Replica Exchange Molecular Dynamics Study of Dimerization in Prion

Jul 8, 2016 - The pathological forms of prions are known to be a result of misfolding, oligomerization, and aggregation of the cellular prion. While t...
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Replica Exchange Molecular Dynamics Study of Dimerization in Prion Protein: Multiple Modes of Interaction and Stabilization Neharika G. Chamachi and Suman Chakrabarty* Physical and Materials Chemistry Division, CSIR-National Chemical Laboratory, Pune 411008, India S Supporting Information *

ABSTRACT: The pathological forms of prions are known to be a result of misfolding, oligomerization, and aggregation of the cellular prion. While the mechanism of misfolding and aggregation in prions has been widely studied using both experimental and computational tools, the structural and energetic characterization of the dimer form have not garnered as much attention. On one hand dimerization can be the first step toward a nucleation-like pathway to aggregation, whereas on the other hand it may also increase the conformational stability preventing self-aggregation. In this work, we have used extensive all-atom replica exchange molecular dynamics simulations of both monomer and dimer forms of a mouse prion protein to understand the structural, dynamic, and thermodynamic stability of dimeric prion as compared to the monomeric form. We show that prion proteins can dimerize spontaneously being stabilized by hydrophobic interactions as well as intermolecular hydrogen bonding and salt bridge formation. We have computed the conformational free energy landscapes for both monomer and dimer forms to compare the thermodynamic stability and misfolding pathways. We observe large conformational heterogeneity among the various modes of interactions between the monomers and the strong intermolecular interactions may lead to as high as 20% β-content. The hydrophobic regions in helix-2, surrounding coil regions, terminal regions along with the natively present β-sheet region appear to actively participate in prion−prion intermolecular interactions. Dimerization seems to considerably suppress the inherent dynamic instability observed in monomeric prions, particularly because the regions of structural frustration constitute the dimer interface. Further, we demonstrate an interesting reversible coupling between the Q160-G131 interaction (which leads to inhibition of β-sheet extension) and the G131-V161 H-bond formation.

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a seed for the creation of highly ordered PrPSC aggregates.3 On the other hand, a certain contrasting study reports a soluble PrP dimer fused to immunoglobulin Fcγ causing an antagonizing effect on the onset of disease by delaying PrPSC accumulation.10 Under fibrillization conditions, the fibril precursor state with partially melted α-helical forms is shown to exist in a monomer−dimer equilibrium with several intermediate structures using CD spectroscopy, analytical ultracentrifugation and chemical cross-linking.11 Amyloid-like β-fibril formation12 and 3D domain swapping involving intermolecular disulfide bond formation13 are two of the widely speculated mechanisms for dimerization. As discerned by Lee et al.,14 the latter facilitates dimer formation such that the intramolecular disulfide bond is broken in order to accommodate an intermolecular disulfide bond. Tompa et al. have developed a kinetic scheme of the prion dimerizationdisulfide rearrangement model.2 In the V129M mutation in prions, the wild-type forms confer as domain swapped dimers, but the nonmutated dimers have showed intermolecular βsheet formation instead of domain swapping.15 Warwicker et

INTRODUCTION Dimerization is a mechanism of self-association adopted by several proteins in order to gain stability, perform certain enzymatic functions, facilitate transport across membranes, restore regulation and/or achieve a pathogenic state.1 There exist some evidence that the prion proteins (PrPC) might exist in a dimer state.2,3 Elucidation of this state is particularly important for prions, because the aggregation of misfolded/ scrapie forms (PrPSC) is known to cause neurodegenerative diseases called transmissible spongiform encephalopathies (TSEs) including Creutzfeldt-Jakob disease, fatal familial insomnia, and Scrapie. Suzette et al. have identified a 60 kDa prion dimer that shows both normal and scrapie (misfolded) characteristics, making it a potential intermediate in prion replication.4 Many such instances of experimental evidence have been recorded for the presence of prion dimers.3,5−7 Friedlander et al. have demonstrated presence of low molecular mass aggregates of protease sensitive PrPSC in prion-infected tissues.8 However, as emphasized by them its role in pathogenesis is yet to be established as either metabolic precursors or off-pathway intermediates. According to the nucleation-dependent polymerization model of prion propagation,9 formation of prion heterodimers could be the first step toward formation of a nucleus that acts as © 2016 American Chemical Society

Received: April 11, 2016 Revised: July 6, 2016 Published: July 8, 2016 7332

DOI: 10.1021/acs.jpcb.6b03690 J. Phys. Chem. B 2016, 120, 7332−7345

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The Journal of Physical Chemistry B al.16−18 have proposed various models for homo- and heterodimers based on theoretical calculations. Cohen et al.19 have elaborated various possible conformations of dimers (assisted by a protein X): (i) conversion of PrPc to PrP* and then the formation of endogenous PrP*−exogenous PrPSC heterodimer; (ii) endogenous PrP*−PrPSC enabling recycling; (iii) combination of two mutant PrPC to form a PrP*/mut− PrP*/mut; and iv) combination of two PrPC to form PrP*−PrP* heterodimer. Different scenarios could lead to any of these dimers being formed yielding a PrPSC−PrPSC dimer, which could then initiate fibril formation. Normal mode analysis by Abraham et al.20 have indicated an elongation of the antiparallel β-sheet into a β-hairpin leading to formation of a β-sandwich with dimer formation. They have also speculated that this could be followed by the step of domain swapping. Derreumaux and co-workers have used 1.5 μs of coarse-grained molecular dynamics simulations to establish that while Helix-1 is retained upon T183A mutation, the Helix-2/Helix-3 subdomain shows slight intermolecular βsheet structures in the dimer intermediate states.21 Sekijima et al.22 have performed the first short simulation study on dimeric form and demonstrated the difference between the conformational dynamics of the monomer and dimer. They have shown that the dimer retains the tertiary structure to a higher extent as compared to the monomer. Moreover, additional structural elements might form at the dimer interface, namely H′ helices (residues 194−197 and 302−305) and S′ β-sheet (residues 191−193 and 299−301). Molecular dynamics simulations by De Simone and co-workers have shown elongation of the βsheet at the residues 133 and 159 in the Q217R mutated prion causing stable dimerization.23 Heterogenous forms of misfolded oligomers instead of amyloid fibrils have also been proposed as intermediates in prion replication.24 There have been indications that this is aided by destabilization of the structural domains formed within the PrPC protein and the loop region between β-sheet-2 and Helix-2, which has been suggested to be critical for misfolded oligomer formation.25 PrPC dimers are shown to have greater tendency toward adoption of a Helix-2/ Helix-3 intermolecular interface with larger binding free energy as compared to β-sheet interface using essential collective dynamics.26 In this work, we have used extensive all-atom replica exchange molecular dynamics (REMD)27 simulations to understand the molecular mechanism, modes of interactions, and altered stability pattern in a PrPC−PrPC homodimer as compared to the monomeric form. We have investigated the conformational changes mainly in terms of the changes in secondary structure, hydrophobic surface exposure and intermolecular interactions. Further, we have explored the energetics concerned with mechanism of misfolding on dimerization with the help of free energy calculations. We have identified the major interfaces of interaction in the dimer form, which coincided with the structurally frustrated regions leading to dynamical instability in the monomeric prions.28,29

efficiency due to united atom treatment of the nonpolar hydrogen atoms (as compared to fully atomistic force fields). Moreover, we have compared the propensity toward secondary structure formation (α-helical and β-sheet) between GROMOS 54a7 and AMBER99SB-ILDN force fields using REMD simulations of the monomer to find qualitatively similar trends but the overall secondary structure content seems to be higher in the GROMOS force field (Figure S1). The target dimer system has been created by replicating two monomers with the distance between the centers of mass (COM) being ∼5.5 nm and surface-to-surface minimum distance being ∼1.5 nm. The monomer and dimer systems have been put in a dodecahedron box with 9311 and 34330 water molecules, respectively. Energy minimization has been carried out using steepest-descent algorithm before starting the molecular dynamics runs. We have used REMD to enhance the conformational sampling by allowing crossing of energy barriers when conformations at lower temperatures may travel to higher temperatures through a Monte Carlo exchange protocol.27 We have used 51 (for monomer) and 96 (for dimer) replicas within the temperature range of 300−400 K and attempted exchange at every 2 ps in order to achieve an average exchange rate of 20%.34 For all these simulations, position restrained NVT equilibration has been run for 100 ps using V-rescale thermostat,35 followed by NPT equilibration for the same duration using the Berendsen barostat.36 Production runs have been carried out using Parrinello−Rahman barostat37 for 400 ns for each replica. In all the simulations, particle mesh Ewald method has been used to describe the long-range electrostatic interactions with 0.16 nm grid spacing.38 Leap frog integrator with the integration time step of 2 fs has been used to solve the Newton’s equation of motion and trajectories have been saved at every 10 ps. We have also performed a set of regular MD and REMD simulations with AMBER 99SB-ILDN force field for the monomer as benchmark studies with identical protocol. Multistate Bennett acceptance ratio (MBAR) method has been used for calculation of free energy from the multicanonical ensemble of the REMD data using the PYMBAR software.39 Conformational free energy surfaces have been calculated for the combined reaction coordinates of number of native contacts and the Cα RMSD with respect to the native structure. An intermolecular contact has been defined to be present for any pair of Cα atoms with the distance below 0.8 nm. For the intramolecular contacts we have used an additional constraint that the residue pairs i and j must be at least 3 residues apart, that is, |i − j| > 3. If the same intramolecular contact is present in the native structure as well, it has been defined as a native contact. Contact maps (relative frequency of occurrence) have been calculated based on the same definition of intermolecular contacts by accumulating statistics for all contact pairs over the whole trajectory. A standard geometric criteria for hydrogen bonds has been used, where the distance between the donor and acceptor atoms is less than 3.5 nm and the hydrogendonor−acceptor angle is less than 30°. Salt bridges have been defined as contacts present between oppositely charged residues (ASP/GLU with ARG/LYS). Hydrophobic solvent accessible surface area (H-SASA) has been defined in terms of the solvent accessible surface area (SASA) of the hydrophobic core of PrPC based on hydrophobic interactions in human PrP as defined by van der Kamp et al.40 It is comprised of the following residues: Met134, Pro137, Ile139, Phe141, Tyr150, Tyr157, Pro158, Val161, Phe175,

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COMPUTATIONAL DETAILS The NMR structure of C-terminal domain (residues 124−226) of mouse prion protein30 has been used for studying both the monomer and dimer forms (PDB code: 1AG2). We have used GROMACS software to perform all the molecular dynamics simulations31 with GROMOS 54a7 force field32 for the protein and SPC water model. The choice of GROMOS force field was dictated by prior literature23,33 as well as the computational 7333

DOI: 10.1021/acs.jpcb.6b03690 J. Phys. Chem. B 2016, 120, 7332−7345

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Figure 1. Structural parameters depicting the initial stages of spontaneous dimerization process: (a) Surface-to-surface minimum distance between the monomers (black line) and distance between the centers of mass (COM; red line), (b) total SASA for the combined system, (c) number of intermolecular salt bridges, and (d) number of intermolecular hydrogen bonds (H-bonds). Because the dimerization is complete before 40 ns, we have focused on the first 100 ns. The inset figures depict the full 400 ns trajectory in case of (a, b, and d) to highlight that the dimers remain stable during rest of the trajectory.

completes within the first 40 ns of the REMD trajectory (Figure 1a). Because the REMD trajectories are accelerated by exchanges between the replicas, we cannot conclude about the real time scale of the events. So we have tested this observation independently in a regular MD simulation starting from the same configuration, where the dimerization is found to complete in a shorter time scale (10−20 ns) as shown in Figure S2. In Figure 1, we have followed multiple structural parameters that depict the initial stages of the dimerization process at 300 K: (i) both the surface-to-surface distance (minimum distance) and COM-COM distance between the monomers (Figure 1a), (ii) total SASA of the combined system, (iii) number of saltbridges formed between the charged residues (ASP, GLU, ARG, and LYS), and (iv) number of intermolecular hydrogen bonds (H-bonds) between the monomers. The initial fluctuations in the surface-to-surface minimum distance between the monomers indicate the formation of many transient dimer states (Figure 1a). However, after ∼40 ns we observe that the dimer state gets stabilized and persists for the rest of the simulation until 400 ns (see the inset diagrams in Figure 1). Further, up to ∼40 ns a gradual decrease in SASA is associated with dimerization after which the SASA continues to fluctuate around an equilibrium average value (Figure 1b). It is somewhat expected that the overall solvent accessible area would reduce upon formation of the dimer interface. In Figure S3, we have shown that the total hydrophobic SASA decreases as well, which signifies the involvement of the hydrophobic

Val176, Cys179, Val180, Thr183, Ile184, Thr188, Thr191, Phe198, Val203, Met205, Met206, Val209, Val210, Met213, Cys214, and Val215. Principal component analysis (PCA) has been performed on the trajectories in order to identify the major modes of conformational fluctuations in the structural ensemble. In order to compare the principal components of fluctuations between the monomer and dimer trajectories, we have performed PCA over the combined trajectory. First, a covariance matrix has been built and diagonalized using the combined trajectory (both the isolated monomer and the individual monomers from the dimer trajectory). Eigenvectors with highest eigenvalues have been used to compute the projections (principal components). The components depicting the projection of the trajectory along the first two eigenvectors have been compared to identify the conformational differences between the monomer and individual monomers in dimer.

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RESULTS AND DISCUSSION Spontaneous Dimerization in Prions. The REMD simulation for the dimer has been started from a configuration where the monomers are separated by a large distance (∼5.5 nm) between their centers of mass (COM). The objective has been to observe the dimerization process and also to allow enough space around the individual monomers so that they can freely rotate around each other without interacting with their periodic images. Remarkably, we have found the process of dimerization to be spontaneous and quite rapid, which 7334

DOI: 10.1021/acs.jpcb.6b03690 J. Phys. Chem. B 2016, 120, 7332−7345

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The Journal of Physical Chemistry B

Figure 2. Time evolution (in REMD) of various structural parameters at replica T = 300, 350, and 400 K to highlight the convergence of their equilibrium fluctuations: (a) number of native contacts for each monomer (Prion 1 and Prion 2), (b) Cα RMSD, (c) total number of residues involved in β-sheet/bridge formation, and (d) total SASA for the combined system.

system would sample the low probability (high free energy) regions of the configurational space. Before computing the average structural properties across the temperature space, we have first tested the convergence of the sampling in an individual replica. In Figure 2, we have shown the time evolution (in REMD) of (a) number of native contacts, (b) CαRMSD, (c) number of residues forming β-sheets/bridge, and (d) total SASA. All the observables have been shown for three different replicas, T = 300, 350, and 400 K, and for two monomers (Prion1 and Prion2) wherever applicable. We notice that all structural parameters are well converged at 300 K throughout the 400 ns trajectory beyond the first 50 ns of the trajectory (corresponds to the initial stages of dimerization). At 400 K, we observe large structural reorganization initially due to the thermal denaturation, and in this case the equilibrium is achieved after the first 200 ns. For each replica, we have discarded the initial part of the respective trajectories corresponding to the drift in structural parameters (e.g., first 50 ns for 300 K) and used the rest of the trajectory for subsequent analysis. In Figure 3, we have summarized the temperature dependence of a wide variety of structural parameters, namely (a) CαRMSD, (b) radius of gyration, (c) number of intramolecular, intermolecular, and native contacts, where native contacts are those present in the reference PDB structure, (d) number of intra- and interprotein hydrogen bonds, (e) fraction of residues forming β-sheet/bridge, and (f) H-SASA. Because of partial thermal denaturation, increasing departure from the native structure with temperature is observed for both the prions, as is apparent from the increasing average Cα-RMSD (Figure 3a),

residues in stabilization of the dimer interface. We would show later that the misfolded states of the monomers, which form at higher temperatures, have considerably large hydrophobic SASA, which might promote the dimer formation, in order to bury the solvent exposed hydrophobic regions. In addition to the hydrophobic interactions, other electrostatic interactions seem to be stabilizing the dimer configuration as well. For example, the number of intermolecular salt bridge contacts (Figure 1c) formed between the charged residues (we find significant involvement of 164ARG, 167ASP, 194LYS, and 221GLU) and intermolecular H-bonds (Figure 1d) keep steadily increasing during the dimer formation process. All these factors contribute toward the higher thermodynamic stability of the dimer as compared to the isolated monomers leading to the spontaneous and rapid dimerization process. We must clarify here that the REMD trajectories are not strictly continuous in time, and the time axis (X-axis) shown in Figure 1 (and subsequent figures) is for indicative purposes only. One may find this time axis to be analogous to Monte Carlo steps, where the directionality is associated with the irreversible “equilibration” process (dimerization here). Nevertheless, as mentioned before we have tested the actual time scale of the dimerization process using normal MD trajectories as shown in Figure S2. Characterization of Structural Parameters: Temperature Dependence. The full set of REMD trajectories at multiple temperatures has enabled us to explore the temperature dependence of the structural properties. This would provide us additional insight into the underlying conformational energy landscape because at higher temperatures the 7335

DOI: 10.1021/acs.jpcb.6b03690 J. Phys. Chem. B 2016, 120, 7332−7345

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Figure 3. Temperature dependence of average structural parameters for Prion 1 (black) and Prion 2 (red): (a) Cα RMSD; (b) radius of gyration; (c) number of native contacts (black: Prion-1 and red: Prion-2), number of intramolecular contacts in Prion-1 (green) and Prion-2 (blue) (including both native and non-native), and number of intermolecular contacts between Prion-1 and Prion-2 (magenta); (d) number of intramolecular hydrogen bonds in Prion-1 (black) and Prion-2 (red) and number of intermolecular hydrogen bonds (blue); (e) fraction of residues involved in βsheet/bridge formation in dimer; and (f) H-SASA of the dimer.

content. This signifies that such misfolded states with higher βcontent are metastable in nature (higher in the free energy landscape) and their population may significantly increase with temperature. Furthermore, the total H-SASA increases rapidly at higher temperatures (Figure 3f) implying that these conformational states with exposed hydrophobic (nonpolar) regions would have significantly higher propensity to aggregate or oligomerize. These observations hint toward the presence of metastable β-rich conformational states that may have higher hydrophobic exposure leading to amyloid-like aggregation. Interestingly, our results closely resemble the findings of Simone et al.23 as they have identified a few structurally conserved hydration sites in the C-terminal region of PrPC for stabilization of the native fold, and thus dehydration of these sites (e.g., at elevated temperatures) would lead to an increased

decreasing the average number of native contacts (∼175 to ∼125; Figure 3c) and the average number of intraprotein hydrogen bonds (Figure 3d). However, for the same range of temperatures there is only a minor change in radius of gyration (Figure 3b) and number of intramolecular contacts (Figure 3c). This observation implies that the compactness of the structure is maintained even though there is extensive conformational change with formation of new contacts. Also, the average number of intermolecular contacts (Figure 3c) and average number of interprotein hydrogen bonds (Figure 3d) increase with temperature signifying that the dimerization propensity is considerably higher at elevated temperatures (with implications for temperature-induced aggregation processes). In Figure 3e, we have shown the temperature dependence of the secondary structure content, specifically the β-sheet/bridge 7336

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The Journal of Physical Chemistry B propensity toward misfolding. Thus, the hydration state of PrPC is intimately connected to their propensity to misfold and aggregate. Free Energy Surfaces (FES): Structural Diversity and Multitude of Pathways. Our next objective is to perform a comparative analysis of the conformational stability (both thermodynamic and dynamic fluctuations) between the isolated monomers, and the individual monomers in the dimeric state. We would first like to study the effect of dimer formation on the conformational free energy landscape of the monomers and then we would explore the coupling between the intermolecular and intramolecular reaction coordinates in this process. In Figure 4, we have demonstrated the conformational free energy landscapes with two reaction coordinates: Cα-RMSD

of their interconversion signifies the conformational heterogeneity, as well as the ease of misfolding in prions. We have shown a few representative metastable intermediate states with higher β-sheet content (up to 20%), slightly lower α-helical content and increased hydrophobic exposure as opposed to the native monomers. Most of the β-rich states in the isolated monomer correspond to extension of the existing β-sheet region, as well as partial melting of H2 and H3 as suggested in prior simulation studies.28,29 In contrast, the number of metastable states in the dimer system is much lower. We observe very similar conformational states/features near the broad global minimum for both monomer and dimer forms. The metastable states, existing far away from the native state for the isolated monomer, are not observed in case of the dimer under similar sampling conditions. This signifies the overall increase in the thermodynamic and kinetic stability of the monomeric prions upon dimerization. As mentioned before, unfolding of H2 and H3 due to their inherent instabilities have been confirmed as the initiation sites for misfolding in prions. 29,41 The dimerization process seems to alleviate these structural frustrations leading to both thermodynamic stabilization of the dimer (as compared to isolated monomer), as well as reduction in local dynamic instabilities.28 Effect of electrostatic interactions and salt bridge formation on the stability of dimers in a GCN4 leucine zipper have been studied by Liu et al.,42 where they find signatures of intermediate states acting as kinetic traps, particularly if the salt bridge strength is beyond certain optimal values. While we do not observe signatures of such kinetic traps in prions, where the dimerization occurs spontaneously, the significant increase in salt-bridge formation adds to the stabilization as demonstrated before. The aforementioned work by Liu et al.42 has also highlighted an interesting aspect of the coupling between the intermolecular contact formation and the changes in internal structures of the monomers. Derreumaux and co-workers have developed a novel method to characterize both the intramolecular and intermolecular degrees of freedom separately in the context of amyloid tetramer formation.43 On a similar spirit, we have constructed the FES using a combination of intermolecular and intramolecular structural reaction coordinates to elucidate the coupling between them, if any. This would help us to understand the role of intermolecular contact formation upon dimerization on the intramolecular structure of the individual monomers. We have computed the FES of (i) the total number of native contacts versus number of intermolecular contacts (Figure 5a), and (ii) the fraction of residues forming β-sheet/ bridge versus the number of intermolecular contacts (Figure 5b). We observe that the global minimum on both the FES occur when the number of intermolecular contacts is ∼40. The shape of the contour diagrams clearly indicate that the increase in the intermolecular contacts leads to (i) reduction in the native contacts of the monomers, and (ii) increase in the βcontent. These 2D FESs very nicely bring out the coupling between the intermolecular interaction coordinate with the intramolecular structural changes in the prion. We may conclude from these results that the thermodynamic global minima in the dimeric state may not involve large structural changes in the monomers, but the shape of the FES (minimum free energy pathway) shows that the tightening of the interactions between the monomers (increase in intermolecular contacts) would induce eventual disruption of the native intramolecular structure.

Figure 4. FESs involving the number of native contacts versus the CαRMSD (nm) for (a) isolated monomer, and (b) Prion-2 in the dimer state. Representative structures have been shown for each minimum of the respective FES. The contour lines have been drawn at every 1 kcal/mol.

(nm) and the number of native contacts both for the isolated monomer (Figure 4a) and the monomers in the dimer state (Figure 4b). We observe that the overall FESs for both the systems have qualitatively similar features. The landscape looks quite shallow, thus allowing large scale conformational fluctuations and heterogeneity. Unlike normal globular proteins, prions do not have a very deep folding funnel corresponding to the native state. There exist multiple metastable minima on the FES, and the relatively low barrier 7337

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Figure 5. FES depicting the coupling between the intermolecular and intramolecular coordinates: (a) number of intermolecular contacts versus the total number of native contacts in the dimer, and (b) number of intermolecular contacts versus the fraction of residues forming β sheet/bridge (%). The contour lines have been drawn at every 1 kcal/mol.

Figure 6. Root mean square fluctuations (RMSF) of the residues for the replica corresponding to 300 K temperature: (a) Monomer and Prion-1 of dimer and (b) monomer and Prion-2 of dimer. The horizontal bar on the top marks the secondary structures (B1, H1, B2, H2, and H3) present in the original NMR structure.

Structural and Dynamical Differences between Monomer and Dimer. So far we have discussed and compared the thermodynamic stability of the conformations adopted by the monomer and dimer. Now we turn our attention to the dynamic properties and specific conformational motions. We have first compared residue-wise root mean square fluctuations (RMSF) between the monomers and dimer to investigate the differences in region-wise dynamic stability (Figure 6). The residues corresponding to the coil regions between β2 and H2, between H2 and H3, terminal regions, and the C-terminal ends of H2 and H3 demonstrate large conformational fluctuations. This observation is in agreement with earlier simulation and experimental studies suggesting that the inherent structural frustration and dynamic instabilities in these regions make them the hotspots of misfolding or actively involved in oligomerization.21,28,29,41,44−50 The comparison of RMSF between monomer and dimer clearly indicates that even though the regions of higher fluctuations are mostly similar, the magnitude of dynamic instability is considerably lower in the case of the dimer as compared to monomer. Thus, the formation of the dimer interface may alleviate much of the structural frustration and instability by formation of favorable protein−protein intermolecular contacts as will be further demonstrated below. In order to fully understand the conformational complexity, as well as the important modes of conformational dynamics, we have used PCA. Our objective here is to define the unique conformational states in both monomer and dimer. In order to

define a common set of eigenvectors, we have performed the PCA over the combined trajectories for (i) isolated monomer, (ii) Prion 1 in dimer, and (iii) Prion 2 in dimer. The same set of eigenvectors obtained from the combined trajectory has been used to identify and compare the principal components for individual trajectories. Figure 7 shows a visual representation of the first three modes (highest eigenvalues) and the corresponding RMSF to elucidate the involvement of specific residues in those modes of motion. For subsequent analysis, we use projections of the first (PC1) and second (PC2) components along the eigenvectors 1 and 2 for (a) monomer, (b) Prion 1 of dimer, and (c) Prion 2 of dimer. We have shown in Figure S4 that inclusion of the third component (PC3) does not provide any additional resolution in the clustering because it has high degree of similarity with the PC1. Thus, we continue to use the 2D projection in (PC1, PC2) space for our purpose. According to Figure 7a,b, the PC1 involves movement of the terminal regions away from each other, exposing hydrophobic regions in H2 and H3 along with a large fluctuation in the coil region between β2 and H2. On the other hand PC2 (Figure 7c,d) shows additional large motion in the C-terminal region of H2, which is widely considered to be the Achilles heel in Prions.29,41 In Figure 8, we have presented an extensive comparison of the (PC1, PC2) projection between the isolated monomer and the monomer (Prion 2) in the dimer system for three different temperatures, T = 300, 350, and 400 K. In all of the cases, the 7338

DOI: 10.1021/acs.jpcb.6b03690 J. Phys. Chem. B 2016, 120, 7332−7345

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Figure 7. PCA depicting the residue-wise dynamics of top three modes: (a) PC1, (c) PC2, and (e) PC3. The RMSF for each of these modes have been shown as well, that is, (b) PC1, (d) PC2, and (f) PC3.

between H2 of both the prions. It can thus be inferred that the formation of dimers facilitated by this region prevents it from adopting conformations that involve its inner movement and thus increase the stability of dimer prions as compared to the monomer prion. The unique structures appearing for the dimer states at higher temperatures are mostly due to the increase in intermolecular interactions by cross β-sheet formation or formation of dimer interface in the regions with exposure of hydrophobic surfaces. Conformational Heterogeneity in Dimer Structures: Multiple Modes of Interaction. Although the process of dimerization is spontaneous and irreversible, we observe a surprisingly large structural heterogeneity within the dimer state, i.e. even though the monomers remain in close contact with each other, they can rotate with respect to each other while sampling various possible modes/faces of interactions. In order to characterize the hotspots of interactions between the monomers, we have computed the relative frequency of intermolecular contact formation for all the residue pairs between the monomers (Figure 9) for three kinds of

appearances of unique clusters have been marked by red rectangles. For example, the marked cluster for 300 K (Figure 8A(i)) for monomer is absent in the dimer state. Representative structures of all such clusters have been shown side by side with a transparent overlay of the native structure. For the unique misfolded state appearing in the monomeric system at 300 K, the loop region between H2 and H3 along with the C-terminal end of H2 deviates largely from the native state along with outward movement of H1. Interestingly, this region also appears to participate the most in intermolecular contact formation as will be shown later. Knaus et al. have compared the dimeric and monomeric studies using solution NMR and they found major structural differences in H3 and switch region containing residues 189VAL−198PHE in human prion protein.3 “Peeling-off” of the interactions between H2 and H3 and the rest of the globular domain has been implied in exposing these helices and making them aggregation-prone.46 We see this mechanism in our monomer structures leading to an increase in the β-sheet content, while for dimer structures this leads to interactions 7339

DOI: 10.1021/acs.jpcb.6b03690 J. Phys. Chem. B 2016, 120, 7332−7345

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Figure 8. Principal component analysis depicting projection on eigenvector 1 (PC1) and 2 (PC2) at three different temperatures: (A) 300 K (i) monomer (ii) Prion-2 of dimer and (iii) a conformation belonging to the distinct cluster (absent in prion-2 of dimer but present in monomer), (B) 350 K (i) monomer (ii) Prion-2 of dimer and (iii) a conformation belonging to the distinct cluster (absent in monomer but present in prion-2 of dimer), and (C) 400 K (i) monomer, (ii) Prion-2 of dimer and (iii) a conformation belonging to the distinct cluster (absent in monomer but present in prion-2 of dimer). The red dot depicts the position of crystal structure on PC 1 and PC 2. The red box on (a−c) is used to point the distinct cluster.

residues in PrPC plays a major role in dimerization, as they have been widely speculated as aggregation prone sites in prions.51 The two monomers orient in a manner such that most of the H2 part is exposed to other prion in the dimer with bending of the terminal tails around these regions. The 3D domain swapping between H2 and H3 would require bringing these regions closer to each other.1,2,14 Even though it would not be possible to study the disulfide bond formation/breakage through classical MD simulations, we see that these helices from both the prions are arranging themselves adjacent to each other, thus revealing a possibility toward formation of disulfidelinked dimers. In Figure 10c,d, the intermolecular contacts for the second cluster have been highlighted. We have observed contacts between N-terminal (154MET-160GLN) and C-terminal (188THR-197ASN) ends of H2 of Prion-1 with N-terminal of Prion-2 (124GLY-130LEU). Even though not clearly apparent for the structure shown in Figure 10d, we have seen intermolecular β-sheet formation between these contact regions. Structures with intermolecular β-sheets between the terminal end of Prion-1 (222SER-226TYR) and natively present β-sheet regions in their extended forms (124GLY-

interactions: (a) Cα contacts defined by a distance cutoff of 0.8 nm, (b) intermolecular H-bonds, and (c) intermolecular saltbridge formation. Remarkably, the contact map looks quite heterogeneous signifying a multitude of possible modes of interactions between the monomers. We have also performed PCA over the dimer trajectory, and the (PC1, PC2) projection of the same has been shown in Figure S5. The presence of large number of unique clusters highlights the extensive conformational heterogeneity possible in the dimer state, and few of the representative structures have been shown. In order to identify the most populated modes of monomer− monomer interaction, we have analyzed the three most dominant clusters and characterized the various types of contacts in a similar manner as described above. In Figure 10, we demonstrate the contact maps and representative structures for the three most populated modes of dimer formation. Figure 10a,b highlight the residues involved in intermolecular interactions for the most populated dimer cluster. The highly hydrophobic H2 residues (182ILE-194LEU) participate in these interactions with N-terminal regions (125LEU-127GLY) and H2 (182ILE-194LEU) of the other prion. It is interesting to note that the VTTTT region along with hydrophobic 7340

DOI: 10.1021/acs.jpcb.6b03690 J. Phys. Chem. B 2016, 120, 7332−7345

Article

The Journal of Physical Chemistry B

The last cluster shows contacts between H2 C-terminus of Prion-1 (181ASN-194LYS) and coil region before H2 and its N-terminal of Prion-2 (167ASP-178ASP) but the contacts formed do not involve major participation from the terminal residues in these dimer modes (Figure 10e,f). This cluster is also characterized by melting of intrinsically unstable H2 and involves higher contribution toward intermolecular interactions by means of salt-bridges (e.g., Figure 9c). In PrP dimers observed by Derreumaux and co-workers,21 H2 and H3 are found to show slightly higher intermolecular β-sheets as compared to the helically stable H1. Role of Q160-G131 Interaction in Inhibition of βSheet Extension. Intermolecular β-sheet formation is a hallmark of fibril-like structures. At higher temperatures, we observe frequent appearances of intermolecular β-sheet rich structures, often associated with large extension of the natively present β-sheets as shown in Figure 11a. Another representative snapshot (Figure 11b) shows a different mode of intermolecular β-sheet formation. Both of these modes involve (either partially or fully) participation of the existing β-sheet region. Simone et al. have shown that there exists a negative structural design element involving an interaction between the side chain of Q160 and G131 backbone carbonyl, which inhibits the further extension of the β-sheet by formation of backbone Hbonds between G131 and V161.23,33 Because our study seems to demonstrate considerable extension of the existing β-sheet as well as participation of G131 in intermolecular β-sheet formation, we have explored the role of the Q160-G131 interaction in inhibiting such interactions. In Figure 12, we have presented a 2D scatter plot of (i) the H-bond distance between G131 backbone O and Q160 side-chain N, and (ii) the H-bond distance between G131 backbone O and V161 backbone N. This 2D distribution clearly shows two distinct clusters signifying the “mutually exclusive” nature of these two interactions. For the structures with the G131-Q160 H-bond present (distance