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Disordered versus Fibril-like Amyloid β (25-35) Dimers in Water: Structure and Thermodynamics Madeleine Kittner and Volker Knecht* Department of Theory and Bio-Systems, Max-Planck-Institute of Colloids and Interfaces, 14424 Potsdam, Germany ReceiVed: July 14, 2010; ReVised Manuscript ReceiVed: September 13, 2010
Alzheimer’s disease is associated with the precipitation of the amyloid β (Aβ) (1-40) peptide in the form of fibrils. Among the full length peptide, smaller fragments such as Aβ (25-35) which retains the toxicity of the full length peptide are also present. Aβ’s toxicity is attributed to soluble oligomers which, however, are difficult to study experimentally due to their transient nature. Here we present replica exchange molecular dynamics simulations of Aβ (25-35) dimers in explicit water. Similar to a previous study, dimers are found to exist as disordered compact in equilibrium with ordered extended fibril-like conformations. In addition, our results suggest effects from slight differences in ionic conditions and yield insights on this system in unprecedented detail. In the compact state, the peptides adopt β-hairpin or unstructured U-shaped conformations with different relative orientations. In the extended state, the peptides are outstretched and form antiparallel in- or out-of-register intermolecular β-sheets. In addition to the previous study, we reveal the driving forces governing the equilibrium between the disordered and the fibril-like state. In particular, it is shown that the compact state is favored by a high entropy while the fibril-like state is lower in energy arising from favorable covalent and electrostatic interactions between and within the peptides. Our results suggest that the transition from the compact to the fibril-like state involves reptation, i.e., a change in register of an intermolecular β-sheet without dissociation of the peptides. Introduction Neurodegenerative diseases such as Alzheimer’s disease (AD) are associated with the aggregation of proteins into insoluble fibrils which have a specific cross β-structure and are referred to as amyloid fibrils.1 Compared to the mature fibrils, it is difficult to gain information about early aggregation intermediates using common experimental techniques. This is mainly caused by the rather disordered nature of the early intermediate states. On the other hand, oligomers are believed to be the main toxic components influencing cell viability. In order to design therapeutic agents against AD, it is therefore essential to study small oligomers in terms of conformational diversity and major interactions driving the transition from disordered oligomers to fibril-like structures. In the case of AD, amyloid fibrils are found in the brain tissue of patients. The primary component of the fibrils is the amyloid β-peptide (Aβ). This pathogenic fragment is derived by cleavage of the amyloid precursor protein (APP), composed of 40-42 amino acids, and can be toxic already in dimeric form.2 Besides the full length Aβ, also other fragments are present. Among them, the 11-residue fragment Aβ (25-35) is the shortest which forms β-sheet-rich fibrils and exhibits the toxicity of the full length peptide.3-5 The amino acid sequence of Aβ (25-35) is shown in the Supporting Information in Figure S1. Several experimental groups studied the nature of Aβ (25-35) aggregates in solution. For example, CD and FT-IR spectra of Aβ (25-35) solutions at pH 7 and temperatures ranging from 281 to 310 K measured immediately after mixing showed the occurrence of β-turn as well as, presumably antiparallel, β-sheet conformations. Further incubation of the solutions for several * To whom correspondence should be addressed. E-mail: vknecht@ mpikg.mpg.de.
hours yielded a decreased β-turn but increased β-sheet content.4,6-8 Obviously, the smallest detectable aggregates already contain a significant β-sheet structure but to a lesser extent than mature fibrils. H/D exchange NMR measurements revealed insights on the peptide arrangement within Aβ (25-35) fibrils.9 The determined protected core region (K28-M35) suggests either an out-of-register antiparallel or in-register parallel alignment of the peptides.9 Observing the preaggregated state of Aβ (25-35) in water experimentally is unfeasible as at concentrations necessary for analysis Aβ (25-35) is already aggregated. However, threedimensional structures of Aβ (25-35) in mixtures of water with hexafluoroisopropanol (HFIP) as a membrane-mimicking environment have been solved by NMR.10 In a 80:20 (v/v) HFIP/ water mixture, Aβ (25-35) adopts an R-helical conformation, while in 20:80 HFIP/water a type I β-turn centered on residues S26 and N27 is formed. Likewise, Wei and Shea performed all-atom replica exchange molecular dynamics (REMD) simulations in a 80:20 HFIP/water mixture yielding an R-helical conformation similar to experiments.5 Their MD simulations of Aβ (25-35) in pure water showed the formation of collapsed coil structures coexisting with two types of β-hairpin conformations, which were characterized as type II′ β-turn structures differing in the twist of the strands to one another. According to recent results of the same group, Aβ (25-35) dimers mainly adopt disordered conformations. The rarely populated ordered conformations contain partly extended peptides forming planar parallel or antiparallel β-sheets, and parallel β-sheets within V-shaped conformations.2 They also showed that protofibrils with peptides in these conformations remain stable for about 50 ns. It would be interesting to study the stability of these protofibrils depending on the peptide concentration. Similarly, short MD simulation by Ma and Nussinov showed that
10.1021/jp1065264 2010 American Chemical Society Published on Web 10/22/2010
Amyloid β (25-35) Dimers in Water protofibrils containing extended peptides in parallel in-register or antiparallel out-of-register alignments are stable over several ns.11 Starting from extended parallel or antiparallel β-sheet conformations, the stability of dimers and small oligomers was also studied using impulse-docking and short MD simulations.12 Here, the most stable dimer contained extended peptides forming an antiparallel β-sheet and salt bridges between the termini. We have studied Aβ (25-35) using a similar approach as Wei and Shea, reproducing the existence of disordered compact in equilibrium with ordered extended fibril-like conformations, suggesting effects of differences in ionic conditions, and yielding insights on this system in unprecedented detail. Compared to the study by Wei and Shea, we give a detailed structural analysis of the different conformational ensembles of Aβ (25-35) dimers. We also reveal the driving forces governing the equilibrium between the disordered and the fibril-like state. In particular, we find that the compact state is favored by a high entropy while the fibril-like state exhibits lower energy. Our results give evidence for the transition from the compact to the fibril-like state to involve reptation, i.e., a change in register of an (intermolecular) β-sheet without dissociation of the peptides. Methods Simulation Setup. The dimerization of Aβ (25-35) was studied in explicit solvent under periodic boundary conditions using REMD simulations. The amino acid sequence and initial configuration of the peptide are shown in Figure S1 in the Supporting Information. The protonation was chosen to mimic neutral pH. The main β-hairpin conformation of the monomeric state served as initial configuration of the peptides.5 To simulate spontaneous dimerization, two peptides in random mutual orientation separated by 1.1 nm were placed in an octahedral box. The dimensions of the box were chosen such that the minimum distance between the solute and the boundaries of the box was 1.2 nm for the initial configuration. To counterbalance the positive charge of the peptide, one chloride ion per peptide was added. The remaining space was filled by 4003 water molecules. To remove residual overlaps of atoms, the system was energy minimized using the steepest descent method. This was followed by a simulation of 1 ns at 293 K and 1 bar, where the atom positions of the peptide were kept close to their initial positions by harmonic potentials to equilibrate the solvent. In addition, the system was simulated for 1 ns without restraints at the same temperature and pressure providing the initial configuration of the REMD simulation. The temperature REMD method was introduced by Sugita and Okamoto and is described in detail elsewhere.13 In brief, in REMD simulations, copies (replica) of a system are simulated simultaneously but independently at different temperatures. In regular time intervals, pairs of replica at neighboring temperatures are exchanged according to a Metropolis criterion. This method accelerates crossing of energy barriers at high temperatures and thereby allows fast sampling of conformational space. In this work, 50 replicas of the system were simulated at constant volume and temperatures forming a geometric sequence between 290 and 374 K for 200 ns each. To avoid correlated back exchanges, swapping of replica between neighboring temperatures was attempted every 5 ps. The chosen exchange rate is large compared to the integrated autocorrelation time of the potential energy of the system at 290 K (0.61 ps).14,15 All simulations were carried out using the GROMACS 3.3 software package.16 The peptide was described using the GROMOS96-43a1 force field, which handles CHi (i ) 1, 2, 3)
J. Phys. Chem. B, Vol. 114, No. 46, 2010 15289 groups as united atoms.17 The water molecules were described using the three-site simple point charge (SPC) water model.18-20 Covalent bonds were constrained using the LINCS method21 for the peptide and the SETTLE algorithm22 for the water molecules. In addition, the masses of atoms attached to hydrogens were redistributed so as to increase the mass of the hydrogen atoms simulated explicitly. This eliminates highfrequency motions of the hydrogens which allows the use of a time step of 4 fs.23,24 Noncovalent interactions (i.e., electrostatic and van der Waals interactions) were treated using a twin range cutoff, distinguishing between short-range interactions for interatomic distances up to 1 nm and long-range interactions for distances between 1 and 1.4 nm. Short-range interactions were calculated every step while long-range interactions together with the neighbor list were updated every five steps. Electrostatic interactions were modified so as to include the mean reaction field of the region beyond the cutoff distance, modeled as a continuum with a dielectric constant of 54, the self-consistent value for SPC water.25,26 In all simulations, peptides and solvent were separately coupled to an external heat bath using a Berendsen thermostat with a relaxation time of 0.1 ps.27 In the case of constant pressure simulations, the pressure was coupled to 1 bar with a relaxation time of 1 ps using the Berendsen method.27 Snapshots of the system were saved every 20 ps and only data of the final 100 ns were used for analysis. Analysis Methods. From the equilibrated canonical ensemble the free energy along an order parameter x was determined from
∆F(x) ) -kBT ln
[ ] P(x) Pmin
(1)
Here, T ) 293 K denotes the temperature of interest, P(x) the probability of the system to be in state (x), and Pmin the minimum probability for which ∆F ) 0. The reaction coordinate chosen was the radius of gyration averaged over both peptides (Rg). Evident from Figure S2 (in the Supporting Information), Rg was equilibrated within the statistical error after the first 100 ns of the simulation. If shown, errors were obtained from block averages dividing the remaining trajectory into four segments. Dimer configurations were analyzed based on the root-meansquare deviation (rmsd) in structure between backbone atoms of residues N27-G33 of both peptides. Using the method by Daura et al.,28 two configurations with an rmsd below 0.15 nm were considered to belong to the same cluster representing a “conformation”. The determination of the rmsd cutoff is shown in Figure S3 in the Supporting Information. The secondary structure was calculated based on an analysis of hydrogen bonds within the main chain of the peptides using the DSSP program.29 The secondary structure content of the individual residues was obtained as an average over both peptides and over all configurations within a certain Rg range. Contact maps based on main-chain hydrogen bonds were calculated as described in detail elsewhere.30 In short, two residues i and j were considered to be in contact if at least one out of two possible main-chain hydrogen bonds was formed. The contacts of all pairs of residues are illustrated in a twodimensional matrix H(i,j). If a contact is formed, H(i,j) equals 1, otherwise it is zero. Averaging H(i,j) over an ensemble of configurations yields the contact frequency P(i,j), denoted as contact map. The occurrence of specified main-chain hydrogen bonds or side-chain interactions was calculated along the radius of gyration, NX(Rg). Here, data were obtained in 0.02 nm intervals of Rg and averaged over the configurations within such an
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Figure 1. Central configurations of the nine largest out of 442 clusters denoted by A to I with populations given in parentheses. Together, these clusters contain 25% of all configurations. The colors distinguish between polar (blue), positively charged (red), and apolar (yellow) residues. The number in the upper left of each box refers to the Rg region in which the conformations were found.
interval. A side-chain contact was considered if the minimum distance between the center of mass of two side chains was smaller than 0.4 nm. Results Conformational States of Aβ (25-35) Dimers in Water at 293 K. Studies of the monomeric Aβ (25-35) in water by Wei and Shea and in our lab (see Supporting Information for setup) reveal an equilibrium between collapsed coil conformations and two β-hairpin conformations, differing in the twist of the β-strands.5 Both β-hairpin conformations are characterized by a type II′ β-turn involving residues G29 and A30, and two short antiparallel β-strands consisting of residues N27-K28 and I31-I32. These β-strands are stabilized by three interstrand main-chain hydrogen bonds: I31:HN-K28:O, K28:HN-I31:O and G33:HN-S26:O (see Figure S1 in the Supporting Information). In this work, we study the conformational states of Aβ (25-35) dimers in explicit water at 293 K starting from the main β-hairpin conformation. We performed a cluster analysis based on the backbone atoms of residues N27-G33 using an rmsd cutoff of 0.15 nm which resulted in 442 poorly populated clusters. The central structures of the nine most populated clusters (A-I) together with their populations are shown in Figure 1. Eight out of nine clusters show rather ordered conformations in terms of β-sheet content. In four of these ordered conformations, corresponding to 13% of all configurations, the individual peptides are partly or fully extended forming antiparallel intermolecular β-sheets. In three clusters, corresponding to 7% of all configurations, one or both of the peptides adopt a β-hairpin-like conformation. In the ordered cluster (F) the dimer contains a parallel intermolecular β-sheet. Similar results were obtained by Wei et al., although their dimers seem to be more disordered which was presumably due to the higher temperature of their system. We chose 293 K for our analysis to mimic the conditions of previous experiments on this peptide. Subensembles of Aβ (25-35) Dimers Based on the Radius of Gyration. On the basis of the observation of compact and extended conformations, we chose the radius of gyration (Rg) as a measure for the linear extension of the peptides as an order parameter to visualize the heterogeneous free energy landscape. In Figure 2 the free energy along Rg is plotted showing two local free energy minima located at Rg ≈ 0.6 nm and Rg ≈ 0.9 nm. The minimum for the smaller radius of gyration is roughly
Kittner and Knecht 3 kJ/mol below that for the larger radius. Both minima are separated by a free energy barrier of 6 kJ/mol at Rg ≈ 0.75 nm. Additionally, the minimum for the smaller radius of gyration is separated in two parts by an energy barrier of roughly 1 kJ/ mol. According to the free energy, five Rg regions can be distinguished; Figure 2 also shows the appearance of the nine main conformations A-I within these Rg regions. First of all, dimers in the free energy minimum at Rg ≈ 0.6 nm adopt rather compact structures consisting of two types. In dimer conformations D, G, H in Rg region I both or one of the peptides are in a β-hairpin-like conformation similar to the monomeric state at 293 K (see Figure S1, Supporting Information). On the other hand, dimer conformations within Rg region III are unstructured (C) or contain a short parallel intermolecular β-sheet (F). In conformations found in region V (A, B, E, I), the peptides are outstretched forming an antiparallel β-sheet. The individual extended conformations differ in the length of the formed β-sheet and the bending of the peptides. Interestingly, the dominant conformations A and B are found to be extended “fibril-like” dimers, although compact conformations are more populated in total; see Figure 2. Secondary Structure and Hydrogen Bond Network within the Subensembles. To investigate the defined subensembles of dimers in more detail, we analyzed secondary structure motifs and intermolecular main-chain hydrogen bonds for the individual Rg regions. Figure 3 shows the secondary structure content of the individual residues averaged over all configurations within a certain Rg region. Plotted are (A) the turn, and (B) the intramolecular and (C) the intermolecular β-sheet content per residue averaged over all configurations within a certain Rg region. For comparison, Figure 3A,B includes data of Aβ (25-35) monomers. Figure 4 shows the corresponding hydrogen bond maps. Plotted is the probability of finding at least one hydrogen bond between residues of peptide A and peptide B. A high probability stands for a particularly stable hydrogen bond. As known from previous work, Aβ (25-35) monomers adopt a β-hairpin conformation.5 Shown by the dotted line in Figure 3A, the turn of the β-hairpin is located at the hydrophobic residues G29 and A30, termed in the following as the turn residues. The intramolecular, antiparallel β-sheet of the β-hairpin is formed between the residues N27-K28 and I31-I32 as shown in Figure 3B. This β-hairpin motif is found for approximately 40% of the monomer configurations which corresponds well to the population of the main β-hairpin conformation found for monomers; see Figure S1 in the Supporting Information. A similar turn and intramolecular β-sheet content was found in dimer configurations within region I. Approximately 30% of the peptides within such dimers adopt a β-hairpin-like configuration as exemplified by conformations D and G in Figure 1. With increasing Rg the turn and intramolecular β-sheet content decreases, indicating a gradual dissolution of the β-hairpin motif. This is illustrated by the conformations C and F shown in Figure 1. None of the extended configurations in region V forms intramolecular β-sheets, and only an insignificant amount of turn content is observed, though turn conformations are more abundant than the specific intramolecular β-sheets. As plotted in Figure 3C, with the loss of the β-hairpin motif the intermolecular β-sheet content for the inner residues increases. Only a few compact dimers in region I form intermolecular β-sheets, preferentially involving the hydrophobic residues I32 and G33. As shown by the hydrogen bond map in Figure 4A, 20-30% of the dimers in this region form a main-
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Figure 2. Free energy profile along the radius of gyration, Rg, and the definition of five Rg regions which are populated by (I) 25%, (II) 8%, (III) 33%, (IV) 10%, and (V) 24% of all configurations. The capital letters A-I refer to the main conformations given in Figure 1 and show their position along the Rg axis. Also shown are representative conformations (a-e) illustrating the transition between compact and extended dimer conformations and their position along the Rg axis.
Figure 4. Intermolecular main-chain hydrogen bond maps of Rg regions (A) I, (B) III, (C) IV, and (D) V, defined in Figure 2. Hydrogen bonds characterizing antiparallel in-register alignment of peptides within extended dimers (D) are marked in red.
Figure 3. Secondary structure content of individual residues averaged over both peptides within a dimer for Rg regions I-V defined in Figure 2 and the monomeric state distinguishing between (A) turn, (B) intramolecular β-sheet, and (C) intermolecular β-sheet content.
chain hydrogen bond between residues I32 of both peptides. In region II only roughly 10% of the configurations show intermolecular β-sheets but here involving also the hydrophilic residues N27-K28. For compact dimers in region III, the intermolecular β-sheet content of the inner residues increases to 15-20%, except for residues G29 and A30 corresponding to the stable β-turn. According to the hydrogen bond map shown in Figure 4B, dimers in this region form main-chain hydrogen bonds rather between N- and C-terminal residues like S26-M35 and N27-M35. At the transition region IV where the β-hairpin
motif is almost lost, the intermolecular β-sheet content increases to 30-50% for the inner residues N27-G33. In approximately 20% of the configurations, peptides are already aligned in an antiparallel manner as indicated by the diagonal contact pattern spanning from the top left to the bottom right of the hydrogen bond map in Figure 4C. The importance of the turn residues G29 and A30 in the course of the transition to extended conformations will be discussed later. In extended dimers (region V), intermolecular β-sheets between the inner residues are formed in 60-90% of the configurations, like in conformations A, B, E, and I illustrated in Figure 1. The hydrogen bond map of extended dimers in Figure 4D indicates the main-chain hydrogen bonds stabilizing the antiparallel β-sheet involving the inner residues K28-G33 that correspond well to the protected core region within Aβ (25-35) fibrils discovered by H/D exchange NMR measurements.9 The formation of in- and out-of-register antiparallel β-sheets is possible, the former being marked in red in the diagram. Most of the revealed hydrogen bonds appear in 40-60% of the extended dimer configurations. Comparing the probability of main-chain hydrogen bonds for the different types of dimers, the ensemble of extended dimers
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Figure 6. Inter-main-chain hydrogen bond patterns of dimer conformations c, d, and e in the transition region shown in Figure 2. Dominant hydrogen bonds are drawn by dotted lines. Color coding of the amino acids as explained in Figure 1.
Figure 5. Probability of selected intermolecular main-chain hydrogen bonds (HB) or side-chain contacts (SC) as a function of the radius of gyration. Plotted are significant contacts at (A) Rg ≈ 0.7 nm, and (B) Rg > 0.7 nm.
seems to be more ordered than the ensembles of compact configurations. Intermolecular Interactions Possibly Promoting the Transition from Disordered, Compact to Fibril-like, Extended Dimers. According to the above-described analysis, the ensemble of Aβ (25-35) dimers at 293 K can be divided in two main subensembles containing either compact or extended conformations. The extended conformations show the highest intermolecular β-sheet content and might serve as precursors for protofibril formation. In order to determine intermolecular interactions promoting the transition from the ensemble of disordered, compact conformations to the ensemble of extended, fibril-like dimers, we studied the configurations within Rg region IV as shown in Figure 2 in more detail. First, configurations corresponding to a certain Rg interval of 0.02 nm within Rg region IV were clustered as described in analysis methods. Representative conformations and their position along the Rg axis are plotted in Figure 2. Starting at the small Rg end of the transition region, dimers form parallel or antiparallel β-sheets between N-and C-terminal residues as illustrated by conformations a, b, and c shown in Figure 2. With increasing Rg the antiparallel alignment of the peptides is preferred while the residues involved in the β-sheets slightly change between conformations c, d, and e. The most significant intermolecular main-chain hydrogen bonds and side-chain contacts were calculated as described in analysis methods. Figure 5 shows the probability of such an intermolecular contact along Rg. According to Figure 5A, intermolecular main-chain hydrogen bonds between residues K28-I32 and A30-A30 are present in 10-30% of the configurations at Rg ≈ 0.7 nm. About 30% of the dimers in this Rg region are also stabilized by a hydrophobic side-chain contact between residues I31-I31. The importance of I31 for the stability and toxicity of Aβ (25-35) fibrils is also indicated from experimental results.4 For radii of gyration between 0.7 and 0.8 nm, intermolecular main-chain hydrogen bonds between residues N27-I31 and both turn residues G29-G29 are the most prominent interactions as shown in Figure 5B. With further
increasing Rg, the most stable main-chain hydrogen bonds are formed between residues G29-G33, I31-I31, K28- I32, and A30-A30. The observed intermolecular contacts are present in the prominent conformations shown in Figure 2. In this transition region, the formation of intermolecular main-chain hydrogen bonds between the turn residues A30-A30 and G29-G29 seems to support the antiparallel alignment of the peptides as found in extended conformations. Additionally, we also observe a shift of the intermolecular hydrogen bond network, which is drawn in a simple sequence pattern shown in Figure 6. Starting with conformation c, where both peptides are in an in-register antiparallel alignment, we find the prominent main-chain hydrogen bonds as shown in Figure 5A. Conformation d shows a slight out-of-register shift stabilized by main-chain hydrogen bonds between residues G29-G29 and N27-I31. With increasing Rg as in conformation e the β-sheet shifts again but in the opposite direction, now forming three hydrogen bonds between the hydrophobic residues G29-G33 and I31-I31 as illustrated in Figures 5 and 6. Note that Rg denotes the average over the radii of gyration of the individual peptides. The change in register is observed for transitions of Rg from 0.7 to 0.8 nm. Dissociated states also present in our simulations correspond to significantly smaller values of Rg (
