Molecular Mechanism of the Inhibition and Remodeling of Human Islet

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Molecular Mechanism of the Inhibition and Remodeling of Human Islet Amyloid Polypeptide (hIAPP ) Oligomer by Resveratrol from Molecular Dynamics Simulation 1-37

Qianqian Wang, Lulu Ning, Yuzhen Niu, Huanxiang Liu, and Xiaojun Yao J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/jp507529f • Publication Date (Web): 12 Dec 2014 Downloaded from http://pubs.acs.org on December 19, 2014

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The Journal of Physical Chemistry

Molecular Mechanism of the Inhibition and Remodeling of Human Islet Amyloid Polypeptide (hIAPP1-37) Oligomer by Resveratrol from Molecular Dynamics Simulation Qianqian Wang,† Lulu Ning,‡ Yuzhen Niu,‡ Huanxiang Liu,†,‡,* and Xiaojun Yao‡,§ †

School of Pharmacy, Lanzhou University, Lanzhou 730000, China

‡

State Key Laboratory of Applied Organic Chemistry and Department of Chemistry, Lanzhou

University, Lanzhou 730000, China §

State Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied

Research in Medicine and Health, Macau University of Science and Technology, Taipa, Macau, China

1

ACS Paragon Plus Environment


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ABSTRACT: Natural polyphenols are one of the most actively investigated categories of amyloid inhibitors, and resveratrol has recently been reported to inhibit and remodel the human islet amyloid polypeptide (hIAPP) oligomers and fibrils. However, the exact mechanism of its action is still unknown, especially for the full-length hIAPP1-37. To this end, we performed the all-atom molecular dynamics simulations for hIAPP1-37 pentamer with and without resveratrol. The obtained results show that the binding of resveratrol is able to cause the remarkable conformational changes of hIAPP1-37 pentamer, in terms of secondary structures, order degree and morphology. By clustering analysis, two possible binding sites of resveratrol on the hIAPP1-37 pentamer were found, locating at the grooves of the top and bottom surfaces of β-sheet layer, respectively. After the binding free energy calculation and residue energy decomposition, it can be concluded that the bottom site is the more possible one, and that the nonpolar interactions act as the driving force for the binding of hIAPP1-37 to resveratrol. In addition, Arg11 is the most important residue for the binding of resveratrol. The full understanding of inhibitory mechanism of resveratrol on the hIAPP1-37 oligomer and the identification of its binding sites on this protein are helpful for the future design and discovery of new amyloid inhibitors.

KEYWORDS: type 2 diabetes, islet amyloid polypeptide, resveratrol, molecular dynamics simulation

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INTRODUCTION Amyloid, a general term for typically β-sheet-rich, ordered, insoluble and misfolded protein aggregates, is associated with a wide variety of human neurodegenerative diseases.1-3 Classical examples of intrinsically disordered sequences that can form amyloid are β-amyloid (Aβ) peptide related with Alzheimer’s disease (AD)4,5 and human islet amyloid polypeptide (hIAPP, also known as amylin) that’s responsible for type 2 diabetes6-8. IAPP is a 37-residue hormone (KCNTATCATQ10RLANFLVHSS20NNFGAILSST30NVGSNTY), which is composed of multiple function regions9,10 including the N-terminal region (residues 1-19) that involves membrane and insulin binding, the amyloidogenic region (residues 20-29) and the C-terminal region (residues 30-37) that involves peptide self-association. The amyloid formation of hIAPP is believed to contribute to the death or loss of β-cells and to graft failure after islet transplantation.11-13 At present, for type 2 diabetes, there is still no effective drug to be entered clinical trial stage. A lot of researches indicate that stabilizing the native-unfolded monomers, inhibiting the growth of oligomers or remodeling the oligomers are crucial for the inhibition of amyloid aggregation.14-18 Therefore, finding an effective inhibitor that’s able to alter the pathway of hIAPP fibrillation is to be considered as a promising strategy in the treatment of type 2 diabetes. Presently, natural polyphenols are one of the most active categories of amyloid inhibitors.19-22 Apart from the common advantages of small-molecule inhibitors including the easy crossing of the blood-brain barrier, stability in biological fluids and 3



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not involvement in immunological response, they are of natural origin, are present in many foods and are usually assumed by humans with the diet in variable amounts and continuous way.19,23 Given their strong inhibitory activity and the reliable safety, natural polyphenol compounds have gained more and more attention of amyloid researchers in recent years. Resveratrol (trans-3,4’,5-trihydroxystilbene) is a small natural polyphenol and is particularly abundant (130-220 µM) in red wine. This compound has recently been reported to be capable of directly interfering with the amyloid aggregation of different peptides and reducing their neurotoxicity.24-29 Using the different biophysical methods (ThT fluorescence, atomic force microscopy (AFM), attenuated total reflection (ATR) FTIR spectroscopy and a cytotoxicity assay on a pancreatic cell line), Winter’s group showed that resveratrol could not only inhibit the IAPP amyloid formation both in solution and on the aggregation-fostering lipid membranes, but also have remarkably enhanced effects on the survival of the INS-1E cell line treated with IAPP.24 Then, considering the importance of heterogeneous membranes in biological process, the same group further extended their study to model raft membrane systems.25 Via the fluorescence microscopy techniques, they investigated the interaction of IAPP with lipid model raft mixtures and testified the efficient inhibition of resveratrol against IAPP amyloid formation once again. Since resveratrol is nontoxic to pancreatic β-cells, this natural polyphenol is thought to have the potential to be developed as drug candidate for type 2 diabetes. However, no mechanistic evidence is presented to explain how resveratrol or other natural polyphenol compounds exert their effects. 4


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To study the molecular mechanism of inhibition and remodeling of hIAPP1-37 oligomer by resveratrol and identify the possible binding sites of this inhibitor on the hIAPP, in this study, we performed all-atom molecular dynamics (MD) simulations for hIAPP1-37 pentamer in the presence and absence of resveratrol in explicit solvents (Figure 1). The obtained results will provide the valuable information for the discovery of inhibitors against the aggregation of hIAPP.

METHODS Simulation Setup. The initial pentameric coordinates of hIAPP1-37 were kindly provided by the Tycko lab based on the solid-state NMR-based methods.30 Before MD simulations, the C-terminal of each chain of hIAPP1-37 pentamer was capped by N-methyl (NME) group to mimic its structural state in physiological condition. Resveratrol molecule was taken from the PubChem Compound library. And its force field parameters were created using Antechamber program in Amber 10.0 package31 and described by the General Amber Force Field (GAFF)32. The partial atomic charges of resveratrol were determined by the restrained electrostatic potential (RESP) fitting technique33,34, and geometric optimization and the electrostatic potential calculation were performed at the hf/6-31g* level of Gaussian 09 suite35. The Tleap module of Amber 10.0 package31 was first used to add all the missing hydrogen atoms. The protein and waters adopt the Amberff99SB force field36. Then, the appropriate number of chloride counterions was added to maintain the electro-neutrality of systems and each system was immersed into a cubic periodic box of TIP3P waters37 with at least 10 Å distance around the solute. For the 5



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hIAPP1-37-resveratrol system, 10 resveratrol molecules were randomly placed around the hIAPP1-37 pentamer. The molar ratio of resveratrol to hIAPP is 2:1, which is equal to that used in another computational study38 and slightly higher than that used experimentally by Winter et al24. Overall, the system of hIAPP-resveratrol complex consists of 48,072 atoms, and the hIAPP system has the 39,037 atoms. In our work, all the molecular dynamics simulations were performed using the Amber 10.0 package31. Initially, to remove the bad contacts between the solute and solvent, five stepwise rounds of minimization were carried out in which positional restraint forces applied to all the atoms of the solute were 5.0, 2.0, 1.0, 0.5, 0 kcal/(mol·Å2), respectively. Each round consists of the 2500-step steepest descent and the 2500-step conjugated gradient calculations. Afterwards, we heated the systems from 0 to 310.0 K over a period of 50 ps in the NVT ensemble, and adjusted the solvent density to equilibrate the system under 1 atm pressure in 100 ps in the NPT ensemble by restraining all the atoms of the solute with the harmonic restraint force of 2.0 kcal/(mol·Å2). Four additional separated MD equilibrations of 100 ps were then performed with the decreased restraint forces of 1.5, 1.0, 0.5, 0.1 kcal/(mol·Å2) to all the atoms of the solute, respectively. These were followed by a further equilibration step of 500 ps by releasing all the restraints. In the production phase, 300 ns MD simulations were carried out without any restraint in the NPT ensemble at a temperature of 310.0 K and a pressure of 1 atm. Coordinate trajectory was recorded every 2 ps for the subsequent analysis. Initial velocities were assigned from a Maxwellian distribution at the initial temperature. The temperature of systems was 6


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regulated using the Langevin thermostat. Bond lengths involving hydrogen atoms were constrained using the SHAKE algorithm39, and the equations of motion were integrated with 2 fs time step. The non-bonded cutoff distance was 10.0 Å, and Particle Mesh Ewald (PME) method40 was used to calculate long-range electrostatic interaction. In total, we performed four tasks of molecular dynamics simulations, including three parallel runs for the hIAPP1-37-resveratrol systems (300 ns) and one hIAPP1-37 system without resveratrol (200 ns). Binding Free Energy Calculation. The binding free energy of resveratrol and hIAPP1-37 was calculated based on the MM/GBSA method41-44 implemented in Amber 10.0 package31. Herein, a single trajectory protocol was adopted, and a total of 500 snapshots were extracted from 250 to 300 ns with a time interval of 100 ps. For each snapshot, the binding free energy was obtained as follows:

∆G = Gcomplex − (GIAPP + Gresveratrol)

Gtotal = Egas + Gsol − T ∆S

Egas = Evdw + Eele

(1) (2) (3)

Gsol = Gsol_np + Gsol_polar Gsol_np = γ ⋅ SAS

(4) (5)

where Gcomplex, GIAPP and Gresveratrol are the free energies of IAPP-resveratrol complex, IAPP and resveratrol, respectively. G was calculated based on an average over the extracted snapshots, and each state was estimated from the gas-phase energy Egas, solvation free energy Gsol. Egas contains an electrostatic term (Eele) and a van der Waals term (Evdw) including the 1-4 interactions. The solvation energy is further 7



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decomposed into the polar (Gsol_polar) and nonpolar solvation energy (Gsol_np). The former was calculated by solving the Generalized Born (GB) model45. Dielectric constants for solute and solvent were set to 1 and 80, respectively. The latter was estimated by the solvent accessible surface area (SASA) determined using a water probe radius of 1.4 Å. The surface tension constant γ was set to 0.0072 kcal/(mol⋅ Å2).46 Here, the effect of conformational entropy was omitted based on several aspects of considerations. Firstly, our aim is to identify which binding site is more possible one and the rank of binding free energy not its absolute value is more important. Many researches indicate that without considering conformational entropy, MM/GBSA and MM/PBSA could still achieve satisfactory accuracy of ranking ligand affinities.41,47-49 Furthermore, the entropic calculation is time-consuming and computationally expensive, and the computed entropy will have a large fluctuation if relative small number of snapshots were used.41 To obtain the contribution of individual residue of hIAPP1-37 pentamer to the total binding free energy, the residue energy decomposition analysis was also performed with the MM/GBSA method. Simulation Analysis. In order to characterize the fibril-like state of hIAPP1-37 pentamer, the nematic order parameter P250,51 was calculated. Considering that the full-length hIAPP1-37 is not a linear molecule and includes the turn structure, we calculated the P2 values based on the Cα atoms of two linear β-stands (residues 7-17 and 28-35) of each monomer. Here, the cutoff value of P2 was set to 0.5 as used in Li’s study18. That is, if P2 is > 0.5, the system has the propensity to be in an ordered 8


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state. Otherwise the system tends to be disordered. Principal component analysis (PCA), an effective tool for investigating the dominant motion of proteins over a MD simulation, can filter out all other unimportant motions by projecting the trajectory along the direction described by a selected eigenvectors.52 The standard PCA adopts the Cartesian coordinates. However, this method can’t discriminate the internal motion from the trivial overall motion very well.53,54 To avoid this problem, in this study, we performed the PCA using the backbone dihedral angles of hIAPP1-37 peptide (dihedral PCA). The obtained two largest principal component eigenvectors (dihedral PC1 and PC2) from dihedral PCA were used as the reaction coordinates to construct the free energy landscape (FEL).55,56 By using this procedure, it is meaningful to directly show the effects of resveratrol on the global dynamics of hIAPP1-37 pentamer. The contact number between two chains of hIAPP1-37 pentamer or between resveratrols and hIAPP1-37 pentamer was defined as the numbers of heavy atom pairs with a distance < 6.0 Å. The dictionary of secondary structure of proteins (DSSP) program developed by Kabsch and Sander57 was used to assign secondary structures of hIAPP1-37 pentamer.

RESULTS Stability of Simulation Systems. The equilibration of MD trajectories obtained from four simulation systems (three hIAPP1-37-resveratrol complexes and one hIAPP1-37) was first monitored by the root mean square deviation (RMSD) of backbone atoms of hIAPP1-37 pentamer (Figure 2A and Figure S1A). As can be seen from Figure 2A, the RMSD values for hIAPP1-37-resveratrol complexes go up 9



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gradually in the first 75 ns, and become converged from 230 ns. RMSDs of hIAPP1-37 pentamer without resveratrols keep stable from 100 ns (Figure S1A). Furthermore, the hIAPP1-37 pentamer with resveratrols has the bigger average values (6.3 Å for the first complex) than that (5.1 Å) without resveratrols, indicating that the binding of resveratrol molecules causes the large structural fluctuations of hIAPP1-37 pentamer. The smaller RMSDs for the third trajectory of hIAPP-resveratrol complex compared to that for the first two imply that the hIAPP1-37 pentamer in this run may alter little. Then, we calculated the contact numbers between two chains of hIAPP1-37 pentamer and between ten resveratrols and hIAPP1-37 pentamer during the whole MD simulations, respectively, as shown in Figure 2B-2C and Figure S1B. From Figure 2B and Figure S1B, the total number of interchain contacts of hIAPP1-37 pentamer for all runs keeps constant from 150 ns, suggesting that the peptide-peptide interactions of this pentamer are stable. From Figure 2C, the contact number between the inhibitor and hIAPP1-37 pentamer fluctuates stably in the last 50 ns, which suggests that these ten resveratrols surrounding around hIAPP1-37 pentamer may find their stable binding positions on or nearby the pentamer. The large fluctuation for the third run of hIAPP-resveratrol complex relative to other two complex runs suggests that resveratrols in the third complex do not form the stable interactions with hIAPP, and may have not found their proper positions around the pentamer like in other two runs. In addition, it seems that just the unstable interactions lead to the little conformational changes of hIAPP1-37 pentamer of the third complex, as reflected from its small RMSD values and the few interchain contacts (Figure 2A and 2B). 10


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Effects of Resveratrol on the Structure of hIAPP1-37 Pentamer. The nematic order parameter P250,51 was used to discriminate between the ordered and disordered conformations and characterize the order degree of hIAPP1-37 pentamer. The calculated results were plotted in Figure 3. From Figure 3, it can be seen that in the absence of resveratrols, the P2 values of both C-terminal and N-terminal β-strands always keep more than 0.5, indicating that the pentamer maintains the initial ordered conformation during the whole simulation. However, upon the presence of resveratrols, the distribution of P2 values of hIAPP1-37 pentamer moves to the left. For example, the large probability of P2 values of C-terminal β-strand locates at < 0.5, meaning that the C-terminal ordered β-strand of this pentamer becomes disordered and unstable. The reduction of P2 values of N-terminal is smaller than that of C-terminal suggests that the former has more stable structure than the latter. Figure 3 also shows that the conformational changes of hIAPP1-37 pentamer in the third trajectory of hIAPP-resveratrol complex are not relatively notable, with both the large P2 probabilities larger than 0.5. To study the effects of resveratrol on the conformational space of hIAPP1-37, principal component analysis52 was then performed based on the last equilibrated 50 ns trajectory. First, we calculated the covariance matrix using the backbone dihedral angles of hIAPP1-37 peptide, and diagonalized them to obtain the principal component eigenvectors. Then, the first two largest principal components from dihedral PCA were used as the reaction coordinates to construct the free energy landscape,55,56 as shown in Figure 4. From Figure 4, the conformations of hIAPP1-37 pentamer in the 11



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absence of resveratrols tend to form two well-defined basins. But upon the presence of resveratrols, more disperse basins appear on the free energy surface, indicating that the initially ordered conformation of hIAPP1-37 pentamer is broken by resveratrols and then the conformational space becomes larger. Compared to the first two complexes, hIAPP1-37 pentamer of the third complex has the smaller conformational changes. As we all know, the stability of amyloid oligomers or fibrils is closely associated with the β-sheet content, and the changes of ordered state of an oligomer are usually accompanied with the changes of its secondary structures.18,58-60 To investigate whether the inhibition of resveratrol could induce the changes of secondary structure composition, we further calculated the contents of different secondary structures of hIAPP1-37 pentamer (Table 1). It can be seen from Table 1 that the hIAPP1-37 pentamer without resveratrols has no helical structure, and is mainly composed of β-sheet (51.16%) and coil (41.81%) structures. However, the presence of inhibitors causes the great reduction of β-sheet content from 51.16% to 36.31% and 38.94% in the first two complexes. Meanwhile, for the first trajectory of hIAPP-resveratrol complex, the contents of helix and coil structures of hIAPP1-37 pentamer increase from 0 to 3.68% and 41.81% to 56.82%, respectively, suggesting that part of β-sheet structure converts into the helix and disordered coil structures. Furthermore, the dominant secondary structure also changes from β-sheet to coil. In the second complex trajectory, we observed the similar phenomenon. For the third run of hIAPP-resveratrol complex, the changes of secondary structure contents of hIAPP1-37 pentamer also have the similar trends, but on less degree. Finally, by further monitoring the evolution of 12


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secondary structure of each residue over time (shown in Figure 5), we found that the β-sheet content of hIAPP1-37 pentamer in the C-terminal region of edge chains (chain A and E) had a significant reduction, resulting in a less-fibril state. The obvious reduction of β-sheet content of C-terminal is in good accordance with the reduction of C-terminal order degree in Figure 3. Combining the above analysis, the addition of small-molecule inhibitor resveratrol indeed has much impact on the overall structure of hIAPP1-37 pentamer, mainly reflecting in the reduction of order degree and β-sheet contents. However, in the third trajectory of hIAPP1-37 with resveratrol, the inhibitor produced smaller impact on this pentamer compared to the first two trajectories, suggesting that resveratrol still has not found its proper binding site in the simulation process of 300 ns. The inconsistent phenomena from three trajectories were caused by the intrinsic randomness of molecular dynamics simulation method, which is why multiple trajectories are used generally to obtain the reliable results. From the principle, through the long molecular dynamics simulation, multiple trajectories should be able to gain the consistent results. Actually, on one side, due to the limitation of computational source, we only can simulate the studied system at the finite time. On the other side, sometimes, one trajectory may fall into one local minimum and need a long time to jump out of this point. These reasons lead that each trajectory may display different features. Despite this, from the long time molecular dynamics simulation, we still can obtain the valuable and instructive information by extracting the consistent information from multiple trajectories and averaging over the 13



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corresponding trajectory. Therefore, the following analysis about the binding sites of resveratrol on the hIAPP1-37 is based on the first two consistent trajectories. Identification of the Possible Binding Sites of Resveratrol on hIAPP1-37 Pentamer. Clustering analysis is generally used to extract the representative structures from the molecular dynamics trajectory. Here, the conformational clustering analysis for the first two runs of hIAPP-resveratrol complex was carried out with the Ptraj module of Amber 10.031 using the last 50 ns trajectory. Snapshots were collected at 4 ps interval. SOM algorithm based on the pairwise similarity (measured by RMSD) was applied to generate clusters. Prior to the clustering analysis, each conformation of hIAPP1-37-resveratrol complex was superposed onto the initial coordinate to remove rigid-body motions, including the rotation and translation. During the calculation process, three conformational clusters were generated, which occupy 52.16%, 43.67%, 4.17% and 50.29%, 47.43%, 2.28% for the first and second trajectories, respectively, indicating that the first two clusters account for most of all the conformations. Then, we aligned the representative structures of the first two clusters of each run, as shown in Figure 6. From Figure 6A and 6B, it’s notable that after the long-time simulation with resveratrol, the morphology of hIAPP1-37 pentamer has great changes. The initial ordered β-sheet layer of hIAPP1-37 pentamer is no longer planar and has a certain degree of torsion. In addition, most of the β-sheet structure of edge chains (chain A and E) is broken by resveratrol. These phenomena indicate that hIAPP1-37 pentamer became less stable again when resveratrol was added. As for the distribution of resveratrol around the hIAPP1-37 pentamer, most of them undergo the 14


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extensive movement around the protein, whereas the small molecules at two positions keep very stable at its position at the last equilibrated trajectory. The consistency of two runs indicates that these two sites are likely to be the possible binding sites of resveratrol on hIAPP1-37 pentamer. Further structural analysis shows that the two binding sites, Site I and II, locate at the grooves of the top and bottom surfaces of β-sheet layer of hIAPP pentamer, as shown in Figure 7. The groove of Site I is composed of residues Ser29, Thr30 and Asn31 of each chain of hIAPP1-37 pentamer. On this site, resveratrol is slightly close to chain E, whose aromatic rings are perpendicular to β-sheet layer and can form the strong van der Waals interactions with the side chain of Asn31 of chain C and D. The phenolic hydroxyl of resveratrol forms the hydrogen bond (H-bond) interactions with the side-chain –OH and main-chain –C=O of Ser29 of chain D, respectively. The bottom groove (Site II) also mainly consists of three residues of each chain, namely residues Arg11, Leu12 and Ala13. Different from Site I, this site approaches chain A of β-sheet layer. Relative to Asn, Arg has the longer side chains and is likely to form the stronger van der Waals interactions with aromatic rings of inhibitor. As a residue rich of positive charge, Arg can also form H-bonds with the –OH of resveratrol. Additionally, another H-bond between the main-chain –NH of Ala13 and the –OH of resveratrol also exists. In order to compare the binding affinities of resveratrol and hIAPP1-37 pentamer on these two sites, we then calculated their binding free energy in both trajectories using the MM/GBSA method41-44. The obtained average binding free energy and the 15



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detailed contributions of various energy components were shown in Table 2. As can be seen from Table 2, the nonpolar interactions (∆Gnonpolar) including van der Waals (∆Evdw) and nonpolar solvation (∆Gsol_np) terms are the driving force for the binding of resveratrol to hIAPP1-37, and the total polar contributions (∆Gpolar) are unfavorable for their binding. In addition, the binding free energy of resveratrol and hIAPP1-37 on Site II (-18.17 kcal/mol) is obviously lower than that on Site I (-3.98 kcal/mol), indicating that resveratrol has the stronger binding affinity with hIAPP1-37 on Site II. Then, Site II is more likely to be the binding site of this inhibitor on hIAPP1-37 pentamer. The similar results of two runs imply the reliability of our calculations. The decomposition of binding free energy is helpful for us to identify the key residues of hIAPP1-37 for the binding to resveratrol. For Site II, the results of energy decomposition in both two runs were shown in Figure 8. Apparently, the binding of resveratrol and hIAPP1-37 pentamer mainly relies on chain A, B and C. And residue Arg11 provides the larger contributions than Leu12 and Ala13, who provides 38.45% and 40.22% energy contributions for the binding to resveratrol in two runs, respectively. Finally, the contribution sources of three key residues were further analyzed and the obtained results were shown in Figure 9. From Figure 9, all the three residues (Arg11, Leu12 and Ala13) interact with resveratrol mainly through van der Waals and electrostatic interactions, and the total solvation contributions are unfavorable for the binding of resveratrol.

DISCUSSION Amyloid aggregation is one of the hallmarks of many neurodegenerative diseases, 16


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whose intermediates (oligomers, protofibrils) and mature fibrils display different toxicity. Accordingly, the discovered small-molecule inhibitors counteracting amyloid aggregation were shown to possess different abilities:61 (1) stabilizing the toxic amyloid precursors; (2) hindering the growth of toxic oligomers or speeding that of fibrils; (3) inhibiting fibril growth and deposition; (4) disassembling performed fibrils; (5) increasing amyloid clearance. For natural polyphenol inhibitors, most of them can inhibit the formation and growth of both oligomers and fibrils, such as our studied resveratrol. From the perspective of geometric structure, all the efficient natural phenols share the common functional characteristics: two aromatic rings with two to six atom linkers and a minimum number of three -OH groups on the rings. The former can generate the aromatic-hydrophobic (aromatic) interactions with the hydrophobic residues of amyloid peptide, and the phenolic hydroxyls of inhibitors are capable of forming hydrogen bonds with the polar groups of amyloid. Since the hydrophobic and hydrogen bond interactions are the driving force of amyloid assembly, the breaking of either of them is unfavorable for the stability of amyloid aggregates. In our study, two aromatic rings of resveratrol form the strong aromatic-hydrophobic interactions with the side chain of Arg11/Asn31 of hIAPP, and the –OH groups on the rings also form H-bonds with the side-chain/main-chain –NH of several residues. The formation of these interactions explains the reason why the stability and order degree of hIAPP1-37 pentamer decrease in the presence of resveratrol to some extent. In addition, we find that even though the inhibitors belong to the same category, they are also likely to have the different binding modes with amyloid. EGCG62, 17



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another natural phenol inhibitor, has the bigger structure than resveratrol. Our previous study indicates EGCGs may bind to the hIAPP1-37 pentamer on three main sites, and the most possible one locates at the C-terminal amyloidogenic region of hIAPP.59 When interacting with hIAPP, the two aromatic rings of EGCG form π-π stacking interactions with the aromatic residues of hIAPP on the surface of β-sheet layer, and the third aromatic ring is away from the surface and stretches into the solvent. However, for the relatively small polyphenol resveratrol, due to the special planar structure, it lies in the groove on the surface of hIAPP1-37 pentamer with its aromatic rings perpendicular to β-sheet layer. The more possible Site II for the resveratrol binding lies at the N-terminal of hIAPP1-37 rather than the C-terminal. Considering that the toxicity of hIAPP is partly derived from the breaking of β-cell membrane and the N-terminal function region of hIAPP is responsible for binding to cell membrane, the addition of many molecules of resveratrol has some protective effects on the β-cell. As we know, amyloid oligomers or fibrils grow in two directions, by lateral association and longitudinal elongation. Although the binding sites of EGCG and resveratrol on hIAPP1-37 are different, they all locate at the surface of β-sheet layer rather than the end of the layer, indicating that they may inhibit the growth of hIAPP oligomers by blocking the lateral association. For our studied resveratrol, despite of not interacting with the amyloidogenic region of hIAPP, it can inhibit the amyloid formation by inhibiting the lateral association of amyloid fibrils. From the in vitro study, there is no indication that typical fibrils formed when full-length hIAPP was incubated with resveratrol,63,64 implying that this inhibition of 18


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lateral growth of resveratrol is sufficient to fully abolish the formation of amyloid fibrils. Actually, this possible inhibitory mechanism was also mentioned in the previous study.38 Differently, the goal of that study is to investigate the effect of resveratrol on the early steps of aggregation, rather than the interaction of resveratrol with the preformed fibril in our work. Furthermore, the peptide model used in that study was a 6-residue segment of hIAPP (hIAPP22-27), and the initial conformation of the preformed tetramer was extracted from the hIAPP22-27 fibrils65. In addition to the potential problem of the stability of this unnatural tetramer, whether the obtained conclusion based on the short hIAPP22-27 fragment can be applied to the full-length hIAPP1-37 remains to be determined.

CONCLUSION In this study, by performing the all-atom molecular dynamics simulations, we investigated the inhibitory mechanism of resveratrol on human islet amyloid polypeptide (hIAPP1-37) oligomer. A series of analysis show that the presence of resveratrol can interrupt the structural stability of hIAPP1-37 pentamer and induce the great conformational changes. For instance, upon the addition of resveratrol, both the β-sheet content and order degree of hIAPP1-37 pentamer notably decrease. By clustering analysis, we found two possible binding sites of resveratrol on the surface of hIAPP1-37 pentamer, and they existed on the top and bottom of β-sheet layer, respectively. The obtained results of energy calculation show that resveratrol has the stronger binding affinity with hIAPP1-37 pentamer on Site II than on Site I, indicating that Site II is the more possible binding site of resveratrol. Additionally, the nonpolar 19



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interactions including van der Waals and nonpolar solvation terms provide the dominant contributions for the binding of hIAPP oligomers and resveratrol. Our work can deep the understanding of the inhibitory and remodeling mechanism of resveratrol on hIAPP1-37 oligomers and further provide the valuable information for the discovery of new and effective inhibitors against the aggregation of hIAPP.

AUTHOR INFORMATION Corresponding Authors *

E-mail: [email protected].

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grant No: 21375054) and the Fundamental Research Funds for the Central Universities (Grant No: lzujbky-2014-k21). We would like to thank Dr. Robert Tycko for providing the atomic coordinates of the hIAPP1-37 fibrillar models.

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29



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Table 1. Contents of Different Secondary Structures of hIAPP1-37 Pentamer in All Four Systems (Three hIAPP1-37-Resveratrol Complexes and One hIAPP1-37). systems

helix(%)

sheet(%)

turn(%)

coil(%)

0

51.16±0.02

7.02±0.02

41.81±0.05

IAPP+Resv01

3.68±0.02

36.31±0.01

3.16±0.01

56.82±0.02

IAPP+Resv02

2.10±0.01

38.94±0.01

10.62±0.03

48.28±0.03

IAPP+Resv03

1.57±0.02

47.89±0.02

6.31±0.05

44.25±0.01

IAPP

30


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Table 2. The Average Binding Free Energy and Its Components Obtained from the MM/GBSA Calculations for hIAPP1-37-Resveratrol Complex (kcal/mol).

contributions

IAPP+Resv01 Site I Site II

IAPP+Resv02 Site I Site II

∆Eele

-3.21±0.35

-12.77±0.67

-4.26±0.35

-10.51±0.53

∆Evdw

-5.16±0.22

-28.49±1.66

-6.38±1.01

-18.31±2.26

∆Eint

0

0

0

0

∆Egas

-8.37±0.20

-41.26±2.91

-10.64±0.59

-28.82±2.69

∆Gsol_np

-1.37±0.06

-4.04±0.50

-1.09±0.43

-2.93±0.48

∆Gsol_polar

5.75±1.12

27.12±3.56

5.08±1.22

19.88±0.33

∆Gsol

4.38±0.98

23.08±1.34

3.99±0.65

16.95±0.64

∆Gpolar

2.55±0.64

14.35±0.29

0.82±0.07

9.37±0.50

∆Gnonpolar

-6.53±0.05

-32.53±0.96

-7.47±0.55

-21.24±0.82

∆Gbind

-3.98±0.69

-18.17±1.25

-6.65±0.62

-11.86±1.32

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Figure Captions: Figure 1. The starting (left) and ending (right) conformations of hIAPP1-37 pentamer in the MD simulation, and the geometric structure of resveratrol (middle). Figure 2. Time series of (A) the RMSDs of backbone atoms of hIAPP1-37 pentamer, (B) the total interchain contact number of hIAPP1-37 pentamer, and (C) the total contact number between ten resveratrols and hIAPP1-37 pentamer in three complexes. Figure 3. Probability distribution of the order parameter (P2) values of C-terminal (left) and N-terminal (right) β-strands. Figure 4. Free energy landscapes (in kcal/mol) of hIAPP1-37 pentamer in the presence and absence of resveratrol. Figure 5. Time series of secondary structure changes of each residue of hIAPP1-37 pentamer calculated by DSSP algorithm. Here, we labeled a coil by “C”, a pi-helix by “I”, α-helix by “H”, a 310-helix by “G”, a turn by “T”, an anti-parallel β-sheet by “B”, a parallel β-sheet by “b”. Figure 6. Two identified binding sites of resveratrol on the hIAPP1-37 pentamer in (A) the first and (B) second systems, respectively. Yellow and medium blue structures are the representative structures from the first and second clusters, respectively. Figure 7. The binding modes of resveratrol and hIAPP1-37 pentamer on (A) Site I and (B) Site II, respectively. Figure 8. Residue contribution of hIAPP1-37 pentamer to the resveratrol binding on Site II in the first (A) and second (B) systems, respectively. Figure 9. Contribution sources of three key residues for the binding to resveratrol. 32


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The energies were averaged by chain A, B and C.

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Figure 1. The starting (left) and ending (right) conformations of hIAPP1-37 pentamer in the MD simulation, and the geometric structure of resveratrol (middle). 173x49mm (300 x 300 DPI)


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Figure 2. Time series of (A) the RMSDs of backbone atoms of hIAPP1-37 pentamer, (B) the total interchain contact number of hIAPP1-37 pentamer, and (C) the total contact number between ten resveratrols and hIAPP1-37 pentamer in three complexes. 70x76mm (300 x 300 DPI)



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Figure 3. Probability distribution of the order parameter (P2) values of C-terminal (left) and N-terminal (right) β-strands. 70x80mm (300 x 300 DPI)


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Figure 4. Free energy landscapes (in kcal/mol) of hIAPP1-37 pentamer in the presence and absence of resveratrol. 170x155mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 5. Time series of secondary structure changes of each residue of hIAPP1-37 pentamer calculated by DSSP algorithm. Here, we labeled a coil by “C”, a pi-helix by “I”, α-helix by “H”, a 310-helix by “G”, a turn by “T”, an anti-parallel β-sheet by “B”, a parallel β-sheet by “b”. 150x140mm (300 x 300 DPI)


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Figure 6. Two identified binding sites of resveratrol on the hIAPP1-37 pentamer in (A) the first and (B) second systems, respectively. Yellow and medium blue structures are the representative structures from the first and second clusters, respectively. 173x70mm (300 x 300 DPI)



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Figure 7. The binding modes of resveratrol and hIAPP1-37 pentamer on (A) Site I and (B) Site II, respectively. 174x67mm (300 x 300 DPI)


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Figure 8. Residue contribution of hIAPP1-37 pentamer to the resveratrol binding on Site II in the first (A) and second (B) systems, respectively. 70x76mm (300 x 300 DPI)



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Figure 9. Contribution sources of three key residues for the binding to resveratrol. The energies were averaged by chain A, B and C. 70x49mm (300 x 300 DPI)


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Molecular Mechanism of the Inhibition and Remodeling of Human Islet

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