 3). From the Table 2, we noticed the number of hydrogen bonds to be more in WT α-syn and α-syn 95 (11) and WT α-syn and α-syn 120 (11) complex than in the WT α-syn and α-syn 123 (7) complex. Among the C-terminal truncated conformers α-syn 95, α-syn 120 and α-syn 123, we observed α-syn 120 show more association with WT α-syn. This is because in α-syn 120, more amino acids have been removed in comparison to α-syn 123. However, α-syn 95 shows less interaction with WT α-syn because of the absence of the entire C-terminal region.

For this reason, we can suggest that there is a strong inter-molecular interaction between the WT α-syn and C-terminal truncated α-syn (95/120/123) that can accelerate the aggregation propensity and form dimer easily.

Computation of potential of mean force

To carry out the association study of WT α-syn and C-terminal truncated α-syn (95/120/123) hetero-dimer complexes, we have combined MD simulation along with umbrella sampling methodology [79] to examine the various interactions between WT α-syn and Cterminal truncated α-syn (95/120/123) hetero-dimer in terms of PMF. The PMF for WT α-syn and C-terminal truncated α-syn (95/120/123) hetero-dimer interaction in water at room temperature as a function of reaction co-ordinate is shown in Figure 4. We defined the reaction coordinate as the distance between the COMs of the synuclein monomeric units. From Figure 4, we see presence of a minimum of WT α-syn and α-syn 95 at a separation of 24 Å with a barrier to dissociation of 1.3 kcal/mol. From the association of WT α-syn and α- syn 120 and 123, we found the presence of a minimum at a separation of 15 Å and 30 Å with a barrier to dissociation of 2.5 and 1.5 kcal/mol respectively. We thus noticed that the energy barrier for the WT α-syn and α-syn 120 was larger when compared to WT α-syn and α-syn 95 and WT α-syn and α-syn 123. Hence we observed a predominant transient interaction between WT α-syn and α-syn 120 that can be further used to understand the fibrillation propensity. Our observations suggest that WT α-syn and α-syn 120 can easily form a dimer and can aggregate at a faster rate than the corresponding heterodimer complex.

Figure 4: PMF plot for the hetero-dimer of αS and C-terminal truncated (Syn 95/120/123) showing the varying optimal distance and the attractive and repulsive forces that are holding the dimers.

In this study, we have investigated the inter-molecular interactions between the WT α-syn and C-terminal truncation (α-syn 95/120/123) to understand the mechanism by which it affects the aggregation propensity of α-syn using the PMF study. We compared the association of WT α-syn with its C-terminal truncation (α-syn 95/120/123). From the PMF study, we observed C-terminal truncation 120 (α-syn 120) to bind strongly to WT α-syn than the other truncated forms of α-syn. We also noticed number of hydrogen bonds to be larger, high geometrical shape complementarity score, larger number of interface residues and interface area for the hetero-dimer (WT α-syn - α-syn 120) than other hetero-dimers (WT α-syn - α-syn 95/123). Thus, we can infer that among α-syn 95, α-syn 120 and α-syn 123, α-syn 120 show more association with WT α-syn. Our findings suggest that more the truncation on the C-terminal region of α-syn more will the effect on its aggregation.

We thank the Tezpur University and UGC for the start-up grant. We also thank the DBT funded Bioinformatics Infrastructure facility in the Department of Molecular Biology and Biotechnology at Tezpur University for providing us computational facility for carrying out this research work.