The role of the acidic domain of α-synuclein in amyloid fibril formation: a molecular dynamics study

The detailed mechanism of the pathology of α-synuclein in the Parkinson’s disease has not been clearly elucidated. Recent studies suggested a possible chaperone-like role of the acidic C-terminal region of α-synuclein in the formation of amyloid fibrils. It was also previously demonstrated that the α-synuclein amyloid fibril formation is accelerated by mutations of proline residues to alanine in the acidic region. We performed replica exchange molecular dynamics simulations of the acidic and nonamyloid component (NAC) domains of the wild type and proline-to-alanine mutants of α-synuclein under various conditions. Our results showed that structural changes induced by a change in pH or an introduction of mutations lead to a reduction in mutual contacts between the NAC and acidic regions. Our data suggest that the highly charged acidic region of α-synuclein may act as an intramolecular chaperone by protecting the hydrophobic domain from aggregation. Understanding the function of such chaperone-like parts of fibril-forming proteins may provide novel insights into the mechanism of amyloid formation.

The aggregation of α-synuclein into amyloid fibrils is a nucleation-dependent process with an initial lag phase, an elongation step, and a steady-state phase (Han, Weinreb, & Lansbury, 1995;Wood et al., 1999). In the lag phase, the nucleus of aggregation is formed. It has been suggested that this nucleus can be as small as a dimer of α-synuclein (Fernández et al., 2004). The nucleation process, which involves the formation of partially folded intermediates , is usually rather slow. Once the nucleus is formed, the aggregates grow rapidly until a thermodynamic equilibrium is established between aggregates and monomers. During the aggregation process, the protein forms a secondary structure containing mainly an antiparallel β-sheet (Lin et al., 2006;Narhi et al., 1999).
The kinetics of α-synuclein aggregation can be affected by various factors. One of the interesting findings was that the lag phase was reduced in vitro in the presence of FK506 binding proteins (FKBPs) (Gerard et al., 2006(Gerard et al., , 2008. FKBPs exhibit a peptidyl-prolyl isomerase (PPIase) activity, which accelerates the cis-trans isomerization of a proline residue (Göthel & Marahiel, 1999). FK506 binds to most FKBPs and is a potent and specific inhibitor of their PPIase activity (Gerard et al., 2006(Gerard et al., , 2008. Several immunophilin ligands such as FK506 and members of the FKBP family have been associated with Parkinson's disease . Several studies have shown that the neurotrophic activity of FK506 is mediated by the interaction with FKBPs (Fischer, Wittmann-Liebold, Lang, Kiefhaber, & Schmid, 1989;Harding, Galat, Uehling, & Schreiber, 1989;Siekierka, Hung, Poe, Lin, & Sigal, 1989;Takahashi, Hayano, & Suzuki, 1989). Furthermore, several reports have suggested the role of the PPIase in neurodegeneration, but a conclusive evidence of this has not been presented so far (Fischer et al., 1989;Gerard et al., 2006Gerard et al., , 2008Gerard et al., , 2010Göthel & Marahiel, 1999;Harding et al., 1989;Siekierka et al., 1989;Takahashi et al., 1989). Pertinent to the subject of the present study, these reports suggested that the cis-trans isomerization of a proline residue may be linked with the kinetics of the α-synuclein aggregation.
Some studies have shown that α-synuclein suppresses the aggregation of the thermally denatured alcohol dehydrogenase and chemically denatured insulin . The chaperone-like activity of α-synuclein is suppressed completely by the removal of its C-terminal residues 98-140 Hoyer et al., 2004;Souza, Giasson, Lee, & Ischiropoulos, 2000). It is interesting to note that the acidic region of α-synuclein has five proline residues. The presence of proline residues reduces protein flexibility. In the absence of an enzyme-catalyzed acceleration of the proline isomerization (Lu, Finn, Lee, & Nicholson, 2007;Wedemeyer, Welker, & Scheraga, 2002), proline residues lock the structure of the peptide backbone in a rigid configuration. Recently, the aggregation kinetics of several α-synuclein mutantsin which one or more proline residues in the acidic region were mutated to alaninehave been studied to elucidate the role of proline residues in the conformation and aggregation of α-synuclein (Meuvis, Gerard, Desender, Baekelandt, & Engelborghs, 2010). It has been shown that the mutation of proline residues to alanine accelerates the aggregation kinetics of α-synuclein, while all proline mutants formed fibrils similar to those composed of the wild-type α-synuclein. It has also been demonstrated that α-synuclein that bears a quintuple mutation of all five proline residues being replaced with alanine is more structured than the wild-type α-synuclein. This observation suggested that proline residues may act as potential helix breakers in the inhibitory conformation of the Cterminus (Meuvis et al., 2010).
At low pH, α-synuclein has been shown to aggregate faster than at neutral pH . It was suggested that the compaction of the protein into a partially folded conformation is responsible for the increased rate of aggregation at low pH. Structural reorganization of α-synuclein at low pH was also studied by NMR spectroscopy and molecular simulations (Wu, Weinstock, Narayanan, Levy, & Baum, 2009). It was observed that there was a significant structural reorganization within the low pH ensemble relative to that at neutral pH in terms of long-range contacts, hydrodynamic radius, and the amount of heterogeneity within the conformational ensembles.
In the present study, we performed systematic simulations of α-synuclein under various conditions in order to provide plausible scenarios for the role of the acidic region in the formation of amyloid fibrils by α-synuclein. Our main aim was to investigate structural changes of the acidic region with relation to its possible interactions with the NAC region under different conditions.

Computational methods
Replica exchange molecular dynamics (REMD) simulations of wild-type and P-to-A mutated α-synuclein were performed in an attempt to understand the role of proline residues in the structure of the C-terminal region and subsequent aggregation of α-synuclein. We carried out simulations of the acidic domain (residues 101-140) and the combined sequence of the NAC and acidic regions (residues 61-140). For the mutated α-synuclein, all proline residues (P108, P117, P120, P128, and P138) were substituted by alanine residues. In order to model the acidic condition of pH 2, the negative charges on the side chains of the acidic residues are neutralized. The all-atom AMBER FF96 force field with modified hydrogen bonding radii was used in this study (Kollman, Cornell, Fox, Chipot, & Pohorille, 1997). A modified GB/SA model was used to describe solvation (Fan, Hwang, & Warshel, 1999;Onufriev, Bashford, & Case, 2000Still, Tempczyk, Hawley, & Hendrickson, 1990). Initial configurations of the monomers prepared with the LEAP program of AMBER10™ were minimized using the steepest descent and conjugate gradient algorithms, and then monomers were heated up to 500 K using the weak-coupling algorithm (Berendsen, Postma, van Gunsteren, DiNola, & Haak, 1984). Several replicas (24 replicas between 290.0 and 621.2 K for the acidic domain only and 34 replicas between 282.8 and 750.0 K for the combined sequence of the NAC and acidic domains) were created. Their temperatures formed a geometrical series, and all the replicas were equilibrated at their respective temperatures. Next, REMD simulations with an exchange interval of 400 fs were performed using the replicas described above (Cheng, Cui, Hornak, & Simmerling, 2005;Mitsutake, Sugita, & Okamoto, 2001;Nymeyer, Gnanakaran, & García, 2004). Distributions of potential energies for the replicas were well overlapped and the swapping probability was~0.6. The convergence of individual MD simulation was confirmed by examining the time evolutions of the secondary structure contents and the root mean square deviation (RMSD). The simulation times for eight different simulations ranged from 26 to 73 ns and the total simulation time for all of the replicas comprised~13 μs. All analyses were done for the last 9 ns of the production simulation. The REMD simulations were done using the SANDER program of AMBER10™ with the SHAKE algorithm (Case et al., 2008;Ryckaert, Ciccotti, & Berendsen, 1977).
As one of the intrinsically disordered protein, it is difficult to determine a single structure representing structural properties of α-synuclein. We can define representative structures for ensemble of structures occurring during the overall trajectory in order to examine the conformational characteristics of the segments of α-synuclein (Yoon, Park, Jang, Lee, & Shin, 2008). Configurations obtained from the trajectories of simulations were clustered into four groups based on relative RMSD values. For each cluster, the representative configuration was chosen as the configuration whose RMSD value is the closest to the average RMSD of the configurations belonging to the particular cluster. The representative configuration for the cluster with the largest population can be considered as the representative structure for the simulations. Cluster analysis was also performed using analysis tools supplied in the AMBER™ software package.

Results and discussion
Proline residues, which have a cyclic structure, may play important roles in determining biological functions of proteins because their conformational changes are restricted by the ring strain. Using the density functional theory (B3LYP) with the basis of 6-31G(d), the activation barrier for the cis-trans isomerization reaction of an isolated proline residue was calculated to bẽ 12.4 kcal/mol (Becke, 1993;Lee, Yang, & Parr, 1988). Furthermore, activation barriers for the cis-trans isomerization of peptidyl-prolyl have been calculated to com-prise~30 kcal/mol (Lu et al., 2007). High activation barriers cause regions with proline residues to become rigid, and the reduction in the flexibility of the protein domain may lead to structural changes associated with a particular function.
The acidic domain of the wild-type α-synuclein (amino acid sequence 101-140) has five proline residues, which indicates a reduced flexibility of this domain. Changing proline residues into alanine residues is expected to make the mutant acidic domain more flexible than the same region of the wild-type α-synuclein. We calculated the radius of gyration as a function of temperature for the wild-type and P-to-A mutant of the acidic domain in neutral and acidic (pH 2) conditions ( Figure 1(a)). Under the neutral condition and at low temperatures, the averaged Rg, <Rg> of the P-to-A mutant was smaller than that of the wild-type α-synuclein. As the temperature was increased, <Rg> of the P-to-A mutant showed larger changes and had larger values at high temperatures compared to the wild-type α-synuclein. Under the acidic condition, the acidic domain for both the wild-type α-synuclein and P-to-A mutant showed a much smaller <Rg> compared to the neutral condition. This is consistent with the fact that most of the negative charges in the acidic domain are neutralized at low pH, which allows the acidic domain to be more compact (See Figures S1 and S2 in Supplementary material).
We also calculated the Rg as a function of temperature for the wild-type α-synuclein and P-to-A mutant of the combined sequence of the NAC and acidic region in neutral and acidic (pH 2) conditions (Figure 1(b)). For the wild-type α-synuclein, at low temperatures <Rg> for the combined sequence of the NAC and acidic region was smaller than that of the acidic region alone. The configurations of the acidic region, in the absence of NAC domain, showed rather extended structures (See Figures S1). For the combined sequence of the NAC and acidic region, the two domains seemed to show strong interaction between each other and the resulting structure was somewhat more compact compared to the structure of the acidic region alone (See Figures S3). The value of <Rg> for the combined sequence showed large changes at approximately 350 K and became much larger than that of the acidic region alone at higher temperatures. It can be argued that the NAC and acidic domains were close to each other at lower temperatures but they became separated as the temperature was increased. As shown in Figure 1(b), when the P-to-A mutation was introduced or when pH was reduced, the value of <Rg> of the combined sequence of the NAC and acidic regions was larger than that of the wild-type α-synuclein at neutral pH. We attribute this difference mainly to the increase in the relative distance between the two regions.
The representative configurations of the combined sequence of the NAC and acidic regions for the wildtype α-synuclein and the P-to-A mutant at neutral and acidic conditions were obtained by performing a cluster analysis of trajectories of the corresponding simulations at 300 K (See Figures S3 and S4 in Supplementary material). Figure 2 shows the representative structures of the simulations under different conditions. For the wildtype α-synuclein at neutral pH, the NAC and acidic domains seem to show extensive contacts between them. After the P-to-A mutation is introduced, the interaction between the two domains appears to be reduced with the end of the flexible C-terminal region flanking away from the NAC domain. Similar conclusions can be made also for the case of the acidic pH. As the acidic region becomes more compact at lower pH, the contact region between the two domains is reduced. By comparing relative positions of the acidic region (red) and NAC domain (blue) in these cases, it can be argued that either the P-to-A mutation or lower pH leads to the increase in the average distance between the acidic region and the NAC domain. This observation was supported by calculations of the distribution of distances between the centers of mass for the two regions. Figure 3 clearly illustrates the fact that the relative distance between the NAC and acidic regions increased with either a reduction in the pH level or an introduction of the P-to-A mutation.
During the formation of amyloid fibrils, aggregationprone regions of monomers, usually hydrophobic domains, such as the NAC region of α-synuclein, need to be able to move closer to each other for a fast aggregation rate. One way of examining the availability of the NAC region for the interaction with other aggregation partners is to calculate the solvent accessible surface area (SASA). We calculated the SASAs for the entire structure of NAC + acidic region and for the NAC domain alone. We compared the averaged SASA of α-synuclein at different conditions to the reference SASA calculated for the wild-type at neutral pH. For the NAC region, either the introduction of the P-to-A mutation or lowering of the pH led to an increase in the overall SASA (Table S1 in the Supplementary material). This result is consistent with the increase in the average distance between the acidic region and the NAC domain ( Figure 3).
We constructed contact maps for the combined sequence of the NAC and acidic regions in different conditions (See Figure S5 in the Supplementary material). In representative structures of the wild-type α-synuclein at neutral pH, contact maps showed strong contacts between the middle parts of the NAC and acidic regions. This is consistent with the earlier suggestion that Figure 1. Radius of gyration as a function of temperature for the wild-type and P-to-A mutant of (a) the acidic region alone and (b) the combined sequence of the NAC and acidic region under the neutral condition and the acidic condition. Figure 2. Representative structures of the combined sequence of NAC domain (blue) and the acidic region (red) of α-synuclein, obtained from simulations at 300 K for (a) the wild-type, (b) the P-to-A mutant at neutral condition, (c) the wild-type and (d) the P-to-A mutant at acidic condition. the combined sequence of the NAC and acidic region shows rather compact structure with extensive contacts between the two domains. The strength of these contacts was reduced when the P-to-A mutation was introduced or when pH became acidic. Figure 4 shows the number of contacts for the combined sequence of the NAC and acidic regions at different conditions. It also clearly demonstrates that the interaction between the two regions, as determined by the number of close contacts, is significantly reduced by the P-to-A mutation or a pH change. These results support the idea that the increase in the exposure of the aggregation-prone NAC region results from the reduction in close contacts between the acidic region and the NAC domain due to the P-to-A mutation or acidic pH.
In contrast to the Aβ peptide, α-synuclein, as an amyloid forming protein, has a rather long sequence consisting of three different domains. These three domains exhibit different properties: the membrane-binding N-terminal region is amphipathic, the NAC domain is hydrophobic, and the polar C-terminal tail is acidic. The middle part of the hydrophobic NAC domain is usually considered to be important for the aggregation of α-synuclein. While the N-terminal region is known to form membrane-bound α-helices, the acidic C-terminal region is unstructured. The possible role of the acidic region during the formation of amyloid fibrils by α-synuclein is not known at present. Recent experiments have provided interesting insights suggesting that structural changes in the acidic region may affect the aggregation processes Souza et al., 2000). Most notably, the mutation of proline residues to alanine residues in the acidic region accelerates the aggregation kinetics (Meuvis et al., 2010).
In the present study, we performed systematic simulations of α-synuclein conformations under various conditions in order to elucidate structural characteristics of the acidic region responsible for changes in the aggregation behavior. The results of the present study provide plausible scenarios for the role of the acidic region in prevention of amyloid formation by α-synuclein. We observed that under neutral pH, the acidic C-terminal domain of the wild-type α-synuclein was fairly stretched and rigid owing to multiple negative charges and five proline residues. The results of our simulations showed that the distance between the acidic region and the aggregation-prone NAC domain was relatively small. We also found that there exist close contacts  between the middle parts of the NAC domain and the acidic region. One may argue that the acidic region is locked in a position where it covers main parts of the NAC domain and blocks the approach of other hydrophobic chains such as the NAC domain of the other α-synuclein. Therefore, the natural role of the acidic region is to prevent the aggregation of the hydrophobic NAC domain and maintain the functional form of α-synuclein. This argument is supported by experimental findings that revealed an acceleration of the aggregation following the removal of the acidic region Souza et al., 2000). The acidic C-terminal region, as described above, may be therefore viewed as an "intramolecular chaperone." The chaperone-like role of the acidic region can be affected by environmental changes or mutations. The present study has shown the structural basis for such effects. Lowering the pH to an acidic condition neutralizes negative charges on the C-terminal domain. As a result, the acidic region may form a more compact structure, greatly limiting the interaction with the NAC domain (Wu et al., 2009). Mutations of the five proline residues to alanine make the acidic region much more flexible. The increased flexibility prevents the C-terminal domain from maintaining a close contact with the NAC domain. In either case, i.e. whether the structure is flexible or compact, the acidic C-terminal region can no longer protect the NAC domain from interacting with other chains for aggregation.
It is now generally believed that most of the small peptides or large proteins can form fibrils if they contain a sequence of residues with a strong propensity for aggregation. Under normal conditions, functional proteins avoid being involved in amyloid formation by preventing aggregation. The simplest way to block aggregation is to prevent the aggregation-prone region from interacting with its counterparts from neighboring chains. The results of the present study suggest that the highly charged acidic C-terminal region of α-synuclein may act as an intramolecular chaperone that protects the NAC domain from aggregation. Understanding the function of such chaperone-like parts of fibril-forming proteins should provide novel insights into the mechanism of amyloid formation and suggest new avenues for treatment of diseases caused by protein aggregation.

Supplementary material
The supplementary material for this paper is available online at http://dx.doi.10.1080/07391102.2015.1033016.

Disclosure statement
No potential conflict of interest was reported by the authors.