Controlling in Vitro Insulin Amyloidosis with Stable Peptide

Nov 16, 2015 - Insulin aggregation, to afford amyloidogenic polypeptide fibrils, is an energetically driven, well-studied phenomenon, which presents i...
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Controlling in Vitro Insulin Amyloidosis with Stable Peptide Conjugates: A Combined Experimental and Computational Study Narendra Kumar Mishra,† R. N. V. Krishna Deepak,‡ Ramasubbu Sankararamakrishnan,*,‡ and Sandeep Verma*,† †

Department of Chemistry, DST Thematic Unit of Excellence on Soft Nanofabrication and ‡Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, Kanpur 208016 Uttar Pradesh, India S Supporting Information *

ABSTRACT: Insulin aggregation, to afford amyloidogenic polypeptide fibrils, is an energetically driven, well-studied phenomenon, which presents interesting biological ramifications. These aggregates are also known to form around insulin injection sites and in diabetic patients suffering from Parkinson’s disease. Such occurrences force considerable reduction in hormone activity and are often responsible for necrotic deposits in diabetic patients. Changes in physicochemical environment, such as pH, temperature, ionic strength, and mechanical agitation, affect insulin fibrillation, which also presents intrigue from the structural viewpoint. Several reports have tried to unravel underlying mechanisms concerning the aggregation process taking into account a three aromatic amino acid patch PheB24-PheB25-TyrB26 located in the C-terminal part of the B chain, identified as a key site for human insulin−receptor interaction. The present study describes design and inhibitory effects of novel peptide conjugates toward fibrillation of insulin as investigated by thioflavin T assay, circular dichroism, and AFM. Possible interaction of insulin with peptide-based fibrillation inhibitors reveals an important role of hydrophobic interactions in the inhibition process. Molecular dynamics simulation studies demonstrate that inhibitor D4 interacts with insulin residues from the helix and the C-terminal extended segment of chain B. These studies present a novel approach for the discovery of stable, peptide-based ligands as novel antiamyloidogenic agents for insulin aggregation.

1. INTRODUCTION Protein aggregation is an energetically driven process that plays a crucial role in the onset of several neurodegenerative diseases. Molecular proteinaceous aggregates, either in intracellular or in extracellular space, are formed through a nucleation-dependent process, where a lag phase precedes an exponential growth phase.1−4 In a set of sequential events, protein monomers initially self-assemble into oligomers, which in turn are recruited to form protofilaments, filaments, and other complex polymorphic structures. It is observed that a number of intermediate morphologies emerge during the growth phase of amyloidogenic structures by accessing permissible spatial configurations by polypeptide chains. From the functional standpoint, amyloid protein aggregates are known to impair cell function by interacting with cell membranes, leading to altered intracellular cation content, such as Ca2+ ions, and redox load. Additionally, protein oligomers and fibrils may induce disassembling of cell membrane lipids, modify synaptic plasticity, and induce neuronal cell death.5−8 Many amyloid-based disorders, which include Alzheimer’s (Aβ peptides), Huntington’s (huntingtin, polyQ expansion), and Parkinson’s diseases (α-synuclein) and type-II diabetes (pro-islet amyloid polypeptide), are ascribed to specific protein © 2015 American Chemical Society

and/or peptide sequences, which have a higher propensity to self-assemble in the form of thermodynamically irreversible fibrillar aggregates. The latter are implicated for clinical disease states without any effective treatment in sight.9−14 Insulin has 21 amino acid residues in the A chain and 30 residues in the B chain, which are linked through one intrachain and two interchain disulfide bonds.15 During insulin dimerization, which happens to be a critical step toward its amyloidogenesis, the surface responsible for monomer interaction is almost planar and mainly consists of nonpolar aromatic residues (B22−30). Insulin dimers serve as fibril precursors, which repeat along the fibril length, followed by elongation, stabilized via precursor stacking along the fibril axis.16,17 A study by Jørgensen and co-workers ascribed critical roles to Phe(B25) and hydrophobic interactions in insulin aggregation. It is important to also point out that residues PheB24-PheB25-TyrB26 are part of the β-strand region and support antiparallel β-sheet formation.18 NMR and MD simulations with Phe(B25)-deleted insulin confirmed the Received: August 24, 2015 Revised: November 14, 2015 Published: November 16, 2015 15395

DOI: 10.1021/acs.jpcb.5b08215 J. Phys. Chem. B 2015, 119, 15395−15406

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propyl)carbodiimide hydrochloride (EDC), N,N-dicyclohexylcarbodiimide (DCC), and AMBERLITE IR120 were purchased from Spectrochem, Mumbai, India, and used without further purification. D-Amino acid tryptophan was purchased from Sigma-Aldrich. 1H and 13C NMR spectra were recorded on JEOL-JNM LAMBDA 500 model operating at 500 and 125 MHz, respectively. HRMS mass spectra were recorded at IIT Kanpur, India, on a Waters, Q-Tof Premier Micromass HAB 213 mass spectrometer using a capillary voltage of 2.6−3.2 kV. 2.2. Atomic Force Microscopy (AFM). Imaging of fibrils of insulin sample was done using an atomic force microscope (Molecular Imaging, USA). Newly prepared and aged samples of insulin alone and mixed with D4 were imaged operating under Acoustic ac mode (AAC) with the aid of a cantilever (NSC 12(c) from MikroMasch). The force constant was 0.6 N/m, while the resonant frequency was 150 kHz. The images were taken in air at room temperature with a scan speed of 1.5−2.2 lines/s. The data acquisition was done using PicoScan 5 software, while data analysis was done using of visual SPM. A 10 μL amount of fresh and incubated samples was diluted by 100 μL of 0.1 N HCl water (pH 1.6, 25 mM, NaCl), and out of it 5 μL was deposited onto a newly cleaved mica surface at room temperature and uniformly spread using a spin coater operating at 200−500 rpm (PRS-4000). The sample-coated mica was dried at room temperature in a dust-free space for 30 min and the next 30 min in vacuum, followed by AFM imaging. 2.3. ThioflavinT (ThT) Binding Assay. A freshly prepared stock solution of insulin (170 μM) was incubated alone and with compound D1, D2, D3, and D4 (100 μM) in (pH 1.6, 25 mM NaCl) buffer separately; thereafter, 20 μL of ThT was added from 1 mM stock solution of ThT (prepared in Milli-Q water and stored at 4 °C) to a final concentration of ThT of 20 μM. This sample was allowed to incubate at 65 °C for 20 h. Fluorescence intensity was measured at room temperature with λex 410 nm and λem at 488 nm. The fibrillation/aggregation behavior of insulin alone was also studied separately in the same concentration (170 μM). Fluorescence spectra were recorded on a Varian Luminescence Cary Eclipse with 10 mm quartz cells at 25 ± 0.1 °C. AR-grade hydrochloric acid and GR-grade water were used in these studies. 2.4. Scanning Electron Microscopy (SEM). Field emission scanning electron microscope (FE-SEM) images were acquired on a FEI QUANTA 200 microscope, equipped with a tungsten filament gun operating at WD 10.6 mm and 20 kV. A 10 μL aliquot of fresh and aged solutions of insulin alone and incubated with D3 and D4 were diluted to 100 μL with 0.1 N HCl (pH 1.6), of which 10 μL was mounted on a glass surface. The samples were dried at room temperature for 2 h, followed by vacuum drying for another 30 min and subsequently imaged with (FE-SEM). 2.5. Circular Dichroism Spectroscopy. A freshly prepared stock solution of insulin (170 μM) was incubated alone and with compound D1, D2, D3, and D4 (100 μM) in pH 1.6, 25 mM NaCl at 65 °C separately. All CD experiments were carried out at room temperature, and spectra were collected at a final concentration (17 μM) of insulin for different time intervals using a JASCO J-815 CD Spectrometer and quartz cuvette with a path length of 1 mm. CD spectra were collected between 195 and 270 nm, and each spectrum was the average of 3 scans. The buffer spectrum was subtracted before any measurements to eliminate the contribution of buffer (0.1 N HCl, 25 mM NaCl, pH 1.6).

occurrence of altered conformational signatures, compared to wild-type insulin, causing loss of aggregation behavior.18 Insulin aggregates are formed under clinical conditions around injection sites, and this process could also occur in patients suffering from Parkinson’s disease.19 Such instances lead to reduced biological activity of the hormone and are responsible for necrotic deposits in the patients.20 This behavior has been extensively studied through in vitro studies, where involvement of environmental changes and/or destabilizing conditions such as pH, high temperature, hydrophobic interface, ionic strength, and mechanical agitation, were implicated for inducing insulin fibrillation.21−23 This aggregative propensity is also curious from the structural viewpoint where one would envisage involvement of concerted conformational changes, through the formation of metastable intermediates, leading to fibrillar aggregates, as observed for other proteins. Thus, several focused investigations unraveling underlying mechanisms of this aggregation process are described in the literature.24−29 Experimental evidence support that a three aromatic amino acid patch PheB24-PheB25-TyrB26, located in the C-terminal part of the B chain, is a key site identified for human insulin− receptor interaction.30 In fact, a stretch of amino acids from B22 to B30 is implicated for the formation of a viable hormone−receptor interface. B24 is essential for a hormone “activation process”, and any L-amino acid substitution at this position results in almost complete loss of binding affinity.31 It plays an important role in conformational switching during hormone activation, disulfide pairing, and self-assembly.32 Another interesting feature around this tripeptide patch concerns the presence of “B26-turn”, supported by a B24CO···NHB27 hydrogen bond and trans−cis geometrical isomerization around the B25−B26 peptide bond.33 Various small molecule inhibitors of insulin aggregation are described in the literature.34−45 These molecular entities containing aromatic structures, such as indole, tryptophan, and napthaquinone, are emerging as a new class of inhibitors of amyloidogenic proteins and peptides.46−52 Such biological effects are ascribed to perturbation of aromatic stacking interactions in amyloidogenic fibrils by aromatic inhibitors.53,54 Herein, we present D-Trp-containing peptide conjugates, which target the PheB24-PheB25-TyrB26 site in the insulin B chain and inhibit its aggregation. Such an approach is expected to discover inhibitors that possess proteolytic stability under biological conditions.51 We also studied this inhibition process using molecular dynamics simulations. Several factors such as initial position of peptide conjugates, temperature, and the presence/absence of disulfide cross-links influencing insulin− inhibitor binding were assessed by computational studies to understand folding and unfolding kinetics, especially in the context of insulin aggregation. We propose that the mode of inhibitor binding and its interactions with the PheB24-PheB25TyrB26 motif in the insulin B chain is critical and sheds new possibilities for further optimization of inhibitor structures.

2. MATERIALS AND METHODS 2.1. General. Human insulin and bovine pancreatic insulin (Sigma-Aldrich), ThioflavinT (Fluka), dichloromethane, N,Ndimethylformamide, methanol, triethylamine, and 1,4-dioxane were distilled following standard procedures prior to use. Trifluoroacetic acid, hydrochloric acid, N-hydroxybenzotriazole, di-tert-butyloxycarbonyl carbonate, sodium hydroxide, diethyl ether, adipic acid, taurine, 1-ethyl-3-(3-(dimethylamino)15396

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The Journal of Physical Chemistry B 2.6. Digestion of Peptide in Human Serum. D4 (2 mg) was dissolved in 490 μL of PBS buffer and mixed with human serum (210 μL, 30% serum v/v). Peptide solution in the same concentration, but without added serum, was used as control. Peptide D4 with serum was incubated for 96 h at 37 °C. A 20 μL amount of the incubated sample taken after 0, 24, 48, 72, and 96 h was quenched with TFA and injected in HPLC after centrifugation. 2.7. Molecular Dynamics Simulations. Molecular dynamics (MD) simulations of human insulin in the presence of D4 peptide molecule were performed to explore the preference of D4 binding modes with insulin. A single monomer of human insulin (PDB ID 3W7Y; resolution 0.92 Å) was used as the starting structure for MD simulations. The initial structure of D4 peptide was built using Avogadro 2 0.7.2 molecular editor.70 The general Amber force field (GAFF)71 was applied using the Amber Antechamber program72 implemented through UCSF Chimera73 to generate the topology for the D4 molecule. ACPYPE,74 a python interface for Antechamber, was used to write GROMACS topologies for D4 peptide. On the basis of AM1-BCC75 partial charges, a formal charge of −2.00 was assigned at physiological pH. All MD simulations of insulin−D4 systems were performed using the GROMACS 4.5.6 molecular simulation suite.76 In total, we carried out 14 simulations which included two apo simulations and 12 simulations of insulin−D4 systems in which either the initial position of D4 or the reference temperature was different. Simulation details are described in Table 1. All simulations were performed with the Amber 99SB force field.77 Insulin and D4 peptide were solvated with TIP3P78 water molecules in a cubic box of size ∼69 × 69 × 69 Å. The box size was chosen with the objective of keeping a minimum distance of 15 Å from the solute atom to the edge of the box. Sodium counterions were added to neutralize the system. For all simulations, pressure was maintained at 1 bar with a coupling constant of 2.0 ps for Parinello−Rehman pressure coupling.79Two different simulation temperatures, 310 and 338 K, were considered, and the system’s temperature was coupled using a Nose−Hoover thermostat80 with a 0.2 ps coupling constant. Each system was energy minimized, first by steepest decent and then by conjugate gradient methods. The energyminimized systems were then subjected to 100 ps of equilibration under NVT conditions with positional restraints on the heavy atoms. To attain thorough equilibration, the systems were subsequently equilibrated under NPT conditions for 1 ns with positional restraints followed by 10 ns without restraints. van der Waals interactions were described using a spherical cutoff of 12 Å. The short-range and long-range Coulomb interactions were calculated using a spherical cutoff of 10 Å and the particle mesh Ewald81 method, respectively. Periodic boundary conditions were applied in all three (x, y, and z) directions for all systems. A further 100 ns production run was performed for each system, and the MD trajectories from the last 50 ns were used for further analysis. Interaction Energy between Insulin and D4. MD snapshots saved from the last 50 ns production runs for each simulation were used to calculate the interaction energies between insulin and the bound D4 inhibitor. For calculating the interaction energies, a twin range cutoff of 20 and 25 Å was used to describe both electrostatic and van der Waals interactions. The energy components were extracted using GROMACS’ g_energy energy analysis tool. Interaction energy (Eint) was calculated using

Table 1. Simulation Details of Insulin−D4 Complex Systems simulation set I II

III

IV

system apo I apo II system system system III system IV system system VI system VII system VIII system IX system system XI system XII

a

I II

V

X

initial D4 position;b simulation temperaturec

final D4 positiond

N/A; 37 °C N/A; 65 °C P1; 37 °C P1; 37 °C P2; 37 °C

N/A N/A proximal to B proximal to B proximal to A

3.7 4.0 2.6 2.4 2.8

P2; 37 °C

proximal to B

3.0 (0.5)

P1; 65 °C P1; 65 °C

proximal to B A−B interface

4.3 (0.7) 3.4 (0.4)

P2; 65 °C

proximal to A

5.5 (0.4)

P2; 65 °C

proximal to A

3.0 (0.5)

P1; 37 °C

proximal to B

2.6 (0.4)

P1; 37 °C P2; 37 °C

proximal to B proximal to A

2.7 (0.4) 2.6 (0.2)

P1; 65 °C

proximal to B

3.8 (0.2)

average RMSD (Ǻ )e (0.1) (1.2) (0.4) (0.2) (0.6)

a

The insulin structure corresponds to PDB ID 3W7Y. For pairs of systems, I and II, III and IV, V and VI, VII and VIII, and IX and X, the starting structures were the same but different initial velocities were used when they were simulated. Systems IX−XII were simulated with insulin molecule without disulfide bonds. bD4 molecule was initially placed either in the P1 or in the P2 position 5 Å away from insulin as shown in Figure 7. cSimulation temperatures of 37 and 65 °C correspond to physiological and fibril-forming conditions. dA and B indicate chains A and B of the insulin molecule, respectively. eAverage root-mean-square deviation for insulin molecule was calculated by comparing the initial structure and considering only the Cα atoms of the protein calculated for the last 30 ns of production runs. Numbers shown in brackets are the standard deviation.

E int = ESR − LJ + ESR − Coul + E LR − LJ + E LR − Coul

(1)

where Eint is the interaction energy between insulin and D4 peptide. ESR‑LJ, ESR‑Coul, ELR‑LJ, and ELR‑Coul represent short-range Lennard−Jones energy, short-range Coulomb energy, longrange Lennard−Jones energy, and long-range electrostatic energy of the insulin−D4 complex system, respectively.

3. RESULTS AND DISCUSSION Development of peptide-based inhibitors is attractive due to the ease of established chemistry protocols and possibility of introducing a multitude of chemical modifications and their biocompatibility.55,56 However, one of the drawbacks in using peptides with L-amino acids and regular backbone is their inherent instability due to rapid enzymatic degradation.55 Numerous approaches are employed to enhance the stability of biologically active peptides and peptide conjugates from enzymatic decomposition: use of D-amino acid, making a cyclic analogue from linear, and incorporation of noncoded amino acid such as β-Ala, to name a few.51,55,56 We decided to synthesize conjugates D1−D4 (Scheme 1) with D-Trp amino acid to enhance their serum stability in comparison to corresponding L-Trp.50 Conjugate D4 was incubated with human serum up to 96 h, and HPLC analysis at various time intervals showed that only ∼10% of the peptide degraded between these duration (Figure 1). 15397

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Scheme 1. (A) Chemical Structures of Water-Soluble D-Tryptophan and Taurine-Containing Peptides Conjugate; (B) Ribbon Diagram of Human Insulin Monomer (PDB 3W7Y); (C) Primary Structure of Human Insulina

a

Red-colored superscripts represent those amino acid residues which are different in bovine insulin.

of inhibitors at maxima of 488 nm, where D4 turned out to be the best inhibitor for insulin aggregation (Figure 2A and 2B). The inhibitory potential of conjugate D4 was more than D3, which is reasonably better than D1 and D2, a result similar to what reported for L-peptide conjugates.51 In the case of bovine insulin (BI), ThT fluorescence intensity was reduced up to 80% for D4, while it was 90% reduction for human insulin (HI).50 We conducted a time-dependent fluorescence assay to speculate at what stage peptide D4 interferes with the insulin fibrillation process (Figure 2C). D4 was added to an incubating insulin solution at different hours, time points from 0 to 6 h ,and studied ThT fluorescence at 488 nm. Maximum inhibition was observed if D4 was present at the time of incubation (0 h). The inhibitory effect diminishes as the time of addition of D4 increased. Saturation of fluorescence intensity at 6 h of addition is achieved perhaps due to interaction of peptide D4 with oligomers emerging at the late nucleation phase or secondary nucleation pathway. It has been reported that early oligomers in certain amyloid proteins are more cytotoxic in nature than mature fibrils.20,28 In short, peptide D4 is very likely to interact with the early nucleation phase of insulin aggregation. Concentration-dependent inhibition of insulin fibril formation by D4 was studied over a period of 30 h with both BI and HI (Figures S10−S13). Different concentrations of peptide D4, 12.5, 25, 50, and 100 μM, were incubated with BI and HI under amyloidosis conditions followed across the literature. It was found that 12.5 μM of peptide D4 is sufficient to maintain the native conformation of protein until 30 h and there is no enhancement in ThT emission corresponding to mature fibril as observed over 30 h with BI and HI. However, incubation of insulin with higher concentrations of peptide D4 (200 μM) did not allow formation of longer fibrils until 80 h (Supporting Information). These observations suggest that D4 stabilizes native insulin conformation even under harsh conditions.50 We used atomic force microscopy to monitor formation of insulin fibrils and its inhibition by D4. In both cases, bovine and human insulin at 0 h exhibited tiny spot morphologies,62 which

Figure 1. HPLC traces of peptide D4 alone and coincubated with 30% serum at different time points showed that peptide D4 is stable in serum condition even after 96 h of incubation.

Vassar and Culling described the advantage of Thioflavin T (ThT), a cationic benzothiazole dye, for specific staining of amyloid fibrils leading to a dramatic enhancement in fluorescence.57−61A recent study suggested that ThT forms micelles in aqueous solution, which binds to the surface of amyloid fibrils.57 Binding of ThT micelles to amyloid fibrils causes changes in the excitation spectra and enhanced emission fluorescence. We observed that the fluorescence output of ThT was quite low for bovine and human insulin during the early incubation period, which changed over time to exhibit a sharp increase in the fluorescence intensity. The time course of insulin fibrillation was monitored in the absence and presence 15398

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Figure 2. Thioflavin T fluorescence spectra of aggregation BI and HI and its inhibition by D4 (100 μM): (A) with HI and (B) with BI. (C) Inhibitory effect of conjugate D4 on time-dependent fibril formation of human insulin; D4 (100 μM) was added to the insulin solution (170 μM, pH = 1.6, 65 °C) at different time points from 0 to 6 h ((λex = 410 nm; λem = 488 nm).

Figure 3. Time-dependent AFM micrographs of human insulin alone at (A) 0, (B) 5, and (C) 20 h and coincubation of human insulin with D4 at (D) 0, (E) 10, (F) 20, (G) 30, (H) 40, and (I) 50 h, respectively (scale bar = 1 μm).

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Figure 4. Time-dependent AFM micrograph of bovine insulin alone at (A) 0, (B) 5, and (C) 20 h and coincubation of bovine insulin with D4 at (D) 0, (E) 10, (F) 20, (G) 30, (H) 40, and (I) 50 h.

Figure 5. Far UV-CD spectra of insulin aggregation and its inhibition by D4 (100 μM): (A) with HI and (B) with BI.

seeded the formation of smaller fibrils at 5 h in both samples (Figures 3B and 4B).63 After 20 h of incubation, fibril bundles were observed for BI and HI. Coincubation of insulin with peptide D4 from the very beginning, at 65 °C, arrested fibril formation, which remained such over a period of 50 h. These micrographs for HI (Figure 3) and BI (Figure 4) are consistent with ThT fluorescence spectra. This spherical morphology, formed by peptide D4 after interacting with HI and BI, is different from partially unfolded insulin oligomers spherulites.64 However, BI shows a very small amount of fibrils when incubated with peptide D4 for 50 h. This observation suggests that the inhibitory effect of peptide D4 is more pronounced toward human insulin than bovine insulin for reasons that are unclear at the present time. Scanning electron microscopy also

confirmed the results from AFM experiments (Supporting Information). Circular dichroism (CD) is a versatile tool to assess conformational changes in peptide secondary structures.65 The mature amyloid fibrils exhibited CD spectral patterns suggestive of the formation of cross-β-sheet-like signatures.66−68 We followed the interaction of insulin and D4 by CD measurements: insulin shows α-helical signatures in its native form, with corresponding negative ellipticities at 208 and 224 nm. Both BI and HI adopt cross-β-sheet structure within 10 h, as identified by a peak at 218 nm (pH 1.6 at 65 °C). After fibril formation, insulin solution changes from transparent to viscous or as an insoluble precipitate, depending on the concentrations used. The α-helix structure of HI and BI in the presence of D4 is maintained for more than 50 h of incubation 15400

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Figure 6. Hydrodynamic radius of insulin is analyzed by dynamic light scattering for HI alone (A, B, and C) and in the presence of inhibitors D4 (100 μM) (D, E, and F) at 3, 10, and 20 h, respectively, in pH 1.6 at 65 °C.

Figure 7. Starting structures of insulin−D4 complex. D4 peptide was initially placed 5 Å away facing either chain A (blue; position P2 in Table 1) or chain B (orange; position P1 in Table 1) of insulin. Insulin and D4 are displayed in ribbon and stick representations, respectively. Three disulfide bridges of insulin are shown in yellow.

absence of such structural studies, we carried out extensive molecular dynamics (MD) simulation studies on insulin in which the inhibitor molecule is initially placed away from the protein at about 5 Å distance located in two distinct positions. The systems were simulated at two different temperatures (310 and 338 K), and insulin was considered with and without disulfide bonds. A total of 14 different simulations representing combinations of these conditions (D4 position, temperature, presence or absence of insulin disulfide bonds) were performed in order to find out whether D4 has any preferred mode of binding and interactions with insulin. The details of the various simulations carried out are listed in Table 1. The simulated systems can be divided into four sets. The first set (apo I and apo II) contains MD simulations of insulin in apo form in two different temperatures. In the second set (systems I−IV), insulin was simulated with D4 at the physiological temperature of 37 °C. D4 was initially placed in two different positions facing the chain A or B of insulin (Figure 7). Two independent simulations were carried out for each position by changing the random seed, which assigned different initial velocities. In the third set of simulations (systems V−VIII), the starting structures of insulin and D4

and did not reveal any kind of precipitation (Figure 5), suggesting that D4 is able to stabilize the native structure of insulin. In short, the helicity of insulin was maintained for 50 h with a decrease in their ellipticity. (Tables S3 and S4). Particle size distribution was further investigated by dynamic light scattering (DLS), where aggregation and fibril formation could be correlated to its size.36After 3 h of incubation, HI showed an average hydrodynamic radius of ∼800 nm, which reaches 1.6 μm at 10 h, and the particle size was further increased to 40−70 μm at 20 h of incubation (Figure 6).69However, the average hydrodynamic radius of insulin aggregation, when coincubated with D4, was less than those of insulin alone at every time point, suggesting that D4 is able to interfere with insulin aggregation. Molecular Dynamics Simulation of Insulin−D4 Interactions. The above experimental studies demonstrate that the D4 peptide can inhibit insulin aggregation. The probable binding site of D4 is also revealed in the previous experimental studies.50 However, knowledge about the actual binding site of D4 and the nature of its interactions with insulin will be evident only if the structure of insulin−D4 complex is determined experimentally using X-ray crystallography or NMR. In the 15401

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stable, and the two helices from chain A exhibited some deviation from each other. The most variable region is the Cterminal part of chain B, which also happens to be the preferred binding site for the D4 molecule. In all simulations, the D4 molecule quickly moved closer to the insulin molecule and started interacting with the residues of chain B and/or A. The MD-simulated results for the insulin− D4 system at the end of the production run for each of the three sets II−IV are shown in Figure 9. Two immediate conclusions can be reached from this figure. Although the conformation of D4 was initially in the extended form, we can see that D4 adopts a somewhat folded and compact structure during the course of the simulation in all three sets. This is presumably to bring the hydrophobic indole groups in close proximity with each other. To validate this point, we calculated the radius of gyration of the D4 molecule for each simulation, and MD trajectories of this parameter clearly show that the radius of gyration of D4 decreases quickly from 9 to less than 6 Å in the beginning of equilibration (Figure S27). The second and most important analysis is to find out which region of insulin is preferred to bind for the D4 molecule. For this purpose, we computed center of mass distances between D4 and the helical region(s) of chains A or B of insulin. The two distance trajectories for each of the simulations (Figure S28 in the Supporting Information) indicate that D4 binds to chain B in 7 out of 12 simulations from the three different simulation sets. The inhibitor binds to the region facing the chain B of insulin and mostly in between the helix and the C-terminal disordered region. This happened even when insulin was placed at the opposite side facing chain A of insulin (Figure 7) in one of the simulations (system IV). In one simulation (systems VI) D4 occupied a position at the interface of the two chains of insulin. In four simulations in which D4 was initially placed at the chain A side, the D4 inhibitor remained close to chain A. We then calculated the interaction energy between insulin and the D4 molecule in all 12 simulations from the three sets II− IV. In each simulation, MD snapshots saved every 100 ps were considered, and interactions energies were calculated for each MD-simulated structure as described in the Materials and Methods section. This is a total of 501 structures for each simulation. Then the top 10 structures with the most favorable interaction energies were selected and further energy minimized. Interaction energies for the minimized structures were then determined, and the average energy values with standard deviation are presented in Table 2 for each of the 12 simulations. Average interaction energies between insulin and D4 complex systems in the 12 simulations varied from −159 to

molecules are the same as the second set, but the simulation temperature corresponds to that of fibril-forming conditions (65 °C). Here, again two independent simulations were performed for each D4 position. In the fourth set (systems IX−XII), the insulin molecule was considered without three disulfide bonds and the Cys residues were in their reduced form. The structure in which the D4 molecule was facing chain B was simulated at higher temperature also (system XII). When D4 was placed facing chain B of insulin, an additional independent simulation was carried out (system X). The MD trajectories of RMSD values for each simulation are provided in Figure S26. It is clear that the RMSD values are stabilized in the last 50 ns of 100 ns production runs in all simulations with the exception of system IX. For this simulation there is a sudden transition of RMSD around 80 ns. Hence, we extended the simulation of this system by another 100 ns and found that the RMSD values of this simulation remain stable from 80 to 200 ns. In general, the average RMSD of insulin is 1.5−3.0 Å higher if insulin is in the apo state or when the systems were simulated with D4 at a higher temperature of 65 °C (Table 1). We also calculated the average helical content of each simulation for the last 50 ns of the production run. It ranges from 32% to 47%, which is in reasonable agreement with the percentage helical content calculated from the CD studies reported here (Tables S3 and S4). We also superposed the eight structures saved at the end of the 100 ns production run from sets II and III (systems I−VIII) on the starting structure (Figure 8). It appears that the helix in chain B is the most

Figure 8. MD-simulated structures saved at the end of the production run from 8 simulations belonging to simulation sets II and III (systems I−VIII) are superposed on the starting structure. Different secondary structural regions of insulin molecule from both chains A and B are displayed in different colors. Chain B is shown in the foreground.

Figure 9. Superposition of D4-bound insulin structures saved at the end of the production run for simulation set (A) II, (B) III, and (C) IV. In all structures chains A and B of insulin are shown in blue and orange, respectively. D4 inhibitor is shown in stick representation, and the inhibitor structure is displayed in different colors for different simulations. 15402

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Table 2. Interaction Energy Analysis for Insulin and D4 Complex Systems Presented for the Minimized Top 10 MD Simulated Structures simulation set II

III

IV

system system system system system system system system system system system system system

I II III IV V VI VII VIII IX X XI XII

⟨EVdw⟩a,b (kJ/mol) −236.7 −246.1 −147.7 −305.4 −299.7 −319.4 −172.0 −230.0 −312.3 −267.1 −239.1 −244.3

± ± ± ± ± ± ± ± ± ± ± ±

⟨EElec⟩a,b (kJ/mol) −82.3 −93.8 −11.8 −78.2 −97.9 −118.8 −57.7 −66.7 −84.6 −135.0 −172.1 −61.2

13.1 18.4 10.4 10.7 29.7 19.5 33.5 32.6 15.1 25.2 16.8 20.4

± ± ± ± ± ± ± ± ± ± ± ±

63.9 64.6 81.6 37.2 40.2 59.3 75.8 72.0 51.5 56.4 44.1 37.8

⟨EFFY⟩c (kJ/mol) −78.2 −92.1 7.8 −75.6 −114.2 −54.2 −11.6 −84.4 0.9 −72.1 −101.7 −71.8

± ± ± ± ± ± ± ± ± ± ± ±

⟨ETot⟩a,b (kJ/mol) −319.1 −339.9 −159.4 −383.6 −397.6 −438.1 −229.7 −296.7 −396.9 −402.2 −411.2 −305.5

11.7 5.4 22.9 20.4 30.6 8.1 19.1 25.5 11.1 18.2 16.2 13.5

± ± ± ± ± ± ± ± ± ± ± ±

56.9 69.7 77.0 37.2 63.0 62.1 68.1 76.6 52.6 57.2 50.4 38.8

a

EVdw, EElec, and ETot represent vdw, electrostatic, and total interaction energy between the insulin molecule and D4 inhibitor calculated for each minimized MD-simulated structure. The angular brackets represent the average calculated for the top 10 structures bFor each structure, EVdw = ESR‑LJ + ELR‑LJ; EElec = ESR‑Coul + ELR‑Coul; ETot = EVdw + EElec. See eq 1 for details. cInteraction energy between D4 and the FFY motif of chain B in insulin. Interaction energy between the three residues of FFY motif and D4 is calculated as the sum of vdw and electrostatic energy between these two groups. The angular brackets represent the average calculated for the top 10 structures.

−438 kJ/mol. van der Waal’s (vdw) interactions (ESR‑LJ + ELR‑LJ in eq 1) seem to be the most dominant factor in the interactions between the insulin molecule and D4 inhibitor. In almost all cases the average vdw interaction energies are exceeded by 100−200 kJ/mol compared to the electrostatic component (ESR‑Coul + ELR‑Coul in eq 1). Since the FFY motif has been shown to be important in ligand binding, we also calculated the interaction energies between D4 and the FFY motif of chain B for each simulation (Table 2). It is clear that in 9 out of 12 simulations, interactions of D4 with the FFY motif range from −58 to −172 kJ/mol. Even when the inhibitor molecule D4 binds to chain A of insulin in systems VIII and XI, its orientation is such that it is closer to chain A helices as well as the C-terminal region of chain B which possess the FFY motif. We also examined the top 5 structures with the most favorable interaction energy between insulin and D4 molecule. Their interaction energies lie between −461 and −529 kJ/mol (Table 3). Both vdw and electrostatic interactions are highly favorable between insulin and D4 with vdw interactions being more dominant in almost all of them. We identified interacting residues from insulin and characterized the type of noncovalent interactions between insulin and D4. In four out of five structures, chain B of insulin contributes a majority of the interacting residues. Another interesting observation is that at least two residues from the motif F24F25Y26 from chain B are involved in interactions with the D4 inhibitor in all these structures. Our previous experimental studies suggested that this motif may be important in inhibitor interactions.50 We also noticed that two of the structures (Min-IV and Min-V) belong to the simulations in which Cys residues forming the disulfide were considered in reduced form. One of them also show extensive interactions with chain A residues perhaps due to the opening of the insulin molecule in the absence of a disulfide bond. Many residues from Min-IV and Min-V structures seem to be involved in electrostatic interactions with the D4 inhibitor. A representative structure showing insulin−D4 inhibitor interaction is displayed with interacting residues in Figure 10. Previous simulation studies focused on small peptide segments from the B chain of insulin to understand the nucleation and their capability to inhibit the fibrillation of full

Table 3. Top Five Insulin−D4 Complex Structures with Most Favorable Interaction Energies structure Min-I

system system IV

EVdw, EElec, ETot (kJ/mol) −300.0, −161.1, −461.1

Min-II

system V

−322.1, −184.8, −506.8

Min-III

system VI

−338.0, −190.9, −528.9

Min-IV

system X

−256.6, −227.7, −484.4

Min-V

system XI

−251.5, −244.6, −496.1

interacting residuesc Chain A: I2, V3e Chain B: G8, S9,d,e L11, V12, E13, L15, F24, Y26, T27,d P28 Chain A: I2, V3,e Y19e,f Chain B: L11, V12, L15d, Y16, C19, G20, G23, F24e,f, Y26d, K29h Chain A: V3e Chain B: C7,e,f G8d,e,g, S9, L11, V12, L15, F25,e,f, Y26,e K29,d T30g Chain A Chain B: V12, E13, Y16,d C19,d G20,d E21,d G23,d,g F24,d Y26d,g Chain A: G1,e I2,e,g Q5, Q15, N18,g Y19,d N21d,g Chain B: R22,h F24,d,gY26

a

For details of each system and simulation conditions, see Table 1. For definitions and notations of different energy components, see footnotes a and b of Table 2. cSix different noncovalent interactions between insulin and D4 inhibitor have been identified. These interactions were defined using the criteria given by Jain et al.85 and references within. Specific interactions are mentioned in footnotes d− h. All other interactions can be defined as hydrophobic. dC−H···O. e C−H···π. fπ···π. gN−H···O. hSalt bridge. b

length insulin.27 Small peptide fragments from other proteins have also been used as possible antiamyloid agent to prevent insulin aggregation.45 The influence of pH on the flexibility of insulin and its implication on aggregation has also been investigated using a combined approach of experimental and simulation techniques.83 Computational studies have focused on specific interactions that are likely to promote the fibril formation and shift the balance away from the native structure.83−85 Recently, Hong et al.40 developed a biocompatible molecule which potentially has two applications: to monitor the kinetics of amyloidogenesis and to inhibit fibril 15403

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The Journal of Physical Chemistry B

Figure 10. (A) Insulin−D4 interaction map produced using LigPlot+.82 Residues marked with red semicircles form hydrophobic contacts with D4. Three out of four Trp in D4 are involved in hydrophobic interactions. (B) Representative insulin−D4 complex structure. Insulin residues interacting with D4 are shown in stick representation in green. Chains A and B are displayed in blue and orange, respectively. D4 peptide is shown in gray.

studies present a novel approach for the discovery of the next generation of stable, peptide-based ligands as novel antiamyloidogenic agents, specific for insulin.

formation. The current experimental studies have amply demonstrated the capability of D4 peptide to inhibit the insulin aggregation. Multiple molecular dynamics simulation studies in this study investigated the mode of D4 binding to the insulin polypeptide. The preferred interacting region during binding seems to be the FFY (B24−B26) motif in chain B, and it contributes significantly to the interaction energy in most of the simulations, and this is irrespective of whether D4 binds close to the helix of chain B or the helical regions of chain A. Interaction energies of the top 5 minimized structures with the most favorable interaction energy indicate that several hydrophobic residues of insulin including those from the FFY motif are involved in interacting with D4 peptide conjugate. Another interesting feature is the presence of several C−H···O and C− H···π interactions between insulin and D4. The potential role of C−H···O interactions in stabilizing the amyloid aggregates has been highlighted in our previous studies.85 By forming many C−H···O and C−H···π interactions, D4 inhibits these groups from undergoing a conformational transition which also could prevent these groups from participating in such interactions at the intra- and intermolecular level within and between insulin molecules.

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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.5b08215. Synthetic details of new compounds, their analytical characterization, microscopy and CD spectral data, and figures concerning MD simulation studies (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS N.K.M. and R.N.V.K.D. thank CSIR-India for Senior Research Fellowship. This work was supported by an Outstanding Investigator Award to S.V. from DAE-SRC, Department of Atomic Energy, India, and by DST through a J. C. Bose National Fellowship. High Performance Computing Facility at IIT-Kanpur supported by DST and MHRD is gratefully acknowledged.

4. CONCLUSIONS This work demonstrates the synthesis of water-soluble Dtryptophan and taurine-containing biocompatible peptide conjugates. Out of these, one peptide D4 is found to be preventing the fibrillation of insulin more intensively. This peptide has been found to be stable in serum condition even after several hours of incubation. Using ThT fluorescence we observed that peptide D4 delays the fibrillation of insulin, which is further confirmed by CD spectroscopy, AFM, and DLS results. These experiments illustrate that the peptide conjugate D4 inhibits insulin fibrillation in a concentrationdependent manner. Hydrophobic interactions, aromatic stacking, and hydrogen bonding of peptide D4 with insulin are suggested to inhibit the nucleation phase and slow down the elongation process. Our extensive MD simulation studies demonstrate that the D4 molecule has a preferred binding site in insulin. Residues from the helix and the C-terminal extended segment of chain B are involved in interactions with D4. These

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