Mechanism of Inhibition of Beta Amyloid Toxicity by Supramolecular

Article Cite This: J. Phys. Chem. B XXXX, XXX, XXX−XXX

pubs.acs.org/JPCB

Mechanism of Inhibition of Beta Amyloid Toxicity by Supramolecular Tweezers Sumit Mittal, Kenny Bravo-Rodriguez, and Elsa Sanchez-Garcia* University of Duisburg-Essen, Universitätsstraße 2, 45141 Essen, Germany S Supporting Information *

ABSTRACT: The Aβ1−42 dimer is the smallest oligomer of the 42-residue Aβ peptide (Aβ1−42), which is involved in Alzheimer’s disease. The molecular tweezer CLR01 is a synthetic molecule that selectively binds lysine and arginine residues to inhibit toxic aggregation of amyloidogenic peptides. Here, we performed replica exchange molecular dynamics simulations of Aβ1−42 in explicit water to study, at the molecular level, the effect of CLR01 binding to the lysine and arginine residues in the dimer. We found that CLR01 molecules encapsulate both lysine residues of each Aβ1−42 monomer while only establishing labile interactions with the arginine residues. This encapsulation leads to the noncovalent disruption of interand intramolecular interactions involving the monomers. Additionally, the total β-sheet content in the Aβ1−42 dimer decreases because of this binding. With CLR03, a negative control molecule that shares the charged core of CLR01 but does not form inclusion complexes, Aβ1−42 dimer formation is observed, similar to the reference system without ligands. Our work allows establishing a molecular mechanism for the modulation of protein−protein interactions in Aβ1−42 by CLR01. This mechanism is characterized by an aggregation pathway driven by the encapsulation of lysine residues as well as by the secondary interactions of tweezers with the peptide units and with other CLR01 molecules.

1. INTRODUCTION

The 42-residue Aβ peptide (Aβ1−42) is known to be involved in Alzheimer’s disease.9 While the exact neurotoxic form of the peptide (i.e., the fibril form or the low-molecular-weight soluble oligomers) is still debatable, structural information on the small oligomers is very much needed. Given the high aggregation propensity of Aβ1−42, there is limited experimental data on its soluble aggregates.10,11 The molecular tweezer CLR01 is able to remodel Aβ1−42 fibrils and to regulate the oligomerization process of Aβ1−42, favoring the formation of oligomers of small size.4 Therefore, CLR01 is a potential therapeutic agent against Alzheimer’s disease.4,6 In silico studies can provide insights into the structure and dynamics of amyloid monomers and small oligomers.12−15 Allatom molecular dynamics (MD) simulations indicated that Aβ1−42 dimerization involves the loss of monomer solvation free energy followed by a structural rearrangement of the dimer.16 Another study predicted that the Aβ1−42 dimer primarily adopts coil/turn conformations (∼80%) with 11% α-helix and ∼8% β-sheet contents.17 Although lower, the latter value agrees with circular dichroism (CD)-derived results (∼12% β-sheet content) on the disordered character of earlier aggregates.18 Hamiltonian and temperature replica exchange MD (REMD) simulations of Aβ dimers using a coarse grained (CG) model revealed that both, Aβ1−40 and Aβ1−42, adopt primarily turn or random coil conformations, with an increased

Synthetic molecules that bind to protein surfaces in a predictable manner allow tuning protein−protein interactions and have thus the potential to inhibit aberrant aggregation. In this context, Klärner and Schrader introduced molecular tweezers that are able to bind specifically to lysine and to arginine residues via combined hydrophobic and electrostatic interactions.1−3 Molecular tweezers feature a highly electronrich interior and are therefore effective hosts for electron-poor residues (Figure 1). Experimental studies have shown that the tweezer CLR01 is able to inhibit aggregation of amyloidogenic peptides without toxic side effects,4−6 highlighting this molecule as a promising candidate for antiamyloid therapeutic intervention.6−8

Figure 1. Chemical structures of the molecular tweezer CLR01 (left) and of the control molecule CLR03 (right), which lacks the hydrophobic arms of CLR01. © XXXX American Chemical Society

Received: October 24, 2017 Revised: March 7, 2018

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DOI: 10.1021/acs.jpcb.7b10530 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B β-sheet propensity at the C-terminal region compared to the respective monomers.19 Extensive discrete MD simulations also indicated the disordered nature of the Aβ1−42 dimer with some β-strands connected by turns and loops.20 Additionally, that study proposed a higher β-sheet content in the L17-A21 region (known as central hydrophobic cluster or CHC) and in the Cterminal region of Aβ1−42 with respect to Aβ1−40. Several small molecules are reported to interact with the Aβ1−42 peptide and to reduce its cytotoxicity.21−24 These molecules, presumably, bind to Aβ1−42 to alter the aggregation and fibrillization pathways. As discussed above, CLR01 inhibits Aβ1−42 toxicity.4,8 One possible mechanism suggests that binding of CLR01 to exposed lysine residues of Aβ1−42 disrupts key electrostatic and hydrophobic interactions, leading to the formation of nontoxic and nonamyloidogenic structures.4 The supramolecular binding of CLR01 to lysine residues is a crucial step for the inhibition of toxic aggregation. CLR03 (Figure 1), a synthetic molecule lacking the hydrophobic arms of CLR01, is unable to encapsulate the lysine residues and does not prevent Aβ1−42 oligomerization.4,25 The Aβ1−42 monomer has two lysine residues, K16 and K28, and both are reported to play an important role in self-assembly and toxicity. For example, a salt bridge between the side chains of K28 and D23 was experimentally suggested as a crucial stabilizing interaction.26 K28 is also proposed to form a salt bridge with E2227 and to establish hydrophobic interactions with nearby residues such as V24. These hydrophobic interactions were also reported to be crucial for the structural stability of Aβ21−30 fibrils.28,29 K16, on the other hand, is placed in the vicinity of the CHC region of the peptide (residues 17− 21), an important region for the aggregation process.20 Thus, binding of CLR01 molecules to lysine allows modulation of Aβ1−42 self-assembly and toxicity. Although to a less extent than lysine, CLR01 can also encapsulate arginine residues.30,31 As discussed above, the lysines and the arginine of Aβ1−42 are targets for the modulation of self-assembly and toxicity. Accordingly, simulations focusing on the individual influence of K16 and K28 on Aβ oligomerization reported that mutations to alanine in K16 lowered the β-sheet content and reduced the toxicity in Aβ1−42 more than the alanine mutation in K28.32 Low-resolution experimental studies using dynamic light scattering and electron microscopy techniques suggest that CLR01 binding causes subtle conformational changes in Aβ1−42 but do not establish the nature of these changes at the molecular level.4,25 Here, we use advanced sampling simulations (REMD) in combination with multiscale calculations (Quantum Mechanics/Molecular Mechanics (QM/MM)) to investigate the selective binding of CLR01 to arginine and lysine residues in the dimer of Aβ1−42, the smallest reported toxic oligomer.33 As CLR03 shares the charged motif of CLR01 but not the hydrophobic arms, we also studied the effect of CLR03 on the dimer as a negative control. Our work allows establishing a molecular mechanism for the modulation of protein−protein interactions in Aβ1−42 by the molecular tweezer. CLR01 induces conformational changes on the monomers and leads to a different aggregation pathway driven by the encapsulation of lysine residues as well as by the secondary interactions of tweezers with the peptide units and with other CLR01 molecules. The aggregates thus formed are characterized by less interpeptide contacts with respect to the Aβ1−42 reference dimer without ligands.

2. COMPUTATIONAL DETAILS We performed REMD simulations34,35 to study the effect of CLR01 and CLR03 on the aggregation of two Aβ 1−42 monomers. The initial coordinates for the Aβ1−42 monomer were kindly provided by the group of A. E. Garcia and are based on the third centroid obtained from previously published microsecond REMD simulations.36 Those calculated structures are in excellent agreement with the reported experimental NMR parameters of the Aβ1−42 monomer.37,38 For the Aβ1−42 dimer, two copies of the monomer were placed at 18 Å from each other (Figure 2A). The protonation

Figure 2. A. Starting geometry with two Aβ1−42 monomers separated by 18 Å (β-sheet: magenta, turn: blue, random coil: green, and βbridge: cyan). The terminal residues are highlighted: M1 (red) and A42 (green). B. Starting geometry with two Aβ1−42 monomers separated by 18 Å and six CLR01 molecules. CLR01, lysine, and arginine residues are highlighted.

states for the protein residues were set at pH 7. The system was solvated in a truncated octahedron box of water molecules with a minimum distance of 20 Å from the wall. This resulted in ∼15 500 TIP3P water39 molecules. Six Na+ ions were added for charge neutralization. The solvated system was then energyminimized. An NVT (200 ps) equilibration was then performed to relax the solvent molecules while keeping the protein atoms constrained. Subsequently, the full system was allowed to relax for 500 ps within the NPT ensemble (300 K, 1 atm). During the NPT equilibration, the temperature was kept constant at 300 K with the Berendsen thermostat.40 This structure was employed as an initial geometry for the REMD simulations. A total of 75 replicas were used for the REMD simulations with temperatures ranging from 300 to 450 K (Table S1 and Figure S1, Supporting Information).41 All structures were reequilibrated for 200 ps at the corresponding temperature of the replicas in the NPT ensemble. The resulting geometries were subsequently used for the production runs of each replica in the NVT ensemble. The temperature of each replica was set using the velocity-rescaling thermostat.42 The exchanges between the neighboring replicas were attempted every 2 ps, with a target acceptance ratio of 20%. The LINCS scheme was used to constrain the bonds, making it feasible to use a time step of 2 fs.43 The particle mesh Ewald method44 was employed to treat the long-range electrostatic interactions. The cutoff for the van der Waals (vdW) interactions was set to 1.4 nm. The nonbonded pair lists were updated every 0.010 ps. All simulations were performed using the GROMACS 4.5.7 software.45 The same protocol was applied to study four additional systems in which, as described above, two Aβ1−42 monomers were initially placed at 18 Å from each other, this time in the presence of CLR01 or CLR03. The new systems contain B


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The Journal of Physical Chemistry B Table 1. Geometrical Parameters Describing the Encapsulation of Lysine and Arginine Residues by CLR01a

Cp−Cp′

system b

CLR01−Lys CLR01−Argc Aβ1−42 dimer + 6 CLR01

Aβ1−42 dimer + 2 CLR01 Aβ1−42 dimer + 6 CLR03

Aβ1−42 dimer + 2 CLR03

K16A K28A K16B K28B R5A R5B K16A K16B K16A K28A K16B K28B R5A R5B K16A K16B

5.5 5.6 5.6 5.8 5.7 5.7 5.8 6.3 5.6 5.7

± ± ± ± ± ± ± ±

0.5 0.6 0.5 0.5 0.9 1.3 0.5 0.5

Cm−Cm′

P−N (CZ)

C−C1−N (CZ)

3.7 3.8 4.1 4.4 4.1 4.2 4.2 4.9 4.1 4.2

4.1 4.0 4.2 ± 0.7 4.1 ± 0.5 3.9 ± 0.2 4.0 ± 0.5 19.7 ± 15.1 25.4 ± 17.1 4.0 ± 0.3 3.9 ± 0.6 25.4 ± 13.1 24.8 ± 15.7 33.2 ± 13.0 30.1 ± 15.8 27.9 ± 10.8 19.5 ± 18.8 21.4 ± 15.1 26.1 ± 14.6

90.3 86.9 86.5 ± 6.7 86.1 ± 6.4 85.9 ± 6.8 87.3 ± 8.2 72.5 ± 32.8 97.9 ± 37.6 85.5 ± 6.7 86.7 ± 7.4 86.9 ± 32.7 82.8 ± 38.1 88.3 ± 31.4 90.5 ± 38.5 102.9 ± 32.2 81.9 ± 41.9 80.9 ± 42.3 102.1 ± 34.8

± ± ± ± ± ± ± ±

0.5 0.6 0.5 0.5 1.0 1.5 0.5 0.6

The distances Cp−Cp′ and Cm−Cm′ measure if CLR01 is deformed upon formation of the inclusion complex. The distance P−N (P−CZ for Arg) and the angle C−C1−N (C−C1−CZ for Arg) measure how well threaded is the side chain of Lys(Arg) inside the cavity of CLR01. Distances are in angstroms, and angles are in degrees (°). A and B are the monomer chains in the dimer. bExperimental values, crystal structure of the complex 14-33σ−CLR01 (PDB 5OEH).48 cExperimental values, crystal structure of a ternary complex 14-3-3ζ−Cdc25CpS216−CLR01 (PDB 5M37).62 Calculated values are reported for the S1 ensemble. a

of the trajectories was performed using Daura’s algorithm,58 and the peptide backbone was used for the root-mean-square deviation (rmsd) least-square fit with a cutoff of 4 Å between two conformations. Other analyses, such as salt bridges, hydrogen bonds, contact maps, number of contacts, and solvent accessible surface area (SASA), were performed using various tools and plug-ins of the GROMACS and VMD programs. A cutoff distance of 4 Å between atoms of two residue side chains was considered to define a contact. The 2D free energy surfaces were generated using the g_sham tool available in GROMACS. The free energy surface was constructed using −RT ln P(x,y), where P(x,y) represents the probability of two selected reaction coordinates. The collision cross section (CCS) values were calculated using the MOBCAL software59 and the trajectory method.60 The interchain vdW interaction energy values were calculated using the MM-PBSA method.61

CLR01 or CLR03 molecules in a 1:1 or 1:3 proportion. A 1:3 ratio means that all lysine and arginine residues form inclusion complexes with CLR01 in the initial geometry (Figure 2B). For the 1:1 ratio, only K16 is encapsulated by CLR01. Because no inclusion complex can be formed by CLR03, the CLR03 molecules were placed near the corresponding lysine or arginine. For the simulations of the Aβ1−42 dimer in the absence and presence of six CLR01 or six CLR03 molecules, trajectories of 400 ns were calculated. We also performed 100 ns REMD of the systems with two CLR01/CLR03 molecules for a total simulation time of 105 μs (Table S2, Supporting Information). In all simulations, the CHARMM27 force field46,47 was used for the protein. For CLR01 and CLR03, CHARMM force fieldbased parameters, tested and used by us in previous studies,31,48 were employed. Although the CHARMM27 force field is reported to produce biased α-helical conformations,49 we did not observe this behavior (see Results and Discussion). This is in agreement with other reports of a good performance of CHARMM27 for amyloid beta proteins.50−52 Here, the calculated secondary structure content of the Aβ1−42 dimer was found to be in good accord with experimental data on early oligomers18,53,54 as well as with previous simulation studies using different force fields.20,55 The secondary structure characterization was done using the STRIDE program,56 as implemented in VMD.57 The clustering

3. RESULTS AND DISCUSSION For the analysis of the dimer simulations, we focused on two cases: (a) the 310 K replica and (b) an ensemble (termed S1) of 50 000 conformations from the last 100 ns (50 ns in case of simulations with two CLR01 or two CLR03 molecules) of five replicas corresponding to the temperatures 300, 305, 310, 315, and 320 K. Both sets of results generally agree with each other. Therefore, we focus our discussion on the S1 ensemble. The C


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propensity of the Aβ1−42 dimer indicates that, in accordance with its disordered nature, coils and turns are the most prevalent secondary structure motifs in all systems, ∼80% of the total secondary structure content, both in the absence and in the presence of CLR01 or CLR03 molecules (Figures 4 and

results for the 310 K trajectory are presented in the Supporting Information, as well as an analysis of the convergence of the simulations (Figures S2−S6, Supporting Information). 3.1. Binding Sites of the Tweezers. During the REMD simulations, we found that CLR01 molecules bind lysine residues in a conserved manner, as indicated by the P−N interaction distances and the threading angle, C−C1−N (Table 1, S1 ensemble). This interaction is less favored in the case of arginine, as shown by the large values and deviations of the P− CZ distances and C−C1−CZ angles (Table 1). The latter also indicates a more distorted tweezer structure upon binding to arginine. We found the same behavior for the 310 K trajectory (Figure S7, Supporting Information). The larger side chain of arginine (with respect to that of lysine) makes it more difficult to accommodate it inside the tweezer cavity. This is in agreement with the reported higher specificity of CLR01 for lysine residues.2 This observation is also in accord with gasphase ion mobility spectrometry−mass spectrometry (IMS− MS) reports that suggest that CLR01 binds to Aβ1−42 mostly with a 1:2 stoichiometry.25 Interestingly, the CLR01 molecules initially associated with R5 dissociate from the complex to interact with other tweezers via the hydrogen phosphate groups (Figure 3). On the other hand, the interactions with CLR03 are

Figure 4. Average propensity (%) of each secondary structure motif (coil, turn, α-helix, 310-helix, β-bridge, and β-sheet) as calculated for the S1 ensemble. Notice that the propensity of all secondary structure elements follows the same trend in S1 than in the 310 K trajectory (Figure S10, Supporting Information). The standard errors were calculated by dividing the simulations in six intervals of 50 ns (for the last 300 ns) and computing the standard deviation of the average values. For the simulations with two CLR01 and two CLR03 molecules, five intervals of 20 ns were used.

S10, Supporting Information). The α-helical content is below 7% in all cases. This low amount of α-helix, which is in good agreement with the reported CD-derived value of 3−9%,18,54 is in contrast to previous reports suggesting the enhanced α-helix sampling of the CHARMM force field.49 The β-sheet content is 16% in the Aβ1−42 dimer and remains about the same in the presence of the negative control CLR03. These β-sheet propensities for the Aβ1−42 dimer are in good agreement with previous theoretical simulations (16−19%) of the Aβ1−42 dimer.20,55,63 It should be noted that the β-sheet content decreases to 12% in the presence of six CLR01 molecules and to 13% in the presence of two CLR01 molecules (S1 ensemble, Table S4, Supporting Information). The residue-wise secondary structure profiles (Figures S11 and S12, Supporting Information) reveal that the β-sheet motif is dominant in the regions formed by residues 9−12 and 32−35 of both Aβ 1−42 monomers. Residues 3−7 and 26−31 show dominant (approx. 80%) turn conformations. For the 310 K replica, the β-sheet is located primarily in residues 9−12 and 32−35. We note that the later region was also identified in other studies.17,64 However, the analysis of the secondary structure propensities for the S1 ensemble also indicated β-sheet content (5−12%) in the regions comprising residues 16−19 and 36−41, in agreement with previous reports.15,55,64 Importantly, in both cases (310 K and S1), our overall secondary structure values for the Aβ dimer agree with the values reported in the literature (Table S4).18,20,55 The residue-wise probability difference of β-sheet content with respect to the Aβ1−42 dimer indicated a net decrease of the

Figure 3. Cooperative effect of the intermolecular interactions between CLR01 molecules as observed in the REMD simulations of the Aβ1−42 dimer with six tweezers. Here, the inclusion complex involving the tweezer molecule originally associated with R5 (CLR01R5) is lost. Instead, CLR01R5 mediates interactions between the monomers via neighboring tweezers encapsulating lysines. Representative interaction distances are shown in the figure (for the nonlabeled contacts, the values are 1.58 and 1.61 Å from left to right). The population of this motif during the simulation was 33%.

very labile (Table 1 and Figure S8, Supporting Information), in correspondence with the lack of antiamyloid effect of this control molecule.25 We performed QM/MM geometry optimizations of CLR01 on K16 or K28 with the Aβ1−42 monomer (see the Supporting Information for computational details) to determine the preferred binding site of CLR01 on the Aβ1−42 monomer. Our results suggest that CLR01 may slightly favor K16 (Table S3, Supporting Information) as, in this position, the tweezer is able to establish additional stabilizing interactions with Q15 (P−OHCLR01···OCQ15 distance: 1.83 ± 0.23 Å; Figure S9, Supporting Information). 3.2. Effect of the Tweezer on the Secondary Structure of the Aβ1−42 Dimer. The analysis of the secondary structure D


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The Journal of Physical Chemistry B β-sheet character in the presence of six CLR01 molecules (Figures S13 and S14, Supporting Information). Contrarily, the patterns in the presence of CLR03 molecules did not show any net decrease in β-sheet content. The same analysis for the coil conformations showed a net increase in the system with six CLR01 molecules. Our results thus indicate that, albeit the effect is not as pronounced as in other systems studied by us,65,66 the molecular tweezer CLR01 decreases the β-sheet content of the Aβ1−42 dimer and favors coil structures. 3.3. Effect of the Tweezers on the Dimer Structure. 3.3.1. Collision Cross Section (CCS) Characterization. Experimentally obtained CCS values allow commenting on the size and shape of a system. Here, we used the computed CCS values to evaluate the possible effect of CLR01 and CLR03 molecules on the compactness of the dimer (Table 2). Table 2. Weighted-Average CSS values Calculated Using the 10 Most Populated Conformations Obtained from the rmsdBased Clustering for Each Replica in the S1 Ensemblea average Aβ1−42 Aβ1−42 Aβ1−42 Aβ1−42 Aβ1−42

+ + + +

6 6 2 2

CLR01 CLR03 CLR01 CLR03

1291 1470 1342 1354 1329

± ± ± ± ±

55 68 39 60 38

cluster 1

cluster 2

1296 1446 1293 1313 1342

1220 1500 1323 1352 1322

Figure 5. Average interpeptide dCOM values and the standard error for each system in the production ensemble S1.

for the Aβ1−42 dimer in the S1 ensemble is 1.7 ± 0.5 nm, which increases to 2.9 ± 0.6 nm in the presence of six CLR01 molecules. Conversely, the dCOM value in the system with six CLR03 molecules (2.0 ± 0.6 nm) is closer to the reference dCOM value in the Aβ1−42 dimer. We found the same tendency for the 310 K trajectory (Table S6, Supporting Information). As discussed above, the presence of six CLR01 molecules results in the separation of the two monomers. Thus, the number of interchain contacts also decreases. We analyzed the occurrence of two types of conformations: (i) with less than 50 interchain contacts and (ii) with more than 350 interchain contacts (S1 ensemble, Table S7, Supporting Information). For the Aβ1−42 dimer simulation, the probability of more than 350 contacts is about 71% and only 16% in presence of six CLR01 molecules. The same effect was very pronounced in the 310 K trajectory (Table S8, Supporting Information). In addition, the time evolution of the number of interchain contacts in the absence and presence of CLR01 and CLR03 for the replicas at 310, 315, and 320 K also indicates a clear loss of interchain contacts in the system with six CLR01 molecules, unlike in the Aβ1−42 dimer without any ligand (Figure 6). Figure 7 shows the two most populated clusters for the S1 ensemble of each REMD simulation. In the absence of CLR01, the Aβ1−42 monomers form a dimer, the structure of which is in general agreement with reported experimental and computational data.19,51,69 As mentioned above, CLR03 lacks the hydrophobic arms that allow CLR01 to encapsulate side chains inside its cavity, and it is thus not able to establish conserved interactions with the lysine and arginine residues in Aβ1−42 (Table 1). In general, the calculations with CLR03 are similar to those of the reference system without any ligand. However, its bridgelike structure with two negatively charged hydrogen phosphate groups allows CLR03 to establish electrostatic interactions with positively charged residues of each Aβ1−42 monomer, which could aid dimer formation. This finding is in agreement with experimental reports by Zheng et al.25 of CLR03 facilitating early Aβ42 oligomerization. Nevertheless, these interactions are labile as CLR03 molecules are also driven away from the Lys/Arg residues to interact with each other and with the solvent. Furthermore, the most populated clusters from that simulation show no decrease in the β-sheet character of the dimer, unlike in the simulation with tweezers (Figure 4). Taken together, our results indicate that, in the presence of six CLR01 molecules, the Aβ1−42 subunits undergo conforma-

a

The CCS values (in Å2) for the two most populated clusters are also reported.

The weighted-average CCS value for the Aβ1−42 dimer was calculated to be 1291 Å2, which is in line with the CCS values of 1243 and 1256 Å2 reported for the Aβ1−42 dimer based on gas-phase IMS−MS studies.25,67 The CCS values in the presence of the control compound CLR03 or with only two CLR01 molecules are similar to those for the Aβ1−42 dimer without any ligand (average CCS values of 1342, 1329, and 1354 Å2). An experimental study reports a decrease of 119 Å2 in the cross section of the Aβ42 dimer upon addition of two CLR01 molecules.25 However, it should be noted that these measurements were performed in the gas phase and on a time scale that considerably differs from those of the simulations. A recent study68 comments on the effect of different experimental and simulation conditions as well as of different CCS calculation methods on the values of CCS for the Aβ dimer and tetramers. The authors observe that Aβ oligomers simulated in an explicit solvent have larger CCS values than those simulated using an implicit solvent model by approx. 50 Å2, and in turn, simulations in an implicit solvent report larger CCS values than vacuum simulations by up to 70 Å2. Importantly, we found that the average CCS value notably increases in the presence of six CLR01 molecules (1470 Å2). We observed a similar behavior for the 310 K trajectory (Table S5 and Figure S15, Supporting Information). This increase in CCS suggests a more extended conformation of the dimer in the presence of multiple CLR01 molecules, which could be a consequence of the separation of the monomers or, as shown before, the monomers adopting a more disordered conformation. 3.3.2. Inter- and Intramonomer Interactions. To evaluate if the peptide monomers are indeed further away from each other in the presence of the tweezer, we computed the average interpeptide center-of-mass distances, dCOMs (Figure 5 and Table S6, Supporting Information). The average dCOM value E


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Figure 6. Time evolution of the total number of interchain contacts in each system averaged every 500 ps for the 310, 315, and 320 K replicas. A contact is considered when the distance between the chains is less than 4 Å. Notice that we started the REMD simulations after pre-equilibration simulations in which the monomers are free to approach (see Computational Details). Thus, the initial number of contacts can be larger than zero.

pathway, driven by the interactions between CLR01 molecules and by additional CLR01−Aβ1−42 contacts (Figures 3 and 7). Next, we also analyzed the intrapeptide (as well as interpeptide) interactions using residue-based contact maps. The intrapeptide interaction map for the S1 ensemble (Figure S16, Supporting Information) illustrates the interactions between both termini in each Aβ1−42 monomer. In addition, the pattern corresponding to the antiparallel β-sheet is clearly visible in all cases spanning the residues 8−12 and 31−36. Residues 17−24 interact with residues 36−42. However, in the presence of six CLR01 molecules, the interactions between the CHC region and the C-terminus of the peptide are lost or more weakened than in the simulations with two CLR01 molecules or with the control. Furthermore, with six CLR01 molecules, the contacts corresponding to the antiparallel β-sheets involve a lower number of residues. Although the contact maps in the presence of only two CLR01 molecules do not greatly change with respect to the reference, the interactions between residues 4−10 and residues 20−30 are stronger. Overall, in the system with six CLR01 molecules, the interactions between the CHC region and the C-terminus of the monomer are affected and the β-sheet interactions within each peptide are reduced. A similar pattern was observed for the replica at 310 K (Figure S17, Supporting Information). The effect of CLR01 molecules is more pronounced in the case of interpeptide interactions (Figures 8 and S18, Supporting Information). The contact maps for the Aβ1−42 dimer reveal that residues 36−41 of one monomer are in proximity to residues 11−20 and residues 32−39 of the other monomer. We note that the regions formed by residues 17−21 and the Cterminus are reported to be involved in the dimerization process as they are part of the dimer interface.21 Our simulations indicate that in the reference (no ligand) and negative control (CLR03) systems, the dimer interface mostly involves hydrophobic residues. Most of these hydrophobic residues lie in the CHC region (Figures 8 and S18, Supporting

Figure 7. Most populated clusters from the REMD simulations of the reference system Aβ1−42 (top), Aβ1−42 with six CLR01 (middle), and the negative control Aβ1−42 with six CLR03 (bottom). CLR01 and CLR03 as well as lysine and arginine residues are highlighted. Aβ1−42 is shown with the same color code as in Figure 2.

tional changes, to feature more disordered and rather extended structures. These monomers follow a different dimerization F


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Figure 9. Interpeptide contacts between the two monomer units (in orange and magenta) of the Aβ1−42 dimer (reference, top left) and the Aβ1−42 dimer with six CLR03 molecules (negative control: bottom left) are mainly driven by hydrophobic interactions, although CLR03 seems to promote additional polar interactions. The addition of CLR01 results in the loss of intermonomer contacts and a different distribution of hydrophobic residues (top, right). The aggregation process is then mediated by tweezer−tweezer interactions (bottom, right). The structures shown are representative snapshots where the amino acids at the interface are colored according to the type of residues (nonpolar residues: white; basic residues: blue; acidic residues: red; and polar residues: green).

S9, Supporting Information). Although a small population of the S1 ensemble has lower SASA, the probability distribution of the hydrophobic SASA (Figures 10 and S20, Supporting

Figure 8. Interpeptide contact probability maps for the Aβ1−42 dimer (reference: top), the Aβ1−42 dimer with six CLR01 (middle, left), the Aβ1−42 dimer with six CLR03 (negative control: middle, right), the Aβ1−42 dimer with two CLR01 (bottom, left), and the Aβ1−42 dimer with two CLR03 (negative control: bottom, right). The contact maps correspond to the production ensemble S1 and are averaged over all 50 000 frames of the ensemble.

Figure 10. A. Percentage distribution of SASA of the hydrophobic residues (nm2). B. Average values of the vdW interaction energy (kcal/ mol) between the two peptides for the Aβ1−42 dimer in the absence and presence of CLR01 or CLR03 molecules. All values are calculated for the production ensemble S1.

Information), in agreement with previous studies.17,20,21 Interestingly, the presence of six CLR01 molecules disrupts many interpeptide contacts, indicating the lack of a dimer interface (Figures 9 and S19, Supporting Information). To a less extent, a similar effect is found with only one CLR01 molecule around K16 of each monomer. There, residues 10−15 are in contact with residues 8−14 and residues 34−39 of the interacting monomers. Interestingly, in the system with two CLR03 molecules, more interpeptide contacts are established with respect to the reference. There, the CHC residues of one monomer are in contact with both the CHC region and residues 26−32 of the other monomer. Taken altogether, CLR01 affects both intra- and intermolecular contacts, especially so the latter. 3.3.3. Effect of the Tweezer on the Solvent Accessible Surface Area (SASA). Because the dimer interface is mostly hydrophobic, we estimated the values of SASA for the hydrophobic residues, averaged over two chains. We found that the SASA for the hydrophobic residues is increased by approximately 4 nm2 in the presence of six CLR01 molecules, signifying more exposure of the hydrophobic residues (Table

Information) clearly indicates that the presence of six CLR01 molecules disrupts interpeptide hydrophobic interactions, which are crucial for dimer formation.70,71 This is in line with the calculated vdW interaction energies, (modular values) which decrease in the system with six CLR01 molecules with respect to the reference and control simulations (Figure 10, Table S10, Supporting Information). We further investigated the effect of CLR01 and CLR03 molecules on the structural properties of the Aβ1−42 dimer by estimating the potential of mean force (PMF) for each system as a function of the radius of gyration (Rg) and the number of interchain contacts (Figures 11 and S22, Supporting Information). For the S1 ensemble, the PMF plot of Rg versus the number of interchain contacts reveals that the representative values at the minimum-energy basin were centered at (1.28 nm, 497 and 1.28 nm, 529) in the Aβ1−42 dimer. The corresponding PMF plot in the system with six CLR01 molecules changed drastically, with a minimum-energy basin at 1.65 nm and 34 interchain contacts. This is an indication of the G


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approximately 80% of all secondary structure motifs. β-sheet regions account for 16% of all elements, in good agreement with previous computational studies (16−19%).20,55 Computational reports17,19,20,64 also indicate that the dimer interface in Aβ1−42 comprises C-terminal residues and residues 17−21 in the CHC region. Here, the network of interchain contacts involves primarily C-terminal residues and residues 10−17 in the Aβ1−42 dimer. We note that, although these computational studies of the Aβ1−42 dimer are a valuable reference, the exploration of the full conformational space of dimers is beyond the scope of the present work. Thus, we cannot entirely compare our results to those from computational studies that focused on the sampling of equilibrated Aβ1−42 dimers. Instead, our aim is to provide insights into the molecular basis for the effect of a supramolecular ligand on the Aβ interactions that drive together two monomers initially placed apart. This way, the analysis of the REMD simulations allows us to propose a mechanism of regulation of Aβ1−42 aggregation by the molecular tweezers. The addition of six CLR01 molecules leads to a decrease in the β-sheet content of the dimer (∼4%). The binding of six CLR01 molecules also results in a less compact form of the aggregate with less interchain contacts than the reference dimer (Tables S7 and S8). The hydrophobic interactions are less favored, thus reducing the probability of dimer formation (by “dimer”, we mean the structure formed in the absence of CLR01). Secondary tweezer−protein and tweezer−tweezer interactions lead to a different aggregation pathway of the Aβ1−42 monomers with respect to the reference system. Interestingly, we also observed interactions between ligands in the simulations with the inactive control CLR03, albeit these interactions were not conserved. This suggests that, not only the interaction between tweezers but also their unique ability to thread lysine side chains inside their cavity while establishing secondary interactions with the protein environment are the keys for the antiamyloid effect. To the best of our knowledge, this is the first all-atom and explicit-solvent computational study of the interactions between the Aβ1−42 dimer and the important class of supramolecular ligands represented by the tweezer. By providing mechanistic insights of how the molecular tweezer CLR01 affects the association of two Aβ1−42 monomers, we contribute to the structural characterization of the influence of ligands on oligomerization, which is a key step for the future development of supramolecular agents with therapeutic potential against amyloid.

Figure 11. Two-dimensional PMF plots as a function of the radius of gyration (Rg) and the number of interpeptide contacts for each system, ensemble S1. Each contour level corresponds to 0.5 kcal/mol.

low interchain contacts in the presence of six CLR01 molecules. The PMF plots for other systems, that is, with two CLR01 or CLR03 molecules, are very similar to that of the Aβ1−42 dimer. The representative values at the minimum were found to be (1.49 nm, 104 and 1.42 nm, 180), (1.38 nm, 244 and 1.38 nm, 273), and (1.31 nm, 281) for the Aβ1−42 dimer with two CLR01, with six CLR03, and with two CLR03 molecules, respectively. This suggests that in the presence of six CLR01, the Aβ1−42 dimer adopts an extended form (large Rg) and dimer formation is not energetically favorable. Similarly, we analyzed Rg versus the number of hydrogen bonds in the dimer and Rg versus the interchain vdW interaction energy (see free energy landscape discussion, Figures S22 and S23, Supporting Information). Taken together all PMF plots, we concluded that the formation of the Aβ1−42 dimer is less favorable in the presence of six CLR01 molecules. CLR01 influences both intra- and intermonomer interactions and leads to a less compact dimer structure with less interpeptide contacts. The presence of only two CLR01 molecules around K16 of each monomer does not significantly influence the dimer assembly.

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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.7b10530. Extended computational details including convergence analysis, QM/MM calculations, and free energy landscapes (PDF)

4. CONCLUSIONS The calculated Aβ1−42 dimer is, in general, consistent with experimental data.18,67,72 The CCS value for the Aβ1−42 dimer obtained from our REMD simulations (1291 Å2) is in good agreement with the experimental values of 1243 and 1256 Å2.25,67 The secondary structure analysis of the calculated Aβ1−42 dimer indicates that it adopts a mostly disordered structure with coil and turn elements accounting for

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Elsa Sanchez-Garcia: 0000-0002-9211-5803 H


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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank D. Rosenman for providing the initial coordinates for the Aβ1−42 monomer. E.S.-G. acknowledges a Plus-3 Grant of the Boehringer-Ingelheim Foundation and the computational time provided by the Computing and Data Facility of the Max Planck Society and the Max Planck Institute für Kohlenforschung. The German Research Foundation (DFG) supported this work via the Collaborative Research Center SFB1093 (K.B.-R., S.M., and E.S.-G.) and the Excellence Cluster RESOLV EXC1069 (E.S.-G., infrastructure support).

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Mechanism of Inhibition of Beta Amyloid Toxicity by Supramolecular

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