Temperature-Induced Misfolding in Prion Protein: Evidence of Multiple

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Temperature Induced Misfolding in Prion Protein: Evidences of Multiple Partially Disordered States Stabilized by Non-native Hydrogen Bonds Neharika G. Chamachi, and Suman Chakrabarty Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b01042 • Publication Date (Web): 19 Jan 2017 Downloaded from http://pubs.acs.org on January 20, 2017

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Temperature Induced Misfolding in Prion Protein: Evidences of Multiple Partially Disordered States Stabilized by Non-native Hydrogen Bonds Neharika G. Chamachi and Suman Chakrabarty* Physical and Materials Chemistry Division, CSIR-National Chemical Laboratory, Pune 411008, India *Email: [email protected]

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ABSTRACT

The structural basis of misfolding pathways of a cellular Prion (PrPC) into the toxic scrapie form (PrPSC) and identification of possible intermediates (e.g. PrP*) still eludes us. In this work, we have used a cumulative ~65µs of Replica Exchange Molecular Dynamics simulation data to construct the conformational free energy landscapes and capture the structural and thermodynamic characteristics associated with various stages of the thermal denaturation process in human Prion protein. The temperature dependent free energy surfaces consist of multiple metastable states stabilized by non-native contacts and hydrogen bonds, thus rendering the protein prone towards misfolding. We have been able to identify metastable conformational states with high β-content (30-40%) and low α-content (~10-20%) that might be precursors towards PrPSC oligomer formation. These conformations also involve participation of the unstructured N-terminal domain and its role in misfolding has been investigated. All the misfolded or partially unfolded states are quite compact in nature despite having large deviation from the native structure. Although the number of native contacts decreases dramatically at higher temperatures, the radius of gyration and number of intra-protein hydrogen bonds and contacts remain relatively unchanged leading to stabilization of the misfolded conformations by non-native interactions. Our results are in good agreement with the established view that the Cterminus regions of the 2nd and 3rd helices (H2 and H3) of mammal Prions might be the Achilles heels of their stability, while separation of B1–H1–B2 and H2-H3 domains seem to play a key role as well.

KEYWORDS. Prion, protein misfolding and aggregation, Replica Exchange Molecular Dynamics, Free Energy Surface, hydrogen bond


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The misfolding and aggregation of proteins are long known to be associated with each other. Neurodegenerative diseases (NDs) like Alzheimer’s disease, Huntington’s disease and Transmissible Spongiform Encephalopathies (TSEs) including Creutzfeldt-Jakob disease, fatal familial insomnia, and Scrapie are examples of such protein misfolding and aggregation related disorders.1-4 Formation of misfolded structures from the native cellular forms, their oligomerization and the resulting fibril plaques lead to such often fatal disorders.5-8 While considerable progress has been made towards understanding the mechanism of aggregation and fibril formation in β-amyloids9 in the context of Alzheimer’s disease, we have rather limited understanding of the molecular processes associated with misfolding and propagation of Prion related diseases, i.e. TSEs. The cellular prion (PrPC) is majorly found in neurons and participates in anti-apoptotic activity, copper binding, signal transduction, protecting cells from oxidative stress, and many unknown activities.10 It has a long N-terminal tail with octapeptide repeats, three helices (H1, H2 and H3) and two β-sheets (B1 and B2) and is attached to the membrane by a GPI anchor. 11, 12 However, it can adopt a proteinase-K resistant misfolded conformation, commonly called as the scrapie prion (PrPSc) under the conditions of low pH, high temperature and/or mutations.13 In the oligomeric/fibrillar form, PrPSc is known to be the causative agent of TSEs. Even though the structure of PrPC has been elucidated in great detail, the structure of the PrPSc form remains elusive. FTIR spectroscopy and CD experiments by Pan et. al.14 have demonstrated that Syrian hamster PrPC has high (42%) α-helix content and low (3%) β-sheet, whereas PrPSc has 43% βsheet content and lower α-helix content (30%). Various computational15-24 and experimental11, 13, 25-30

studies have attempted to provide a structural basis of this transformation across several

species, but none have been able to provide a globally accepted PrPSc structure. Moreover, the


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molecular mechanism of Prion propagation, i.e. the process of the toxic PrPSc form inducing misfolding into the healthy PrPC form with eventual fibril formation, remains an unsolved problem. Elucidating the molecular mechanism of the transformation of PrPC to PrPSc has been an active area of research. It has been theorized that there exists a large free energy barrier (~20-25 kcal) and great conformational change between PrPC and PrPSc with a PrP* intermediate.24, 31 Many monoclonal antibody studies have pointed out an importance of N-terminal Domain (NTD) in conformational conversion of PrPC to PrPSc as epitopes in this region undergo extensive conformational rearrangement and is thus implicated in binding of PrPC to PrPSc.32-36 The domain between the residues 90–145 is presumed to be involved in beta-sheet rich conversion of PrPC.37 The length of amino terminal deletion affects the reversibility in the pressure induced aggregation behavior.38 The residues in the NTD in one study are shown to be less prone to forming stable stacked β-sheets on their own but they can be interacting with other regions leading to misfolded fibril structures.39 A β0-β1-α1-β2-α2-α3 can be adopted by the full length Human PrPC where β0 is made up of residues 118 to 122 and it forms anti-parallel β-sheet with β1.40 Moulick and Udgaonkar have also highlighted the importance of NTD-CTD interactions through their finding that NTD can significantly alter the Prion stability at pH 7.41 Further, C-terminal domain being the structured segment is proposed to undergo major structural rearrangement upon misfolding17 and forming the β-core.42 The instabilities, fibrillization and oligomerization propensities of the three helices have been studied using experimental as well as computational methods.22-24,

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Helix 1 (H1) is said to be

intrinsically stabilized with salt-bridges, while Helix 2 (H2) and Helix 3 (H3) exist in a frustrated structure due to very unusual pattern of hydrophobic residues22-24,


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that may increase their

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propensity to misfold into a β-sheet rich structure.49 Due to this instability of H2 and H3 they have been proposed to be the initiation site for misfolding, while H1 is shown to dislocate and ease the conformational conversion. Separation of the two subdomains i.e. B1-H1-B2 and H2-H3 of CTD of PrPC has been highlighted in many studies leading to “peeling off” and exposure of H1 when moving away from the H2-H3 core.50,

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The stabilization of PrPC is said to be

influenced by preservation of H1 and it can adopt metastable folded and unfolded states both stabilized by salt-bridges when detached from the H2-H3 hydrophobic core.44 The misfolding may be induced by lowering of pH,15,

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protonation of His18753,

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and exposure of the

hydrophobic core55 that would promote oligomerization. While extension of the natively present β-sheets has been reported as an on-pathway feature of misfolding,47 Barducci et al have shown that its disruption by mutating 164ARG and 128TYR side chains facilitates unzipping of the anti-parallel β-sheet leading to its pathologic form.43 The flexibility of the loop between B2-H2 is also found to affect misfolding as supported by difference in misfolding propensities of various species.56 Mutation of specific residues like 149TYR,57 177ASP and 199GLU58 enhance the dynamical fluctuations of PrPC leading to higher solvent exposure of the hydrophobic core and disruption of interactions between its structural domains. A recent study by Mukhopadhyay and coworkers suggests that β-rich states are off-pathway for amyloid formation rather than being intermediates for PrPSC formation, and on-pathway structures are partially disordered.47 Although, existence of multiple events and pathways towards formation of the toxic state is becoming increasingly evident, the molecular details of these pathways are still far from reaching consensus. In order to understand the thermodynamic stability of the PrPC form with respect to the possible misfolded conformations, and the associated barriers of their interconversion, it is


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extremely crucial to obtain the underlying conformational free energy landscape. Earlier estimates of the unfolding barrier through steered molecular dynamics simulations,24 and metadynamics simulations43, 49 are available. But elucidation of the global free energy landscape identifying the key conformational states, and identifying the minimum free energy pathways connecting the cellular form to the β-sheet rich misfolded forms have been missing. In this work, we have employed atomistic Replica Exchange Molecular Dynamics (REMD) simulations to unravel the free energy landscapes for human Prion protein using a combination of various structural reaction coordinates capturing: (i) a large number of metastable conformational states stabilized by non-native contacts and hydrogen bonds signifying a complex network of many possible pathways and conformational heterogeneity, (ii) appearance of β-sheet rich structures, which is a hallmark of the so-called PrPSc form, (iii) role of NTD in the misfolding associated conformational changes and its interaction with CTD and (iv) possible initiation sites of misfolding, and thermodynamic factors associated with the PrPC to PrPSc transformation.

METHODS

We have used the NMR structure of the Human Prion Protein with both a part of N-terminal domain (residue 90-123) and the C-terminal domain (residues 124-231) (PDB ID: 2LSB).59 First, molecular dynamics simulation was performed using OpenMM60, 61 with implicit solvation (OBC generalized Born model)62 and AMBER99sb-ildn forcefield63 for ~2 µs in order to equilibrate the disordered NTD into a compact state (interacting with CTD). After equilibration of the potential energy, a conformation was selected and used to setup explicit solvent Replica Exchange Molecular Dynamics (REMD) simulations. All the molecular dynamics simulations have been performed using Gromacs software (version 4.6.5)

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with GROMOS96 54a7 force field


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the protein and SPC water model. The protein has been solvated in a dodecahedron box containing 7695 water molecules. Energy minimization was carried out by steepest descent method. Replica Exchange Molecular Dynamics (REMD). REMD can accelerate the conformational sampling by allowing exchange of configurations between multiple replicas being simulated at a wide range of temperatures in parallel.66 While the canonical Boltzmann distribution is preserved at individual replica, the exchange of configurations allow crossing of energy barriers as they travel from lower temperature replicas to the higher temperatures. We have used 93 replicas in the temperature range of 300–600 K with an optimal distribution of temperatures to achieve an average exchange rate of ~20%.67 Exchanges have been attempted at every 2 ps. Initially, NVT equilibration runs were performed using V-rescale thermostat68 for ~63 ns at each temperature within the range of 300 – 600K so as to ensure that the properties of individual replica have equilibrated. Afterwards, the NVT production run has been continued till 700 ns for each replica amounting to a cumulative >65 µs of run length. In all the simulations, particle mesh Ewald method has been used to describe the long range electrostatic interactions with 0.16 nm grid spacing.69 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. Number of contacts and native contacts. In case of intramolecular contact maps, an intramolecular contact between two residues is said to be present if the minimum distance between them is i+3) rij ≤ 0.8nm and if it also exists in the reference NMR structure of the protein.


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RESULTS AND DISCUSSION

Temperature dependence of structural order parameters. As a first step towards characterizing the underlying conformational energy landscape of the Prion protein, we have studied the temperature dependence of a wide range of structural parameters as obtained from individual replica in the range of 300-600K. Fig. 1 summarizes the temperature dependence of the probability distribution of the following quantities: (a) Cα-RMSD, (b) Number of native contacts, (c) Radius of gyration (Rg) and (d) Solvent Accessible Surface Area (SASA). We have shown the convergence of these properties along the REMD trajectories in Fig. S1. As expected, due to thermal denaturation, the protein structure increasingly deviates from the native state as the temperature rises. The shift in probability distribution of Cα-RMSD towards higher values (Fig. 1a and S1a), and towards lower values in case of native contacts (~150 to ~20: Fig. 1b and S1b) signifies the loss of native structure. The distributions have a distinct bimodal nature with two major native and non-native states, where the population gradually shifts to the non-native state at higher temperatures. The temperature dependence of the structural order parameters has characteristics of a first order phase transition, and the transition occurs in the range of 380-400K (see Fig. S2 for the melting curve). Interestingly, both the distributions of RMSD and number of native contacts demonstrate appearance of an intermediate state around RMSD ~0.8-0.9 and number of native contacts ~50, which is particularly populated around the transition temperature range of 380-420K. At the transition temperature (380K) the system goes back and forth between the two extreme folded and unfolded states (Fig. S1b), while visiting through the multiple intermediate states, which will be explored through the underlying free energy landscape in subsequent sections.


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Figure 1. Probability distributions of structural parameters depicting temperature dependence: (a) Cα RMSD, (b) Number of native contacts, (c) Radius of gyration and (d) SASA. All the structural parameters have been calculated only for the CTD of PrPC. On the other hand, the radius of gyration (indicator of effective size) remains relatively unchanged (Figs. 1c and S1c, respectively) or rather slightly decreases with an increase in temperature indicating that the protein assumes a more compact molten globule structure upon the initial melting of the native structure. While native contacts break on raising the temperature, new non-native contacts begin to appear leading to stabilization of the non-native metastable states as will be demonstrated later. The transition from a compact molten globule-like state to a fully unfolded state (with higher radius of gyration) occurs at much higher temperatures (Fig. S3). Moreover, this structural re-arrangement is characterized by a moderate increase in the 9 Environment ACS Paragon Plus

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SASA (beyond 380K) signifying a possible exposure of the hydrophobic core (Fig. 1d and S1d) as the structure moves towards a molten globule or unfolded state. We can see that there is a tail developing at 380K but a major change occurs only beyond that temperature. Interestingly, the average β-sheet content increases with temperature as well indicating that such states appear much higher in the conformational free energy landscape.

Free Energy Surfaces (FES): Conformational heterogeneity and role of non-native interactions. Energy landscape of a protein is highly multi-dimensional. Thus, it is notoriously difficult to quantify the conformational states and the (mis)folding pathways in terms of a limited set of reaction coordinates/collective variables. In this work, we have used a combination of multiple reaction coordinates to capture the lower dimensional free energy landscapes for the structured CTD, namely (i) Cα RMSD with respect to the PrPC form (NMR structure), (ii) number of native contacts, (iii) number of intramolecular hydrogen bonds, (iv) number of residues forming β-sheet/bridge and (v) number of residues forming α-helix. These reaction coordinates characterize the extent of deviation from the PrPC form (CTD only), or secondary structure content. All the FESs have been computed by the Boltzmann inversion of the corresponding two-dimensional (2D) probability distribution of the reaction coordinates.


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Figure 2. (a) FES involving the number of native contacts versus the Cα RMSD at (a) 300K, (b) 380K and (c) 450K: Key minima (basins) in the FES have been marked and overlaid with conformational clusters. Representative conformations for each basin have also been shown.


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In Fig. 2 we compare the conformational free energy landscape with respect to: (i) the number of native contacts and (ii) Cα RMSD at three different temperatures: 300K, 380K and 450K in order to highlight the temperature dependent crossover in the stability of various intermediate conformational states. The positions of the six major basins (1-6) have been marked on the FES and the representative structures corresponding to each basin have been separately shown in Fig. 3. The FES at 300K (Fig. 2a) is closest to the physiological temperature. The native PrPC form belongs very close to the Basin 1, which is characterized by higher native contact and lower RMSD. Evidently, the folded structure of the PrPC form is not at the bottom of a very deep folding funnel as usually found in highly stable globular proteins, rather it belongs to a shallow free energy basin associated with large scale conformational fluctuations towards the Basin 2. The transition between Basins 1 and 2 involves movement of H1 as will be clarified later and Basin 2 seems to have significant population even at the physiological conditions. At very high temperature (450K), the protein is significantly denatured as expected (Fig. 2c) with very high RMSD and low number of native contacts. Thus, the Basin 1 populated at 300K and Basin 6 populated at 450K would constitute the two extremes that would define the intermediate conformational space we need to explore. The FES (Fig. 2b) at the transition temperature (380K) is characterized by presence of all the intermediate conformational states spanning the range between the two extreme stable states (Basins 1 and 6 in Fig.2b) as discussed before. We find that the first two states observed at 380K (Basins 1 and 2) are similar to the first two states of 300K and the last two states at 380K (Basins 5 and 6) are similar to last state of 450K, thus further emphasizing that the study of FES at 380K would be enough to understand the conformations explored by PrPC while undergoing thermal denaturation. Superimposed structures for conformations at these states for different


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temperatures have been shown in Fig. S4. In order to analyze the conformations belonging to each basin, we have performed clustering using the gromos algorithm.70 The cluster members considered for each basin have also been marked (black dots) on the FES at all the three temperatures in Fig. 2. We find that there exist multiple (at least 4) well-defined intermediate metastable conformational states separated by rather small barrier (10%) common (native) and uncommon (non-native) with Basin 1

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Basin 1 (native state): Basin 1 as mentioned before corresponds to a native-like structure and has all the native contacts between the secondary structure domains intact for both 300K and 380K. For example, hydrogen bonds between (i) TYR128 and ASP178 which is known to hold the β-sheet together (78%)43 and (ii) TYR149 and ASP202 which ensures that H1 is attached to the rest of the domain by interaction with H3 (71%).57, 71 We have only highlighted hydrogen bonds which show minimum occupancy of 70% for each of the basin cluster.

Outward

movement of H1 is known to be accompanied with breaking of the afore-mentioned hydrogen bonds. The helical stability of H2 is attributed to hydrogen bonding between residues THR183:TYR162 and THR183:CYS179 (81%).51 We can see these bonds being formed in the structures belonging to Basin 1. Further, the helices are seen to be largely maintained with H1 slightly melted around C-terminal residues while H2 remains stable for residues 172-189 and H3 for residues 199-224. Other than these typical hydrogen bonds, additional H-bonding can be seen between residues of C-terminal of H3 and N-terminal domain (NTD) i.e. GLU219:ASN100


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(81%) and LYS101 (68%) and GLU221:GLY119 (77%) and VAL122 (68%) (Fig. 6a). The contact map (Fig. 5a) thus corresponds to a native like state with NTD frequently interacting with the C-terminal end of PrPC.

Figure 6. Representative Structures shown with residue pairs having hydrogen bond occupancy of more than 70% shown for each basin: (a) Basin 1, (b) Basin 2, (c) Basin 3, (d) Basin 4, (e) Basin 5 and (f) Basin 6. Basin 1 to 2: Transiting from Basin 1 to Basin 2, we can see higher melting of helices with H2 formed between residues 175-186 and H3 between residues 202-226, while H1 has largely unfolded (Table 1 and Figs. 3b,5b). The melting of H1 can be related to non-native like Hbonding between residues SER143(side-chain):ASP147 (71%), HIS140:TYR149 (77%) and ILE138:ARG151 (68%). The H-bond between TYR149 and ASP202 which is known to hold H1 within the CTD of prion is seen to have broken, such that these residues now form hydrogen bonds locally (ASP202:MET205-77% and HIS140:TYR149-77%). We also show that H-bond


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between TYR128:ASP178 (70%) and TYR162, CYS179:THR183 (73% and 74%) are still maintained. Instead of interaction with GLY119, GLU221 is now interacting with ALA118 and a few newer H-bond formations can be seen between H3 and NTD such as GLU211:TRP99 (72%) and GLN212:ILE139 (68%) (Fig. 6b). PrPC can be divided into two structural domains i.e. B1–H1–B2 domain (residues 132– 167) and the H2–H3 bundle (residues 174–230). Using hydrogen/deuterium exchange and disulfide linkage, it is seen that separation of these two domains precedes oligomerization72 and the same observation can be made in this work as well, wherein breaking of hydrogen bond between TYR149 and ASP202 is one signature of this separation. It has been demonstrated earlier that despite having rather short length H1 has unusually high helical propensity due to salt-bridge formations between the i-th and (i+4)-th residues, whereas H2 and H3 serve as the initiation sites due their intrinsic instability.24,

45

On the other hand, NMR and metadynamics studies by

Camilloni et al indicate that the helical conformation of H1 may be destabilized by large conformational diversity when detached from the H2-H3 domain, and the random coil version of H1 can also be stabilized by salt-bridge formations in this state.44 Tau et al have also shown that one of their beta-sheet rich intermediate has H1 rotating away from core with H1 and N-terminus of H2 melting subsequently.73 We have tried to resolve this conflicting view by showing that the conformations in Basin 2 have unfolded H1 where intra-segment helical contacts are compensated through inter-segment contacts with NTD (Fig. 6b). Moreover, at 300K we see that while H1 is largely folded, H2 and H3 start unfolding at the terminal regions (Fig. S4a and S4b). Thus, both pathways seem to have distinct population in our simulations depending on the temperature. We must point out here that the transition from Basin 1 to 2 is not associated with major decrease in the number of native contacts, thus melting/movement of H1 should not be


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considered the key step (rate determining step) towards misfolding, which would correspond to the subsequent transition from Basin 2 to 3. Basin 2 to Basin 3: Basin 3 features conformations with highly unfolded H2, while H1 is reformed and H3 is partly maintained between residues 143-152 and 203-216 respectively (Fig. 3c). This further supports the earlier observation where “peeling of” of H1 leads to higher destabilization and hydrophobic core exposure in the successive misfolded conformations. We can also see a gradual decrease in interaction of H2 with the end residues of C-terminal of CTD as we move away from Basin 1, which completely disappears in Basin 3 (Figs. 3a-c and 5a-c). The stabilizing native contacts are broken with novel interactions being formed between CYS179:ARG164 (69%) and VAL161:TYR149 (72%), TYR149 is also forming i-i+4 helical hydrogen bond with TYR145. Interactions between H1 (and the loop following it) and H2 are dominant in this basin with involvement of two important residues/regions i.e. HIS187 and VTTTT stem region. His187 is seen to hydrogen bond with ASN181 (88%), VAL189 with ASN159 (69%) and THR191/192 with ASN153/GLU152 (80%) residues. The interactions between H3 and NTD in this conformation are present as hydrogen bonding between ALA117 and TYR226 (69%) (Fig. 6c). Basin 4: Basin 4 shows a remarkably β-sheet rich structure (average value of 31%) with only a small part of H3 (202-213) hydrogen bonded to a few residues (THR183 and ASN181) which originally belonged to H2. There are mainly two sets of beta sheet formations which involve residues 109-112, 129-132, 148-149, 160-163 and 213-215 and residues 124-125, 226-229 and 192-193. These β-sheets are accentuated by backbone hydrogen bonding between HIS111:LEU130, ASP147:THR216 and TYR162:ILE215. Nearby side chain interactions between residues ALA113:TYR128 (86%), ALA116:TYR162 (79%) and ASN108:SER132


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(69%) further stabilize this beta-sheet fibril like structure. GLN91, SER103:GLU219 (72%) and MET109:GLU221 (100%) are a few side chain interactions that hold part of NTD with the end of CTD. The formation of these β-sheets can also be observed in contact map for Basin 4 (Fig. 3d and 5d) as high percentage of contacts can be seen for aforementioned regions. Basin 5 and 6 (unfolded): Basin 5 and 6 on the other hand is characterized by largely unfolded and disordered structures with intermittent beta-bridges between certain residues like ARG228:GLY123, ARG151:TRP99 and TYR162:GLY124 for Basin 5 and LEU125:THR216, TYR162:TYR149 and GLN160:ILE139 for Basin 6. Basin 5 is characterized by formation of many transient hydrogen bonds in the largely unfolded conformations present in this basin and thus the cutoff of hydrogen occupancy has been decreased to 40% in this case. Other random residue pairs with H-bond interactions have also been highlighted in the Figs. 5e-f and 6e-f. When comparing these states with the corresponding clusters at 450K, we observe some similar but random unfolded conformations which appear largely compact for both the temperatures (Figs. S4c-d).

Structural basis of the misfolding pathways and regions of instability. The observation that PrPC form belongs to a shallow free energy basin with comparable stability to the misfolded forms can be correlated with the earlier predictions that the 2nd and 3rd helices (H2 and H3) of mammalian prions have unusual pattern of hydrophobic residues with low helical propensity, which leads to the inherent instability of these regions.23, 24, 49 Thus these helices are possibly weakly stabilized by the tertiary interactions, e.g. buried hydrophobic core, salt bridges etc. The other factors stabilizing the misfolded states might be the preservation of number of hydrogen bonds (due to β-sheet formation) and number of intra-molecular contacts as demonstrated earlier,


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whereas there would be destabilization due to the increased solvent exposure of the non-polar residues. Combining these factors together we may conclude that the PrPC form is only weakly stable (possibly metastable), and thus rendering the mammal Prions rather prone to misfolding both thermodynamically and kinetically. Interestingly, the FES for number of residues forming α-helix vs β-sheet (Fig. 7) hints to the possibility that β-content can increase without a major change in the helical content, where the existing β-sheet may extend to the coil regions. This view agrees with the earlier suggestions that certain amount of native structure (e.g. the metastable helices) needs to melt first, followed by further reorganization and formation of the β-rich structures. We must point out here that the experimental clues regarding the secondary structure content of the PrPSc point to a 43% β-sheet and 30% α-helical content.14 Thus, it is not strictly necessary for the helical segments to fully transform to β-sheet. Based on the overlay of clusters on this FES (Fig. 7), we also observe two distinct states for Basin 5 structures: (i) β-sheet/bridge poor state (~10 residues forming βsheet/bridge) and (ii) β-sheet/bridge rich state (~35 residues forming β-sheet/bridge). Both show low amount of α-helical but higher β-sheet content. We must clarify that we are studying the monomeric precursors to the scrapie form here, and the oligomeric forms are likely to form intermonomer in-register beta sheets leading to the scrapie form. Thus, it is quite likely that the experimentally measured β-content of the scrapie form cannot be directly compared to the population observed in the monomeric state. But it is quite evident that the partially disordered molten globule like metastable states observed in our simulations have quite high propensity towards forming β-sheet, which should have strong propensity towards fibril formation upon oligomerization.


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Figure 7. FES involving the number of residues forming α-helix versus the number of residues forming β-sheet/bridge at 380K. Clusters from each basin in Fig. 2b have been superimposed on this FES. For Basin 5 two states can be seen: i) β-sheet poor state and ii) β-sheet rich state. Corresponding snapshots for each of these states have been marked. There exist a metastable region on this FES around helical content of 5-15% and beta-sheet content 20-30%. The relative stability and dynamic nature of the secondary domains in the prion have been explored in great detail, but a consensus view still eludes us. For example, Blinov et al have described that H3 appears to be the most stable domain, while the β-sheet domain and the adjacent parts tend to be dynamically unstable.74 On the other hand, prior theoretical predictions points to H2 and H3 to be the initiation sites for misfolding in Prions,23, 24 and experimentally it has been shown that selective stabilization of the C-terminal end of H2 increases the global stability.30 Conformational changes in NTD have also been predicted such that the epitopes present in this region are either altered/buried in PrPSc.33 One such epitope region is present


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between residues 94-104. Another antibody mapping study shows that residues 93-102 are exposed in PrPC but are buried upon conversion to the PrPSc isoform.32 In order to understand the region-wise stability/instability of various Prion fragments, we have dissected the conformational fluctuations, structural propensities, and formation/breakage of the key native contacts associated with the misfolding pathways. The residue-wise root mean square fluctuations (RMSF), and residue-wise probability of being in a helical or β-sheet/bridge state have been shown in Figs. 8a-c (from the replica at 300K and 380K). The positions of the helices (H1, H2 and H3) and β-sheet (B1 and B2) as found in the original NMR structure have been marked on the top of the figure as a visual guide. Interestingly, in addition to the NTD and unstructured coil regions the C-terminal regions of both H2 and H3 demonstrate higher fluctuations, reduced helical propensity and a slightly increased β-propensity for both the temperatures. Fig. 8b and 8c clearly depicts the tendency of the existing β-sheet to extend towards the H2 region. The NTD residues 110-120, coil region between B2-H2 and C-terminal region of H3 show particularly higher affinity towards β-sheet formation supporting the view that these regions determine the tendency of misfolding.30,

40, 56

It has been shown that the

residues 105-120 are necessary for conversion of PrPC to a β-sheet rich structure.75 β0(118-122)β1 hairpin is found to expose backbone hydrogen bond donor and acceptor sites to solvent and act as a structural nucleus for growth of intermolecular β-sheets.40 A linear epitope is also predicted to be present between residues 119-127 and since it is adjacent to a highly amyloidogenic sequence it is shown to play an important role in prion misfolding.34 Remarkably, in this study too, we find that the residues 113ALA to 122VAL in NTD show higher fluctuations and greater propensity towards β-sheet formation while interacting with the natively present βsheets, especially at 300K.


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Figure 8: (a) Root mean square fluctuations (RMSF) of the residues at 300K (black) and 380K (red), and (b) Probability of being in helical (black line) or beta (red line) conformation of the residues for the replica corresponding to (b) 300K and (c) 380K temperature. The horizontal bar


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on the top marks the secondary structures (B1, H1, B2, H2 and H3) present in the original NMR structure.

CONCLUSION

An exhaustive account of the structure, dynamics and thermodynamics involved in the misfolding of PrPc has been provided in this article. Using a cumulative ~65µs REMD data we have been able to extensively sample the complex conformational energy landscape of a mouse Prion protein which led to observation of multiple intermediate partially disordered misfolded states stabilized by non-native contacts and hydrogen bonds. We have shown that the PrPC form belongs to a shallow free energy basin and surrounded by many low-lying metastable conformational states with comparable stability and small barrier. The misfolded states are characterized by interaction of NTD with CTD, compact shape, comparable number of hydrogen bonds, higher β-sheet content and solvent exposed non-polar residues. The partially disordered structures (putative PrP* state) might be more prone to aggregation as compared to the PrPC form due to solvent exposed hydrophobic core and propensity towards β-sheet formation. We have dissected the structural basis and transitions between the various conformational states associated with the misfolding pathways in detail. The initial stages of misfolding seem to be associated with decrease in helical content (primarily in the C-terminal regions of H2 and H3) and extension of the existing β-sheet, and movement in the B1-H1-B2 domain. The transition between Basin 2 and 3 is associated with the largest decrease in native contacts and characterized by major melting of H2 and H3. Given the overall complexity of the underlying energy landscape and presence of multiple intermediate states, we envisage existence of multiple


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misfolding pathways, where the specific sequence of events might vary, but broad thermodynamic features remain the same as described. We see that the NTD (residues 113-122) plays an active role in adopting misfolded β-sheet rich structures by either intramolecular β-sheet interaction with natively present β-sheets or by interacting with CTD, especially the Helix-3 and C-terminal residues of CTD. The structural rearrangements mentioned in this article are associated with partial/local unfolding of the native Prion structure with formation of large number of non-native contacts. While we observe large population of beta-rich structures (>30%), the majority of the non-native structures have a disordered conformation stabilized by non-native interactions. As recently indicated by Mukhopadhyay and coworkers,47 the β-rich conformations are possibly the off-pathway intermediates, and the toxic entities might have more disordered conformations as also indicated by recent Metadynamics simulations.49 While our current study attempts to identify the monomeric precursors towards formation of the scrapie form, it would be interesting to study the oligomerisation pathways of these misfolded intermediate states in future.

ASSOCIATED CONTENT Supporting Information. Supporting analysis data including convergence of REMD simulations, melting curve, probability distribution of radius of gyration and structural superposition from same basin obtain from different temperatures. AUTHOR INFORMATION Corresponding Author *Email: [email protected]


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ACKNOWLEDGMENT We thank CSIR 4-PI supercomputing facility for the computational resources. FUNDING INFORMATION S.C. thanks the Department of Science and Technology (DST), India for the Ramanujan Fellowship (SR/S2/RJN-84/2012) and CSIR, India for funding from XIIth five year plan project on Multiscale Modelling (CSC0129).


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Biochemistry

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Temperature-Induced Misfolding in Prion Protein: Evidence of Multiple

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