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Amyloid-β42 Oligomer Structures from Fibrils: A Systematic Molecular Dynamics Study Anselm H. C. Horn and Heinrich Sticht* Bioinformatik, Institut fu¨r Biochemie, Emil-Fischer-Zentrum, Friedrich-Alexander-UniVersita¨t Erlangen-Nu¨rnberg, Fahrstraβe 17, Erlangen, Germany ReceiVed: June 8, 2009
Recent experimental data demonstrate that small, soluble amyloid-β42 oligomers play an important role in Alzheimer’s disease because they exhibit neurotoxic properties and also act as seed for fibril growth. We performed all-atom molecular dynamics simulations in explicit solvent of 0.7 µs in total on five Aβ9-42 oligomers (monomer through pentamer) starting from the fibril conformation. The initial conformation proves to be stable in the trimer to pentamer, and the two parallel in-register β-sheets as well as the connecting turn are preserved. The dimer undergoes larger conformational changes in its C-terminus, and the predominant conformation detected exhibits an additional antiparallel β-sheet in one of the subunits. This conformational rearrangement allows efficient shielding of hydrophobic residues from the solvent, which is not possible for a dimer in the fibril conformation. In addition to the presence of the hydrogen bonds in the β-sheets, the larger oligomers are stabilized by interchain D23-K28 salt bridges, whereas a D23-N27 interaction is found in the dimer. The degree of structural similarity to the fibril conformation detected for the oligomers in the simulation may also offer a structural explanation for the experimental finding that trimers and tetramers act as more potent seeds in fibril formation than dimers because only small conformational changes will be required for fibril growth. The fact that the dimer predominantly exists in conformations distinct from the larger oligomers and the fibril is also interesting for the design of anti-Alzheimer drugs, because it suggests that multiple drugs might be required to target the structurally different neurotoxic oligomers. Introduction 1
Since its first description in 1907, Alzheimer’s disease (AD) has evolved as a major threat to public health in modern society. Despite the research efforts of the past decade, the nature of AD is not yet fully understood, which would be crucial for the development of a viable medication. On a molecular level, AD together with Parkinson’s and Huntington’s diseases belongs to a group of neuropathological disorders in which conformational changes of an otherwise soluble peptide lead to a refolded compound that in turn aggregates into oligomers, filaments, and fibrils accumulating in plaques in the brain of the patient.2 Important insight into the composition of the aggregates came from the experimentally determined structures of Aβ403,4 and Aβ425 in the fibril state. With respect to the monomeric subunits, both structures consistently reveal two β-strands connected by a turn, thus forming a U-shaped topology4-6 (Figure 1a). The monomeric subunits form a longitudinal stack, thereby creating two parallel in-register β-sheets (Figure 1b). Amyloid growing can proceed longitudinally (i.e., the adsorption of new monomers along the fibril axis) and laterally (i.e., the interactions with another stack of monomers at the lateral surface of the growing aggregate). Although several alternative modes of lateral association appear feasible,7,8 the hydrophobic side chains of the residues in the C-terminal strand evidently play a key role for quaternary interactions (Figure 1c).4,7,9 The deposition of extracellular fibrillar plaques was originally assumed to be the disease-initiating event,10,11 but today, increasing evidence suggests that small, soluble oligomers are the direct effectors of synaptic and cognitive dysfunction.12,13 A recent experimental study revealed that small Aβ oligomers, * Corresponding author. Phone: +49/9131-8524614. Fax: +49/91318522485. E-mail:
[email protected].
especially dimers, impair synaptic plasticity and memory, whereas no such effect was found for insoluble plaque cores unless they were solubilized to release small oligomers.14 In addition to their role as neurotoxic species, oligomers also act as seeding nuclei for the growth of novel fibrils.15,16 Their aggregation tendency renders experimental studies of small Aβ oligomers inherently difficult, but there exist several computational investigations that focused mainly on the initial steps of Aβ aggregation.17-26 Recent simulations have also analyzed the mechanism of Aβ monomer deposition on fibril fragments27-29 or the early steps of fibril disassembly.30-32 Generally, all studies have revealed that at least the monomer and dimer exhibit a rather large conformational flexibility. Therefore, a complete exploration of the conformational space sampled during the aggregation process of fibrils appears not yet feasible even by using modern simulation techniques such as replica exchange molecular dynamics (REMD) or implicit solvent models. Consequently, most computational studies focus on particular aspects of Aβ aggregation. One topic, which is not yet fully understood, is the structural similarities and differences between Aβ oligomers and the mature fibril. In light of the experimental observation that oligomers both play a role in initiating fibril growth and exhibit neurotoxic properties, it would be helpful to know whether the Aβ conformation detected in the mature fibril also represents a populated energy minimum for small oligomers or whether this topology is stable only in larger assemblies. The aim of the present study was therefore to address the conformational stability of small oligomers starting from the fibril-bound conformation. For that purpose, all-atom MD simulations of at least 100 ns in explicit solvent were performed at physiological temperatures for the Aβ monomer through pentamer.
10.1021/jp100023q 2010 American Chemical Society Published on Web 01/28/2010
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Figure 1. Topology of the Aβ9-42 peptide chain within the fibril structure. (a) Single Aβ layer: The peptide chain adopts a U-shaped conformation, which is mainly stabilized by hydrophobic interactions between residues L17, F19, and A21 of the N-terminal β-strand and residues V36, G38, and V40 of the C-terminal β-strand. In addition, a salt bridge is formed between D23 and K28. Residues are colored according to their type; Ac denotes the N-terminal acetyl blocking group. (b) Pentameric stack of Aβ monomers representing the fibril conformations. The Aβ layers are arranged to form an intermolecular parallel β-sheet. Color coding is as follows: chain A, blue; chain B, red; chain C, yellow; chain D, silver; chain E, gray. Residues L17, F19, and A21, which constitute the central hydrophobic core (CHC) are indicated as yellow sticks. (c) Mode of lateral association via the hydrophobic side chains of the C-terminal β-sheet. Lateral association is required to constitute the mature fibril.
Materials and Methods All simulations are based on the Aβ17-42 protofibril structure (pdb code 2BEG, model 10) that was obtained from NMR spectroscopic data.5 Using Sybyl 7.3,33 residues 9-16 were added to the model in an extended conformation to account for the secondary structure of Aβ1-42 in the fibrillar form.34 Residues 1-8 were excluded from the model, since these residues are dispensable for fibril growth35 and have only marginal effects on the structure and dynamics of the remaining residues.36 An acetyl group was added to the N-terminus to account for the lack of the eight N-terminal residues, and the C-terminus
was kept ionic. The final peptide sequence, thus, was AcGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA, bearing a net charge of -2. The initial structures of the tetramer, trimer, dimer, and monomer were created by deleting the corresponding number of peptide chains from the pentamer, starting from chain E. All five oligomer structures were electrically neutralized by the addition of an appropriate number of sodium counterions, solvated in a TIP3P37 water box with a minimum distance of 15 Å to the border and then subjected to a two-step restrained minimization. Because large conformational changes were expected to occur during the MD simulations, integer instead of fractional multiples of the standard Amber TIP3P water box were used, resulting in large solvent boxes (Table 1). All systems were then minimized and warmed up to 300 K at a pressure of 1 bar in two stages: for 0.1 ns, the peptide heavy atoms and for another 0.4 ns, the peptide CR atoms were held fixed with a force constant of 5.0 kcal mol-1 Å-2. After the density of the systems was close to 1.0 g cm-3, the simulations were run without any restraints for 104.5 ns (O1, O2, O3, O4, O5) in an NPT ensemble, and coordinate snapshots were collected every 50 ps. Because the dimer system showed larger conformation changes, its simulation was extended by another 100 ns (O2+). To obtain an enhanced conformational sampling for the trimer system, an additional trajectory (O3*) was run at 310 K using the same simulation protocol. All simulations were performed under the particle mesh Ewald approximation,38 applying default values for nonbonded cutoffs and using the parm99SB parameter set,39 which includes an updated backbone torsions library.40 Hydrogen atoms were constrained using SHAKE.41 We chose a time step of 1 fs because this has proven suitable in previous studies.42,43 As noted previously,7,21 Aβ oligomers exhibit an enhanced conformational flexibility as compared to globular proteins. To identify the most populated Aβ conformations sampled, hierarchical clustering44 of all snapshots from the trajectories was performed using the ptraj program of Amber10.45 Analysis of solvent accessible surface area (SASA) and hydrogen bonds was performed using the DSSP program.46 For the calculation of the relative SASA, maximum SASA values for all amino acids were taken from the work of Glyakina et al.47 Salt bridges were monitored by measuring the distance between the carboxylic carbon and the ammonium nitrogen using a cutoff of 4.2 Å.48 Structures and trajectories were visualized using the VMD49 program. The molecular dynamics simulations were performed on the Woodcrest cluster of the Regionales Rechenzentrum Erlangen (RRZE) using Amber950 molecular dynamics executables optimized for parallel execution. Results and Discussion Structural Properties of Pentamer, Tetramer and Trimer. Visual inspection of the representative structures reveals that the trimer, tetramer, and pentamer largely retain the stack of antiparallel β-sheets and the U-shaped topology that is also observed in the fibril (Figure 2). In the simulations, the
TABLE 1: Calculation Setup of the Different Oligomer Simulations system
charge
water molecules
O1 (monomer) O2 (dimer) O3/O3* (trimer) O4 (tetramer) O5 (pentamer)
–2 –4 –6 –8 –10
45 468 44 823 44 082 58 923 58 269
bounding box dimensions (x, y, z; Å) 116.0 116.0 116.0 116.0 116.0
78.1 78.1 78.1 78.1 78.1
59.8 59.8 59.8 78.6 78.6
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Figure 2. Representative structures of the most populated clusters of the (a) Aβ pentamer, (b) tetramer, (c) trimer at 300 K, and (d) trimer at 310 K. These clusters cover 67.3, 52.0, 60.6, and 66.9% of the simulation time of the four systems, respectively. Color coding is as follows: chain A, blue; chain B, red; chain C, yellow; chain D, silver; chain E, gray.
Figure 3. Mean β-sheet content detected in the (a) pentamer, (b) tetramer, and (c) trimer at 300 K and (d) trimer at 310 K simulations. The portion of the β-sheet is plotted for each sequence position. Values were averaged over the entire simulation time and over all chains present in the oligomers. A value of 100% denotes that a particular residue adopted an extended conformation in all chains of the respective oligomer over the entire simulation time.
N-terminal β-strand covering residues 10-26 is generally more stable than the C-terminal strand (residues 31-41) (Figure 3). The overall high stability of the N-terminal β-sheet is also interesting in light of previous computational studies, which reported that residues 10-23 play the most prominent role for fibril elongation, as compared to the residues of the turn and C-terminal strand.29,51 In the C-terminal β-strand, residues 33-36 predominately adopt an extended conformation, whereas a rather high conformational flexibility is observed for G37 and G38 (Figure 3). The fluctuations of the backbone angles (exemplarily shown for the φ angle of G37 in Figure 4) induce a kink at the diglycine motif in the trimer to pentamer (Figure 2) and also decrease the stability of the C-terminal half of the
β-sheet. As shown in Figure 3, an extended conformation is generally less populated for residues 39-41 as compared to 33-36. The D23-K28 salt bridge, which represents a key element for stabilizing the U-shaped protofibril fold, is bifurcated in the experimental structure, and D23 of one layer is involved in interactions with K28 of the same layer and K28 of the adjacent layer.5 Both the intra- and the intermolecular interactions are preserved over more than 80% of the simulation time in the trimer to pentamer (Table 2), which is in accordance with the stabilization of the turn and the overall conservation of the U-shaped topology (Figure 2, 5a).
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Figure 5. Comparison of the polar interaction stabilizing the pentamer and dimer: (a) Asp23-Lys28 saltbridge in the pentamer and (b) Asp23-Asn27 interaction in the dimer, shown for cluster representatives O5 and O2+. Color coding of the different oligomer chains is the same as in Figure 2; residues Asp23, Asn27, and Lys28 are depicted in sticks and colored red, yellow, and blue, respectively. The polar interactions formed by Asp23 of chain A are indicated by green dotted lines in both structures. Figure 4. Changes of the φ angle of G37 over the simulation time. Values are shown for the individual chains of the (a) pentamer, (b) tetramer, and (c) trimer at 300 K simulation time. The values for chains A, B, C, D, and E are colored in black, red, green, blue, and yellow, respectively.
TABLE 2: Population of Key Interactions Monomer (O1), Dimer (O2), Trimer (O3), Tetramer (O4), and Pentamer (O5), Detected during a 100-ns Simulation at 300 Ka salt bridge
O1
O2
O2+
O3
O3*
O4
O5
K28-D23 intra K28-E22 intra K28-D23 inter N27-D23 inter
3.9 0.0
69.0 24.9 11.0 11.2
0.0 6.0 15.7 63.4
99.7 14.8 96.8
100.0 0.0 86.7
100.0 3.2 100.0
100.0 15.3 83.6
a
In addition, the same type of analysis was performed for the trimer at 310 K (O3*) and the extended dimer simulation (O2+). All values represent mean percentage values averaged over all chains of the respective oligomer.
With respect to fibril elongation, our findings indicate that due to the high stability of the N-terminal β-strand and the U-shaped topology, the trimer to pentamer adopts a conformation that allows longitudinal elongation along the fibril axis, suggesting that these conformations might serve as nuclei for protofibril growth. The larger fluctuations and the kink observed in the C-terminal strand suggest that the interface involved in lateral fibril association (Figure 1c) is not fully preformed in these small oligomers. This lower stability of the C-terminal strand is also consistent with a study of fibril dissociation indicating that dissociation starts mainly with the disruption of the interactions of the C-terminal strand.30 The different stability of the secondary structure elements suggests that the trimer to pentamer should exist in conformations competent for longitudinal elongation, whereas the lower stability of the C-terminal strand suggests that lateral association might be favorable only for larger oligomers. Structural Properties of the Monomer. In the monomer simulation, the initial topology is already lost in the early steps of the dynamics due to the lack of stabilizing interactions between adjacent layers of the parallel β-sheet. Instead, an
Figure 6. Representative structures of the two clusters (a, b) detected in the Aβ monomer simulations. The clusters cover 63.0% (a), and 37.0% (b) of the simulation time, respectively. For the dimer simulation, the most populated clusters detected during the first and second 100 ns are shown in parts c and d, covering 82.6% and 57.5% of the simulation time, respectively. Color coding is as follows: chain A, blue; chain B, red.
antiparallel β-sheet (Phe19-Val24 and Ile31-Val36) is formed which persists over the entire simulation time. The representative structures shown for both detected clusters exhibit a very similar topology (Figure 6a,b). These structural properties are consistent with previous studies in which a β-hairpin structure has also been found as a stable structural element evolving from different starting conformations.22,23,52,53 However, it should also be noted that several previous MD simulations of the Aβ monomer reported a rather large conformational variability,25,26,54 and also, the dissociation process itself proceeds via multiple stages and pathways.30 A extensive REMD simulation, which investigated monomer folding, identified two distinct energy minima, with substantial either R-helix or β-sheet content.26 This observation suggests that our simulation has detected an energy minimum also found in other simulations, but sampling of the conformational space is not complete. The latter can most likely be explained by the simulation time of 100 ns, which is short compared to the physiological time scales of Aβ aggregation. Experiments show that the time scale for fibril growth is in the range of days.16 In light of this problem, several previous studies used REMD25,26 or high temperature simulations,27,30 which are
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Figure 7. Analysis of the structural properties of the dimer: (a) Total number of hydrogen bonds monitored over the trajectory (black, total; red, parallel; blue, antiparallel β-sheet). (b) Stability of the N-terminal β-sheet monitored by the interstrand V18-F19 hydrogen bond and by the F19-F19 distance. The latter distance was calculated between the centers of mass of the two F19 residues. Distances
