Analysis of Physicochemical Interaction of Aβ40 with a GM1

Mar 14, 2018 - a GM1 ganglioside-containing membrane. Conformational change of Aβ40 due to interaction with the membrane was monitored and compared ...
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Cite This: J. Phys. Chem. B 2018, 122, 3771−3781

Analysis of Physicochemical Interaction of Aβ40 with a GM1 Ganglioside-Containing Lipid Membrane Majid Vahed,† Saburo Neya,† Katsumi Matsuzaki,‡ and Tyuji Hoshino*,† †

Graduate School of Pharmaceutical Sciences, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba 260-8675, Japan Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan

‡

J. Phys. Chem. B 2018.122:3771-3781. Downloaded from pubs.acs.org by UNIV OF SOUTH DAKOTA on 09/02/18. For personal use only.

S Supporting Information *

ABSTRACT: The interaction of amyloid beta (Aβ) peptides with the cell membrane is one of the factors enhancing Aβ aggregation, which is closely related to neurodegenerative disease. In this work, we performed molecular dynamics (MD) simulation to investigate the initial stage of adhesion of Aβ40 to a GM1 ganglioside-containing membrane. Conformational change of Aβ40 due to interaction with the membrane was monitored and compared with that of Aβ42 observed in the previous study. Multiple computational trials were executed to analyze the probability of Aβ binding using a calculation model consisting of a GM1-containing mixed lipid membrane, a water layer, ions, and Aβ40. A single long-time MD simulation was also carried out. It was suggested from the simulation that a cluster of sialic acids of GM1 head groups often caught the side chain of His13 or His14 of Aβ40 in the early stage of the MD simulations. Afterward, the main chain of Leu34 formed many hydrogen bonds with gangliosides. These residues cooperatively work for Aβ40 to be held on the lipid membrane. It is notable that Aβ40 was observed to be deeply inserted into the head group region of the lipid membrane in some computational trials. In the insertion, Aβ40 occasionally formed a hydrogen bond with sphingomyelin. The difference in the secondary structure between Aβ40 and Aβ42 was an important factor for Aβ40 to be deeply inserted into the membrane.

1. INTRODUCTION Amyloid peptides are involved in the pathogenesis of neurodegenerative disorders such as Alzheimer’s disease (AD), Parkinson’s disease, and Huntington’s disease.1,2 AD is a progressive cognitive deterioration and is one of the most widespread forms of dementia.3 Extracellular aggregation of amyloid-β (Aβ) peptides in the brain is a hallmark of AD.4 The most commonly observed forms of toxic Aβs consist of 40 and/ or 42 residues, which are referred to as Aβ40 and Aβ42.5−7 Aβ is a natively unfolded protein, and under some conditions, Aβs aggregate to develop into a heterogeneous mixture of oligomers and fibrils. Numerous studies have suggested that Aβs exhibit affinity to lipid membranes of neuronal cells8 and that Aβ oligomers on the membrane surfaces are deformed into a pathogenic structure in AD.9−11 Furthermore, it has been shown that soluble Aβ monomers are capable of penetrating deeply into the lipid bilayer.11 It has been reported that Aβ insertion into the lipid membrane was strongly influenced by the pH condition and the presence of metal ions.12,13 Many studies have suggested that the aggregation of toxic Aβ peptides on the lipid membrane is enhanced on the glycolipidcontaining microdomain, which consists of, for example, GM1 ganglioside, sphingomyelin (SM), and cholesterol (Chol) in high concentrations.14,15 It has also been suggested that Aβs are likely to be bound to a particular type of microdomain named © 2018 American Chemical Society

the detergent-insoluble glycosphingolipid (DIG)-rich domain.16 GM1 is an acidic glycosphingolipid that is abundant in the plasma membrane of neurons and is involved in the pathology of AD. Detailed experimental studies have suggested that the Aβ−GM1 complex acts as a seed for the aggregation of Aβs.15,17 A series of in vitro and in vivo measurements and several nuclear magnetic resonance (NMR) studies have demonstrated that GM1 had a strong influence on the secondary structure of Aβ in the early stage of aggregation.18−21 It has been shown that Chol enhances the production of Aβ fragments.22,23 Chol also facilitates Aβ binding to the lipid membrane because a cluster of GM1 molecules, which is formed depending on the concentration of Chol, constitutes a binding site that is suitable for Aβ adhesion.24,25 In our previous study,26 molecular dynamics (MD) simulations were performed for two kinds of GM1-containing membrane models: one model consisted of GM1, SM, and Chol in a ratio of 1:2:2 and the other consisted of GM1 and POPC in a ratio of 1:4. The simulations showed a marked difference between the two membrane models; that is, GM1 molecules in the former model were condensed, whereas those Received: January 5, 2018 Revised: February 23, 2018 Published: March 14, 2018 3771

DOI: 10.1021/acs.jpcb.8b00139 J. Phys. Chem. B 2018, 122, 3771−3781

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The Journal of Physical Chemistry B in the latter model were scattered. GM1 clusters were formed only on the GM1/SM/Chol membrane.26 Experimental studies suggested that condensation of GM1 molecules due to the DIG-like environment was critical for the formation of the Aβ− GM1 complex.25 Hence, in our subsequent study,27 we carried out multiple MD simulations based on the GM1-clustered lipid membrane model, in which Aβ42 molecules were incorporated into the model system with a lipid composition of GM1/SM/ Chol = 1:2:2. The simulation clarified that Aβ42 was occasionally bound tightly to the lipid membrane, in which the amine group of Lys28 of Aβ42 was held by the Neu5Ac residues that formed a gathering pit on the GM1 cluster.27 In the present work, we performed MD simulation with incorporation of Aβ40 molecules in the model with a lipid composition of GM1/SM/Chol = 1:2:2. The results of computation provided a picture of the binding of Aβ40 to the GM1-containing membrane. A comparison was made between Aβ40 and Aβ42 in terms of conformational change and stability of Aβ.

2. METHODS 2.1. Construction of a Computational Model. The initial atom coordinate of Aβ40 was extracted from the structure that is available in a protein data bank with the accession code of 2LFM.28 This structure was determined by solution NMR spectroscopy without a detergent. Because the structure, 2LFM, contained 20 conformations, the first NMR conformation was selected. It should be noted that the Aβ40 structure of 2LFM is obviously partly different from the Aβ42 structure used in our previous work,27 which was determined by solution NMR with a detergent. The mixed lipid membrane of the calculation model consisted of 48 GM1, 96 SM, and 96 Chol molecules with a molar ratio of 1:2:2. Chemical structures of these lipid molecules are shown in Figure S1 in the Supporting Information. The initial atom coordinates of the membrane were extracted from the last snapshot structure of the 100 ns MD simulation in our previous study.26 The model system also contains a water layer with sodium and chloride ions, with the ion concentration set to 150 mM. A single Aβ40 molecule was incorporated into the water layer, as shown in Figure 1. The total number of atoms in the model is about 90 000, and the model size is about 70.0 Å × 70.0 Å × 136.0 Å. 2.2. Calculation Conditions of MD Simulation. The NAMD2.9 program was used for MD simulations.29 The calculation procedure was almost the same as that in our previous works.26,27,30,31 In short, the production run was carried out at a temperature of 310 K and a pressure of 1 atm, kept by the Langevin piston using Nosé−Hoover method. A periodic boundary condition was applied to all of the xyzdirections, and the particle mesh Ewald method was employed. The cutoff distance of van der Waals and Coulomb forces in a real space was 12.0 Å, and the integration time step was 2 fs. MD calculations were carried out five times with changes in the initial position of Aβ40 in the water layer. The simulation time for each computational trial was 100 ns. A 1 μs MD simulation was also performed using a graphic processing unit executable code of NAMD2.12.29 The all-atom CHARMM22 force field including CMAP correction32,33 was applied to the Aβ peptide. For lipids, the CHARMM27 force field34 was applied to SM and Chol and our original parameter shown in the Supporting Information of ref

Figure 1. (a) Top view of the equilibrated mixed membrane after the 100 ns MD simulation with a lipid composition of GM1/SM/Chol = 1:2:2. GM1 oligosaccharides are depicted in green. Acyl chains of GM1, SM, and Chol are colored yellow, cyan, and red, respectively. (b) Initial structure of Aβ40, which is adopted as a starting structure of Aβ. (c) Computational model for a single Aβ40 incorporated in the mixed membrane. Sodium and chloride ions are represented by yellow and green spheres, respectively. The periodic boundary box is indicated by lines. Water molecules are not shown for clarity. (d) Amino acid sequence of Aβ40.

26 was applied to GM1. The TIP3P parameter35 was applied to water molecules. 2.3. Analysis of Simulation Products. The hydrogen bond occupancy was calculated by visual molecular dynamics.36 The criteria for hydrogen bond (H-bond) formation is that the distance between acceptor and donor atoms is less than 3.5 Å and the angle made by acceptor, donor, and hydrogen atoms is less than 60°. The binding energy between the Aβ peptide and lipid membrane was evaluated by the MM/GBSA method in the pbsa module of AMBER11.37 The secondary structure of Aβ peptides was calculated by the defined secondary structure of protein (DSSP) program.38 The root-mean-square fluctuation (RMSF) with respect to the residues was obtained by the ptraj module of AMBER11.37 For visual clarity in the drawing of simulation structures, the positions of some molecules were transferred to the x-, y-, or z-direction by the length of the periodic boundary box. All of the structures were visualized by PyMOL.39 Cluster analysis of the conformational diversity of Aβ40 and Aβ42 was performed in a manner similar to that in our previous work.40 The coordinates of main-chain atoms were extracted every 10 ns from the trajectory of all of the computational trials. The averaged coordinate of the extracted structures was obtained. Then, each extracted coordinate was fitted to the average one to calculate the root-mean-square deviation (rmsd). On the basis of the rmsd values, the structures were classified into groups by performing cluster analysis with the nearest neighboring method using R software. Finally, all of the 3772

DOI: 10.1021/acs.jpcb.8b00139 J. Phys. Chem. B 2018, 122, 3771−3781

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The Journal of Physical Chemistry B structures were connected as a tree called a dendrogram, in which the x-axis is the label for the snapshot structures and the y-axis is the distance for the least dissimilarity among the individual structures. To separate the snapshot structures into groups from the dendrogram, the criteria of distance was set to 12 Å for both Aβ40 and Aβ42. In each cluster group, the averaged atom coordinate was obtained from the member structures. The snapshot structure nearest to the average in rmsd among the members was selected as the representative of the cluster group.

3. RESULTS 3.1. Single Aβ Peptide on a GM1-Containing Membrane. The motion of the Aβ40 peptide on the GM1/ SM/Chol mixed membrane was investigated through multiple MD simulations. We carried out 100 ns simulations five times with incorporation of one Aβ peptide in the calculation model. MD simulation reproduces the dynamic movements of Aβ and the membrane, enabling us to examine the possibility of Aβ binding to the membrane. The final structures for the five computational trials are depicted in Figure 2. Snapshot structures were extracted every 20 ns from the 100 ns MD simulation for all trials (Figure S2). Furthermore, the z-axis change of the center of mass of the Aβ peptide was monitored during the MD simulations (Figure S3). The Aβ peptide initially fluctuated at the water layer in every trial. In the 100 ns period of simulation, the Aβ peptide was bound to the surface of the lipid membrane except for in trial 2. In trial 2, Aβ was scarcely attached to the bilayer; that is, Aβ binding to the membrane surface was soon broken and Aβ fluctuated in the water layer for a long time. In the other four trials, Aβ was observed to be firmly bound to one of the leaflets of the bilayer. The primary molecular axis of the helical region was almost parallel to the lipid surface after binding to the membrane. It should be noted that the Aβ peptide was occasionally deeply inserted into the head group region of the lipid membrane in trials 1 and 3. In trial 1, seven residues, Lys28, Ala30, Ile31, Ile32, Gly38, Val39, and Val40 interacted with the deep inside of the membrane. In trial 3, Arg5, His13, and Leu17 interacted with the lipid membrane and made Hbonds with SM (Figures S4 and S5). The helix content of Aβ was slightly changed during the 100 ns simulation, as shown in Figure 3. Conformational change of the secondary structure of Aβ40 was monitored through the 100 ns simulation (Figures 3 and S6). One of the secondary structures among coil (no secondary structure), bend, turn, π-helix, α-helix, 310-helix, β-bridge, and β-sheet, was assigned to every residue of the Aβ peptide for every 1 ns snapshot structure in the trajectory of the MD simulation in the DSSP plot (Figure 3). The most popular secondary structure of Aβ40 was a bend, and the second-most popular structure was a coil. When Aβ40 interacted with the lipid membrane, a slight increase in bend and helix contents was observed. The N-terminal and C-terminal residues, Asp1, Ala2, Val39, and Val40, showed no secondary structure. Val12 and Asp23 were also likely to have no secondary structure. A stable β-bridge was established between Ile32 and Val36 in trial 3 and was observed for most of the period of simulation. The αhelix structure was almost limited to the region from residues 13 to 22, and an α-helix temporarily appeared at the N-terminal region in trials 2 and 5. A π-helix structure was also temporarily observed at the region for 55−65 ns in trial 2.

Figure 2. Final structures of the 100 ns MD simulations for the five computational trials. The Aβ peptide is depicted in a cartoon representation with magenta, and GM1 head groups are depicted in green. Acyl chains of GM1, SM, and Chol are colored yellow, cyan, and red, respectively. Water molecules and ions are not depicted. Only the molecules in the half leaflet of the lipid membrane that Aβ40 is bound to are shown for clarity. Side (left) and top (right) views are shown for every trials in (a−e).

3.2. Lipid−Peptide Interaction. There are four aromatic residues, Phe4, Tyr10, Phe19, and Phe20, at the N-terminal side of Aβ40. The interaction of these four aromatic residues with GM1 head groups was monitored during the simulation for the five trials (Figure 4). In every trial except for trial 2, some aromatic rings were within a distance that enabled CH−π and/or OH−π interaction with GM1. In trial 1, three residues, Tyr10, Phe19, and Phe20, were within the distance for interaction at 60 ns, but the aromatic rings were apart from GM1 after 65 ns. In trial 3, the interaction between aromatic rings of Aβ40 and the lipid membrane was established after 20 ns. In trial 4, almost all of the aromatic rings were within the distance possible for interaction through the 100 ns simulation. In trial 5, the interaction was established at 30 ns and Phe20 was close to GM1 all through the simulation afterward. As observed in Figure 4, Phe20 tends to establish stable contact with GM1. Phe4 and Tyr10 are unlikely to have steady interaction compared to Phe19 and Phe20. To identify specific lipid−Aβ interactions, H-bonds between Aβ40 and GM1 ganglioside were monitored for all of the five trials (Figure 5). The amino groups of Arg5, His14, and Lys16 3773

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Figure 4. Changes in the distances between the center of the aromatic rings of four residues of the Aβ peptide and the closest atom of GM1 head groups during the 100 ns MD simulation. The colors violet, blue, cyan, and green correspond to the residues Phe4, Tyr10, Phe19, and Phe20, respectively.

Figure 3. Change in the secondary structure of the Aβ40 peptide during the 100 ns MD simulation. The 310-helix, α-helix, π-helix, βbridge, β-sheet, turn, and bend structures are represented by brick, red, orange, light green, green, blue, and cyan, respectively. The white regions represent a coil, which is not an identifiable secondary structure. Snapshots were extracted from the simulation trajectory every 1 ns, and the secondary structures were calculated for the respective snapshots using the DSSP program.

Figure 5. Accumulative number of H-bonds in the 100 ns MD simulations for five trials, separately counted for the respective residues of Aβ40. For each trial, the atom coordinate was extracted every 1 ns, and totally 100 snapshot structures were used for the analysis. The number of H-bonds is the sum of the counts for all of the five trials. The number includes H-bonds involved in both the main-chain and side-chain atoms of each residue.

showed a larger number of H-bonds. The main-chain atoms of Leu34 also showed a large number of H-bonds. Glu11, Lys16, and Leu34 were found to be involved in the H-bond formation with the lipid membrane in every trial, whereas no H-bond was observed for two residues: Phe4 and Val18. The numbers of Hbonds observed during the 100 ns simulations were examined again for the respective residues of Aβ40, with separation into the counterparts of the GM1 head group, NEU and GAL (Figure S7). Both NEU and GAL were possible counterparts of H-bonds of Aβ40, whereas NEU had the largest number of H-

bonds on average. The change in the number of H-bonds in the simulation period was also monitored for every computational trial (Figure S8). The motion of Aβ40 in the simulation (Figure S2) was consistently reflected in the change of H-bond numbers shown in Figure S8. To examine the deep insertion of Aβ40 into the membrane, H-bonds of Aβ40 with SM were monitored (Figures S4 and S5). 3774

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with the same calculation model. The motion of Aβ40 during the 1000 ns simulation is shown in Figure 7. Initially, Aβ40 was

Direct H-bonds between Aβ40 and SM were observed only in two trials, 1 and 3. Figure S4 shows the number of H-bonds from the residues responsible for the interaction with SM. Lys28, Ala30, Ile31, Ile32, Gly38, Val39, and Val40 in trial 1 and Arg5, His13, and Leu17 in trial 3 were orientated toward the space unoccupied by the GM1 head groups on the membrane. In trial 1, the largest number of H-bonds was observed at 29 ns, and in trial 3, that was observed at 78 ns (Figure S5). A typical structure of the deep insertion of Aβ40 is shown in Figure 6.

Figure 7. Motion of Aβ40 through the 1000 ns MD simulation. The snapshot structures were extracted every 100 ns. GM1 head groups are depicted in green. Acyl chains of GM1, SM, and Chol are colored yellow, cyan and red, respectively. Sodium and chloride ions are represented by yellow and green spheres, respectively. The Aβ peptide is depicted in magenta. Water molecules are not shown for clarity.

Figure 6. Atom geometry of an Aβ-bound membrane surface at the moment when Aβ40 was deeply inserted in the lipid membrane. Some residues of Aβ40 were observed to form H-bonds with SM. The snapshot structure was extracted at the time point of 28 ns in trial 1. (a) Side view of the Aβ40-bound membrane. (b) Magnified view at the contact area between Aβ40 and SM. A yellow broken line represents a H-bond between the C-terminus of Aβ40 and SM.

put in the midst of the water layer. Aβ40 fluctuated in the water layer for a while and then approached one of the leaflets. After 50 ns, a stable interaction was established between Aβ40 and the membrane surface. Aβ40 was not detached through the simulation after stable contact had been established. While Aβ40 was steadily bound to the membrane, it changes its conformation on the membrane, as seen in the trajectory for 100−1000 ns in Figure 7. The change of the secondary structure is shown in Figure 8a. The α-helix region ranged from residues 14 to 23 for the first 350 ns, whereas the helical area was extended after that. An αhelix region appeared from residues 25−31 at 350 ns, and this region did not disappear through the simulation after 350 ns. Another α-helix region temporarily appeared for 380−500 ns at the N-terminal side. The main α-helix region expanded from residues 9 to 31 at 660 ns. Furthermore, the major secondary structure of the helical area became a π-helix after 660 ns. The z-axis change of the Aβ peptide during the simulation is shown in Figure 8b. Aβ40 soon approached one leaflet of the membrane at the very early stage of the simulation. The contact between the Aβ peptide and the membrane surface was not dissolved after the first contact through the simulation. The

The binding free energy between Aβ40 and the lipid membrane was calculated by the MM/GBSA method. Because the force field parameters are different from those in the MD simulations, the energy changes calculated by the MM/GBSA method may be a rough indication of the binding affinity of the Aβ peptide with the membrane. Energy gain was observed for four trials (Figure S9); that is, the binding free energies between Aβ40 and the lipid membrane were consequently lowered in all five trials except for trial 2. In trial 3, a large stabilization was observed at 75 ns, at which the number of Hbonds of Aβ showed a peak of 14 during the MD simulation. The α-helix content in the secondary structure was relatively high, and a β-bridge was also observed at that time point. 3.3. Computational Results of a 1 μs MD Simulation. To examine the computational adequacy of the 100 ns simulations, MD simulation was also performed for 1000 ns 3775

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Lys16, and Leu34 to the H-bond interaction were remarkable in both the 1000 and 100 ns MD simulations. The H-bond counts were separated into the counterparts of GM1 head groups (Figure S10b). Although NEU and GAL were possible counterparts of H-bonds of Aβ40 in the 100 ns simulations (Figure S7), H-bonds with GLC were slightly observed in the 1000 ns simulation. It was confirmed again from the 1000 ns simulation that NEU was the main counterpart of H-bonds with Aβ40 among the head group residues of GM1. The numbers of H-bonds with SM were separated into the residues of Aβ40 (Figure S10c). H-bonds with SM were observed only at the deep insertion of Aβ40 into the lipid membrane. It is notable that the residues contributing to the H-bonds with SM were different among the simulations, as seen in Figures S4a,b and S10c. To examine the interactions between Aβ40 and the membrane irrespective of aromatic rings and hydrogen bonding, the changes in distance from the respective residues of Aβ40 to the closest atom of GM1 head groups and/or that of the lipid membrane were monitored through the 1000 ns simulation. The changes in the minimum distance to GM1 head groups in Figure S11 showed that residues 1−10 at the Nterminus were not steadily bound to the membrane and they just approached the membrane surface occasionally. In contrast, residues 11−20 were stably bound to the membrane. In particular, the bindings of Lys16 and Leu17 were strong and both residues were attached to the membrane almost all through the simulation. In the C-terminal side, residues 30−34 were steadily bound to the membrane. The bindings of the Cterminus residues were weak as expected. The residues that were stably bound to the membrane were positioned at the midst of Aβ40, that is, the helix region of the Aβ peptide. Several residues that showed strong connections to GM1 head groups were selected from Figure S11, and the changes in the minimum distances are shown in Figure 9. His14 was close

Figure 8. (a) Change in the secondary structure of Aβ40 during the 1000 ns MD simulation. The coloring for the respective secondary structures is the same as that in Figure 3. Snapshots were extracted every 1 ns from the simulation trajectory. (b) Change in the z-axis of the Aβ40 peptide during the 1000 ns simulation. The center of mass of the Aβ peptide is represented by a blue line. The positions of the uppermost and lowermost atoms of the Aβ peptide are indicated by pink and navy lines, respectively. The center of mass of GM1 head groups is represented by red and green lines for the upper and lower leaflets, respectively. (c) Change in the distances between the center of the aromatic rings of four residues of Aβ40 and the closest atom of the GM1 head groups during the 1000 ns MD simulation. See also the caption of Figure 4 for colors.

Aβ peptide was sometimes deeply inserted into the membrane, as seen at 390−540 ns and at 950−1000 ns. Figure 8c shows the changes in distances between GM1 head groups and four aromatic residues of Aβ40. Stable interaction of either of these aromatic residues with GM1 was established at 50 ns. Until 150 ns, the four residues except for Phe4 were steadily bound to the membrane surface. However, the residues bound to the membrane were exchanged during the simulation. Although all of the aromatic residues made contact with the membrane at around 600 ns, the residues were not always bound to the membrane. In most of the simulation time, some residues were close to the membrane, whereas some were apart from the surface. Therefore, CH−π and/or OH−π interaction of aromatic residues with GM1 can flexibly rearrange its conformation to maintain a steady connection. The number of H-bonds between Aβ40 and GM1 head groups was counted with respect to residues (Figure S10a). Arg5, His13, His14, Gln15, and Lys16 showed a large number of H-bonds in their side chains. The main-chain atoms of Ala30, Gly33, and Leu34 also showed a large number of Hbonds. These H-bond formations were consistent with the accumulative number of H-bonds for the 100 ns simulations shown in Figure 5. In particular, the contributions of Arg5,

Figure 9. Change in the minimum distance from several residues of Aβ40 to the closest atom of NEU, GAL, or GLC of GM1 head groups during the 1000 ns MD simulation. (a) Four residues that were involved in the interaction with the membrane in the former half of Aβ40. (b) Four selected residues in the latter half of Aβ40.

to the membrane until 600 ns, which was compatible with the H-bond count in the 100 ns simulation (Figures 5 and S7). Phe20 was also stably in the vicinity of the membrane, which was consistent with the interactions of the aromatic rings in the 100 ns simulation (Figure 4). Notably, Lys16 was stably close to the membrane surface in the 1000 ns simulation. In the C3776

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Therefore, the high coil and bend content of Aβ40 is responsible for the large structural diversity. A representative structure was selected from each cluster group. Representative structures of Aβ40 peptides are presented in Figure 10a to show the structural differences among the

terminal side, Leu34 was also close to the membrane, which was compatible with the H-bond count in the 100 ns simulation (Figure 5). In addition, the minimum distance of Ile31 was almost always low during the simulation. Hence, the hydrophobic area of A30-I31-I32-G33-L34 was suggested to make a stable contact with the membrane surface. Figure S12 shows the minimum distances from the respective residues to the lipid molecules; SM, Chol, and ceramide part of GM1. The residues located in the N-terminal side of Aβ40 were distant from the lipids all through the simulation. In contrast, residues 31−34 were observed to make a contact with the lipids for 280−330 ns. The contact of the area was compatible with the observation of the interaction between Ile31 and SM in the 100 ns simulation (Figure S4). Asn27 also approached the lipid at that time.

4. DISCUSSION 4.1. Conformational Diversity of Aβ Peptides. The results of DSSP analysis shown in Table 1 indicated that Aβ40 Table 1. Ratios of Secondary Structures of Single Aβ40 and Aβ42 Observed in the Simulationsa secondary structure

Aβ40 (%)

Aβ42 (%)

α-helix π-helix 310-helix β-bridge strand turn bend coilb total

17.0 0.4 0.2 1.0 0.1 7.6 33.9 39.8 100

32.6 19.9 0.1 0.5 0.0 6.8 14.7 25.4 100

The ratio is presented as the percentage of each secondary structure in the sum of all of the snapshot structures acquired every 1 ns from MD trajectories of all computational trials. bCoil denotes no secondary structure.

Figure 10. Structures of the representative conformations in the cluster groups seen in the MD simulations for (a) Aβ40 and (b) Aβ42. The amino acid residues of Aβ peptides are colored from blue to red with the residue position from the N- to C-terminal sides.

contained a significantly high ratio of coils and bends and that the helix content was relatively low, suggesting the instability of Aβ40. In contrast, the helix content was high for Aβ42 and the coil and bend contents were relatively low. This means that the rate of structural transformation of Aβ40 is high compared to that of Aβ42 and that Aβ40 is more flexible than Aβ42. Therefore, considerably diverse conformational changes were observed for Aβ40 in the MD simulations, as shown in Figure S6. Cluster analysis provides information on conformational diversity in the motion of a protein peptide. As can be seen in Figures S13 and S14, the dendrograms obtained by cluster analysis showed more cluster groups for Aβ40 than for Aβ42 despite the fact that the total number of snapshot structures of Aβ42 was larger than that of Aβ40 in the cluster analysis. In the simulation, seven groups of structural conformations for Aβ40 and six groups of structural conformations for Aβ42 can be distinguished when structures were classified at a height of 12 Å in the dendrogram. This implies that Aβ40 has more structural diversity than Aβ42 and that the rate of conformational change of Aβ40 is higher than that of Aβ42. Competition between the peptide−peptide H-bonds and peptide−water H-bonds would be one of the factors for protein folding and flexible motion.41 The main-chain N and O atoms of a peptide are likely to be exposed to the solvent when it has no secondary structure.

cluster groups. Trial 2 (1 ns) in Figure 10a is a member of the cluster group that includes trial 1 (1 ns) and trial 3 (1 ns) (Figure S13). Hence, trial 2 (1 ns) is close to the starting conformation for simulation, and a helical region is observed at residues 13−22. Trial 3 (40 ns) is a member of the cluster group composed of the snapshot structures of trial 3. A marked feature of trial 3 (40 ns) is the presence of a kink site in the midst of the helix region at residue 18. Trial 4 (100 ns) is the most common conformation. The cluster group contains snapshot structures of trial 1, trial 2, and trial 4. Aβ40 peptides in trials 1 and 4 are bound to the membrane surface, whereas Aβ in trial 2 is in the water layer in most of the simulation period. This means that the structure of the Aβ peptide does not strongly depend on whether it makes direct contact with the membrane surface or not. Trial 5 (20 ns), trial 5 (60 ns), trial 5 (80 ns), and trial 5 (1000 ns) are representative structures of small fractional cluster groups. All members of the cluster groups are snapshot structures of trial 5. The conformation of the N-terminal side of the Aβ peptide greatly altered in trial 5, resulting in apparently high diversity in the structure. Representative structures of Aβ42 peptides were selected from the dendrogram shown in Figure S14 and are depicted in Figure 10b. Trial g (1 ns) is a member of the cluster group consisting of 1 ns snapshot structures of all trials. Hence, trial g

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bonds inside the peptide. Hence, the peptide backbone of Aβ40 is more flexible than that of Aβ42. The Aβ40 peptide can be separated into two parts, residues 1−28 and residues 29−40, in polarity. The C-terminal side of Aβ is highly hydrophobic because of many nonpolar residues. Therefore, it is easy for the C-terminal region with no secondary structure to access the hollow sites of other molecules. In contrast, the N-terminal region of residues 1− 28 contains 9 polar residues. Six of them are negatively charged, and the other three are positively charged residues.47,48 An important observation in the present simulation was that Aβ40 was deeply inserted into the head group region of the lipid membrane, as shown in Figure 6. This insertion was not observed in the case of Aβ42 in our previous study.27,49 This means that Aβ40 was more likely than Aβ42 to have a strong interaction with the inside of the membrane. The results of a study suggested the possibility of Aβ creating a pore or channel for ions and reactive oxygen species to access the inside of the membrane and attack the nonpolar lipid molecules.50 The results of the present simulation suggested that the C-terminal side of Aβ40 was more likely than the N-terminal side to interact with SM (Figures S4a and S10c). The hydrophobicity and flexibility of the C-terminal side will be effective for deep insertion of the peptide into the membrane. 4.3. Interaction between Aβ Peptide and GM1Ganglioside. In the present simulations, Aβ40 showed a steady interaction with the lipid membrane. Neu5Ac makes an important contribution to the Aβ binding to the membrane. GalNAc also contributes to the binding through the pyranosyl ring of galactose. Galactose has two chemically distinctive faces: one is a nonpolar face with CH groups and the other is a polar face with OH groups. The nonpolar side of galactose acts as a complimentary block for making contact with nonpolar or aromatic residues of a protein.51 Indeed, both hydrophobic and aromatic interactions are involved in protein folding and molecular recognition.52 Four aromatic residues, Phe4, Tyr10, Phe19 and Phe20, have chances for CH−π and/or OH−π interaction with GM1 oligosaccharide. Hence, the distances from the aromatic rings to the closest heavy atom of GM1 head groups were monitored during the simulations (Figure 4). Arunan and Gutowsky reported that the equilibration distance between two mass centers of a gas-phase benzene dimer was 4.96 Å.53 This value is close to an average distance of 5.05 Å between two phenyl rings interacting with each other in proteins. 54 NMR observation suggested that Gal also approached an aromatic ring to produce the CH−π interaction,55 and theoretical calculations with benzene and βfucose indicated that the intermolecular C−C distances were in the range of 3.89−5.04 Å.55 Hence, a distance of 5.04 Å was assumed to be a criterion for judging the formation of an aromatic interaction in the present study. Among the four residues, Phe20 showed interaction with GM1 most frequently. The rates of interactions with Phe4 and Tyr10 were low compared to those with Phe19 and Phe20 (Figure 4). The two residues Phe4 and Tyr10 are located in the hydrophilic region, and the secondary structure was a coil or bend for most of the period of simulation, whereas the secondary structures of Phe19 and Phe20 were mainly an α-helix (Figure 3). Because an α-helix is more stable than a coil, the rates of movement and/or conformational change are low and these two residues are likely to maintain an interaction with the membrane. A hydrophobic interaction will play a primary role for Aβ

(1 ns) is the starting conformation for simulation, and the helical region is bent at the middle. Trial c (30 ns) is the most common conformation of Aβ42 peptides. The helical region seen in the starting conformation is maintained, but its shape has become straight. The cluster group of trial c (30 ns) is composed of both Aβ peptides that are bound to and not bound to the membrane. Therefore, contact with the membrane does not have a strong influence on the peptide conformation. Trial b (40 ns) is a member of the cluster group composed of the snapshot structures of trial b. The presence of a long helix is a marked feature. Trial f (40 ns) is a member of the cluster group composed of the snapshot structures of trial f. A structural feature of trial f is the presence of a kink site in the midst of the helix region at residue 15. Trial i (20 ns) is a member of a single-membered cluster. A short helix at the Nterminal side is observed in addition to the main helix. Trial g (70 ns) is also a member of a single-membered cluster. The helical region at the N-terminal side has become loose. The structural diversity was further examined by calculating the RMSF of Aβ peptides (Figure S15). No noticeable difference was observed in the RMSF between Aβ40 and Aβ42 when the RMSF values were calculated with respect to the residues using the simulation trajectory in which Aβ was in the water layer (Figure S15a,b). However, a clear difference was seen between Aβ40 and Aβ42 when RMSFs were calculated using the partial trajectories in which Aβ was bound to the membrane (Figure S15a′,b′). RMSFs of Aβ peptides decreased because of the binding to the membrane, and the decrease was significant for Aβ42 compared with that for Aβ40. Cluster analysis was also applied to the trajectory of the 1000 ns simulation (Figure S16). Two of the three groups in Figure S16 are single-membered clusters. The two single clusters consist of the first two snapshots of the 1000 ns simulation. The rest of the snapshot structures comprise a big cluster, which is compatible with the z-axis change of the Aβ peptide in Figure 8b. 4.2. Insertion of Aβ40 into the Lipid Membrane. For Aβ pathogenicity, it is important to know what factor is highly toxic to neurons. It has been proposed that the toxicity of Aβ is mainly caused by oligomer structures of Aβ.42,43 In a study on a small bacterial peptide, HypF-N, which is known to form oligomers and amyloid-like fibrils, the presence of two types of oligomers, one being highly toxic and the other not being toxic, was detected by atomic force microscopy.44 The toxic oligomer strongly interacted with cellular membranes and was able to penetrate into cells. It was suggested that the toxicity of the oligomer was a consequence of the structural flexibility and hydrophobic property.45 Accordingly, the Aβ peptide and its interaction with the mixed lipid membrane are of great interest. The conformational diversity of Aβ peptides will be also related to the toxicity. According to solid-state NMR measurements,46 the stabilization by a salt bridge between Lys28 and the carboxyl group of the C-terminus is a reason why a unique S-shaped β-sheet motif is observed only for Aβ42 fibrils. Because the C-terminal side of Aβ40 is shorter than that of Aβ42 by two residues, such a salt bridge structure is not stable for Aβ40. The results of the NMR study also suggested the presence of a Gly29-Ile41 contact.46 Therefore, Aβ42 is likely to form an extended β-sheet compared to Aβ40, resulting in a decrease of transformation rate. Because many residues of Aβ40 have no secondary structure, there are many main-chain NH and CO groups not involved in the H3778

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the helix-poor region of Aβ40 became loose compared with that of Aβ42 as observed in every trial. This is one of the possible reasons for deep insertion of Aβ40 into the membrane surface. As shown in Table 1, the secondary structures showed much higher rates for π-helix (20%) and α-helix (33%) in Aβ42 than in Aβ40. Aβ40, on the other hand, showed a high coil content. The results of secondary structure analysis (Figure 3) and cluster analysis (Figures S13 and S14) showed that Aβ40 had more diverse structural conformations than Aβ42. If the coil content in the secondary structure is increased, Aβ40 is likely to fluctuate and the freedom of peptide conformation is increased, resulting in an increase of the diversity in the binding structure between Aβ and the membrane. The α-helix content of Aβ42 is larger than that of Aβ40, and the structure of Aβ42 is thus more stable than that of Aβ40. This means that Aβ40 has relatively dynamic movement compared to Aβ42. Hence, Aβ40 effectively interacts with the deep inside of the membrane surface and can make H-bonds with SM, which was not observed for Aβ42.

adhesions, and an aromatic interaction can support Aβ binding to the membrane surface. 4.4. H-Bonds between Aβ40 and the Membrane. Although there was no critical residue of Aβ for making the initial contact with the lipid membrane, two residues were observed to play key roles for Aβ40 adhesion. In the sum of Hbonds observed in all of the five trials, His14 and Leu34 gave large numbers of H-bonds to GM1 (Figure S17). Changes in the number of H-bonds during the 100 ns MD simulation are shown in Figure S18 for His14 and Leu34. The H-bond changes in Figure S18 suggested that the Aβ peptide was unlikely to be detached from the membrane surface when His14 was bound to the membrane. In the initial stage of interaction between Aβ and the membrane, His14 works as both a H-bond donor and an acceptor because of the N atom and NH group of its imidazole ring in the side chain. In the neighborhood of His14, there is a positively charged residue, Lys16, that acts as an anchor to the membrane as well as His14. After a while, Leu34 establishes a strong interaction with GM1. The contribution of Leu34 was obvious in the latter half of the 100 ns MD simulations (Figures S17 and S18). N and O atoms in the main chain enable Leu34 to form two H-bonds with GM1, even though Leu34 is positioned in the hydrophobic region of Aβ. These roles of His14 and Leu34 are also conformed from the changes in distances between the residues and the membrane in the 1000 ns simulation in Figure 9. Glu11, Lys16, and Leu34 were the only residues of which Hbond formation with GM1 was observed in all of the five trials. These results indicated that Glu11, Lys16, and Leu34 play important roles as well as His14 for Aβ adhesion to the membrane surface. The residues Ile31, Val39, and Val40 in trial 1 and His13 and Leu17 in trial 3 were observed to form Hbonds with SM with a certain degree of frequency. Most of the residues involved in H-bonds at the N-terminal side formed Hbonds via the side chain in contrast to those at the C-terminal side. 4.5. Comparison with the Findings from the Previous Studies. Solution NMR spectroscopy and circular dichroism demonstrated that the membrane with a specific lipid composition enhanced the conversion of a soluble monomeric Aβ conformation into a helix-rich one.56 A decrease in the αhelical content was observed in trial 2 when Aβ was detached from lipid membrane after 50 ns. That is, a part of the α-helix region from residues 12 to 24 showed a conformational change to a coil or bend, and the region also temporarily converted into a π-helix when Aβ40 peptide was not bound to the lipid membrane (Figure 3). These conformational changes are compatible with the above experimental findings. Furthermore, a β-bridge structure was observed for single Aβ40 as well as Aβ42 (Figure 3). A β-bridge conformation appeared at the Cterminal region, being compatible with the results of the recent NMR analysis for Aβ fibrils.46 A recent in vivo study showed that soluble Aβ oligomers from brain interstitial fluid were bound to GM1 ganglioside on cellular membranes.57 Some experimental studies suggested that the Aβ peptide was bound to the GM1/SM/Chol mixed membrane with an α-helix-rich conformation when the molar ratio of Aβ to GM1 was low but the β-strand-rich conformation became dominant when the ratio was high.25 In our previous study,27 MD simulations were performed for a GM1-containing membrane with a single Aβ42. The calculation model for the lipid membrane was the same as that used in the present study. In the simulations with a single Aβ40, the secondary structure of

5. CONCLUSIONS The following findings were obtained from the MD simulations of a single Aβ40 peptide interacting with a GM1 gangliosidecontaining lipid membrane. Aβ40 can be bound steadily to the lipid membrane within the 100 ns of simulation. A long-time MD simulation for 1 μs also showed a stable binding of Aβ40 to the membrane. His14 and Leu34 showed the largest number of H-bonds with GM1 among the residues. Many H-bonds from His13 or His14 to the lipid membrane were observed in the early stage of Aβ40 adhesion, whereas Leu34 established a strong connection to the membrane in the later stage. In the 1 μs simulation, Leu34 was not always bound to the membrane surface and the neighboring residues such as Ile31 made contact to the membrane in turn. Accordingly, the hydrophobic region around Leu34 can be stably connected to the membrane regularly once a contact with the membrane was established. In the hydrophilic region around His13 or His14, Lys16 kept a stable contact with the membrane in the later stage of the 1 μs simulation when His13 or His14 was apart from the membrane surface. Neuraminic acid had a large number of H-bonds with Aβ40 among the head groups of the lipid membrane. Four residues, Phe4, Tyr10, Phe19 and Phe20, of Aβ40 were related to aromatic interactions. Phe19 and Phe20 were more likely than Phe4 and Tyr10 to form aromatic interactions with GM1. An important feature of our computational study is that Aβ40 showed more diverse structural transformation than Aβ42. This feature of conformational flexibility enabled Aβ40 to be inserted into the space not occupied by head groups of GM1 on the membrane surface. When Aβ40 is attached to the membrane surface, a part of Aβ40 is possibly inserted deeply into the lipid membrane with the formation of H-bonds with SM. The hydrophobicity and flexibility of the C-terminal side were closely related to this deep insertion of Aβ40.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.8b00139. Chemical structures of the lipid molecules; time courses of MD simulation; changes of z-axis Aβ peptides; analysis of H-bonds; change in the conformation of Aβ40 during the simulation; change in the minimum distance between the respective residues of Aβ40 and the membrane and 3779

DOI: 10.1021/acs.jpcb.8b00139 J. Phys. Chem. B 2018, 122, 3771−3781

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change in the binding free energy; dendrogram obtained by cluster analysis; and RMSF of Aβ peptides (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +81-43-226-2936. Fax: +81-43-226-2936. ORCID

Tyuji Hoshino: 0000-0003-4705-4412 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Calculations were performed at the Research Center for Computational Science, Okazaki, Japan, and at the Information Technology Center of the University of Tokyo. A part of this work was supported by a grant for Scientific Research C from the Japan Society for the Promotion of Science. This work was also supported by a grant from the Japanese Agency for Medical Research and Development.

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