Chem Biol Drug Des 2014 Research Article

Study of Orientation and Penetration of LAH4 into Lipid Bilayer Membranes: pH and Composition Dependence Matin Islami1, Faramarz Mehrnejad2,*, Farahnoosh Doustdar3, Masumeh Alimohammadi1, Mahmoud Khadem-Maaref4, Mohammad Mir-Derikvand2 and Majid Taghdir5 1

Department of Cellular and Molecular Biology, Faculty of Science, Azarbaijan Shahid Madani University, Tabriz 53714-161, Iran 2 Department of Life Science Engineering, Faculty of New Sciences & Technologies, University of Tehran, Tehran 14395-1561, Iran 3 Department of Microbiology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran 1985717443, Iran 4 Department of Physics, Faculty of Science, Azarbaijan Shahid Madani University, Tabriz 53714-161, Iran 5 Department of Biophysics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran 14115-175, Iran *Corresponding author: Faramarz Mehrnejad, [email protected] LAH4 is an antimicrobial peptide that is believed to possess both antibiotic and DNA delivery capabilities. It is one of a number of membrane-active peptides that show increased affinity toward anionic lipids. Herein, we have performed molecular dynamics simulations to compare LAH4 effects on anionic palmitoyl–oleoyl–phosphatidylglycerol bilayer, which approximate a prokaryotic membrane environment and zwitterionic palmitoyl–oleoyl–phosphatidylcholine bilayer, which approximate a eukaryotic membrane environment. One particular interest in this work is to study how different kinds of lipid bilayers respond to the attraction of LAH4. Remarkably, our data have shown that the depth of peptide penetration strongly depends on membrane composition and pH. At acidic pH, LAH4 has exhibited a high tendency to interact strongly with and be adsorbed on anionic membrane. We have also shown that electrostatic interactions between His11 and the phosphor atoms of bilayers should have a significant impact on the penetration of LAH4. These results provide insights into the interactions of LAH4 and lipid bilayers at the atomic level, which is useful to understand cell selectivity and mechanism of the peptide action. Key words: anticancer drugs, antimicrobial peptides, electrostatics interactions, membrane, molecular dynamics simulation

ª 2014 John Wiley & Sons A/S. doi: 10.1111/cbdd.12311

Received 26 August 2013, revised 12 February 2014 and accepted for publication 14 February 2014

Cancer is considered one of the main reasons of morbidity and mortality throughout the world and representing about one-eighth of all deaths. However, in recent decades, much progress has been achieved in respect of therapies, such as chemotherapy, radiation, surgery, or hormone ablation therapy, and they are not successful in more than 50% of cases. One of the major challenges is the multiple-drug resistance problem, which is continued to arise particularly with respect to chemotherapy due to resistance to and low specificity of currently available drugs (1–3). Consequently, there is an urgent need for new therapeutic agents. During the last decays, several antimicrobial peptides (2) have received increasing attention as potential candidates not only for antimicrobial agents but also for anticancer drugs (4). These molecules interact with different bilayers depending on the physical properties of membranes consisting of different lipid molecules (1,5). Their mechanism of action usually entails the disruption of cancer cell membranes, which are similar to membranes of bacterial cell with respect to their anionic characteristics. One of the major differences between cancer and normal cell surfaces is the exposure of the negatively charged lipid phosphatidylserine (PS) on the outer leaflet of the cancer cell membrane, while normal cells exhibit an overall neutral charge due to the zwitterionic phosphatidylcholine and sphingomyelin (2,6–8). Because of mechanism of their action, resistance and cytotoxicity are less likely to occur and thus AMPs are also expected to cause fewer side-effects than chemotherapeutic agents (2,8,9). As a result, AMPs are excellent candidates for development as novel therapeutic agents and complements to conventional antibiotics and anticancer therapy. LAH4 is a synthetic histidine-rich amphipathic alpha-helical peptide that has only 26 amino acids (9,10). It has antimicrobial activity (6,8,9,11–13) and nucleic acid transfection capability (7,8,14–17). All these capabilities and biophysical interest of LAH4 have motivated a number of studies aimed at understanding the behavior of the peptide within the lipid bilayers. Notably, these studies have demonstrated that the transfection activities of LAH4 were strongly dependent on the presence of histidine residues

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in the core of the peptide. In fact, LAH4 has revealed pHdependent membrane insertion, and it is more active at low pH. Indeed, the histidine residues in the peptide are uncharged at neutral pH, but in acidic pH when the side chains are become protonated, it can be capable to achieve a conformation and align to the membrane that induces membrane disruption (8,18). The experimental studies have also confirmed that LAH4 has stronger interaction with anionic phospholipids rather than zwitterionic one and this helical peptide disorders anionic lipid fatty acyl chains such as PG (phosphatidylglycerol; 11) and PS (phosphatidylserine; 8,18) over PC (phosphatidylcholine). These findings implicate greater tendency of LAH4 to interact with anionic membrane that can be useful data in order to confirm on its anticancer properties. In spite of extensive studies, the mechanism by which LAH4 discriminates between PG and PC, however, is not studied. Hence, one particular interest in this article is to investigate favor and disfavor interaction on binding selectivity of the peptide for palmitoyl–oleoyl–phosphatidylglycerol (POPG) and palmitoyl–oleoyl–phosphatidylcholine (POPC) bilayers. Herein, we have selected POPG as indicator of anionic lipids and POPC as indicator of zwitterionic lipids to compare different binding affinity of LAH4 for two diverse kinds of lipid bilayer using molecular dynamics (MD) simulations at acidic and neutral pH. Our purpose of this study was to obtaining information in atomic level around of interactions of LAH4 with membranes that cannot be extracted easily from experimental techniques. This work provides theoretical basis and effective means to discussing about affinity binding of LAH4 to anionic lipids in order to understanding mechanism of its interaction with cancer and microbial cells and introducing LAH4 as anticancer peptide.

Computational Methods Preparation of systems The co-ordinate for LAH4 was obtained from the Protein Data Bank (PDB ID: 2KJN; Figure 1; 19). Pre-equilibrated membranes, 128 POPC lipids with 2460 water molecules and 128 POPG lipids with 128 Na+ and 3527 water molecules, were obtained from the Tieleman laboratorya and Lipidbook (20), respectively. About 5000 water molecules were added to the system to enable larger distances between the periodic images of the lipid leaflets in the z-direction. They were further equilibrated in water for 200 ns. The resultant systems were taken to be the starting point for all the MD systems. Two copies of the peptide were placed in the water phase using VMD software (21). The peptide helical axis was parallel to the interface, and the initial distance between the nearest lipid head groups and the center of mass of the peptides was about 5 nm in each case. To eliminate possible conformation bias and to increase the population of the starting configuration in the same simulation, two different peptide starting geometries, one with its hydrophilic side toward the membrane surface (Peptide A) and the other with its hydropho2

Figure 1: Helical wheel diagrams and the starting conformation of LAH4.

bic side toward the membrane surface (Peptide B), were selected (Figure 2). These orientation situations were designed on the basis of the amphipathic nature of the peptide (Figure 2), in which hydrophobic and hydrophilic residues are preferentially found on opposite sides of the peptide. This method is helpful to examine the influence of different initial configurations on the membrane-association process at the same simulation time. To neutralize the systems, counter ions (Cl
and Na+) were added by replacing water molecules at the most positive/negative electrical potential. We have constructed four systems that the details have been summarized in Table 1.

MD simulation system setup All simulations were performed using the GROMACS software package, version 4. 5. 4 (22,23). The GROMOSE96 forcefield parameters were used for the solvent and the peptide, and the modified GROMOS united-atom parameter set was used after downloading from Tieleman laboratory, file lipid.itp (24,25). These force fields have been used in previous works, considering the membrane interactions of a variety of other antimicrobial peptides (26–28). For all simulations, temperature was maintained at 313 K using a Nose–Hoover algorithm with employing a coupling constant of s = 0.1 ps (29,30), and reference pressure of 1 bar was kept with a coupling constant of s = 0.5 ps applying Parrinello–Rahman algorithm in semi-isotropic Chem Biol Drug Des 2014

Study of LAH4–Membrane Interactions

of all atoms (33). A grid search algorithm with a 1.0 nm cutoff was used, and the corresponding neighbor list being updated every 10 steps. Two independent 200-ns MD runs are carried out for each system using different initial velocity distributions, starting from the same initial state.

Data analysis The analyses of the trajectories and visualization were conducted using the GROMACS analysis package tools, VMD, and PYMOL (34) softwares. The ordering of the lipid acyl chains is usually characterized by the deuterium order parameter, SCD (35). The order parameter is defined as: SCD ¼

Figure 2: Initial snapshot of LAH4 peptide–bilayer simulation.

condition (31). Periodic boundary conditions were used in all three dimensions. The short-range electrostatic interactions were calculated with a distance cutoff of 1.0 nm, and long-range electrostatic interactions were computed using the particle mesh Ewald (PME) algorithm (32). The van der Waals interaction was calculated using a cutoff of 1.4 nm. Energy minimization was applied to each system using the steepest descent algorithm and a tolerance of 1000 kJ/ mol/nm. All systems were equilibrated under NVT-ensemble state condition for 100 ps. Position restraints were placed on all peptide heavy atoms employing a spring constant of 1000 kJ/mol/nm2 to allow further equilibration of peptide, lipid, and solvent while keeping the conformation of peptide unchanged. After the positional restraint equilibration, each of four systems was submitted for unbiased MD runs. The time step was considered 2 fs. The LINCS algorithm was used to constrain bond lengths and angles

1 h3 cos2 h
1i 2

(1)

where h is the angle between the CH-bond vector and the bilayer normal, the brackets denote an average over time and over all the lipids, and axial averaging about the bilayer normal is assumed; SCD defined in this way can be directly compared with the order parameter measured by deuterium NMR and is therefore denoted as the deuterium order parameter. In this study, we have used the same method as carried out by previous studies to calculate potential of mean force (PMF)s of the hydrophobic residues (36). The method was applied to all simulations. Potential of mean force was calculated as functions of a pair correlation function g(r) using: WbðrÞ ¼
kb TLnðgðrÞÞ

(2)

where Wb(r) (b = Cb of hydrophobic residues) denotes the PMF, kb refers to the Boltzmann constant, T indicates the simulation temperature, and g(r) is the radial distribution function (RDF) between the phosphate atoms and hydrophobic residue. The lateral diffusion coefficient, D, was calculated from the long-time mean-square displacement of the lipids by: \r 2[ ¼ 4DDt

(3)

where is mean square displacement of a randomly moving tracer, and t is time. The averaging indicated by the brackets was performed over all lipid molecules.

Table 1: Summary of the molecular dynamics simulations

System LAH4 + LAH4 + LAH4 + LAH4 + POPC POPG

POPG POPC POPG POPC

Number of water molecules

pH

Number of lipid molecules

T(K)

6572 5746 6572 5746 6158 7012

5 5 7 7 – –

128 128 128 128 128 128

313 313 313 313 313 313

Duration K K K K K K

2 2 2 2 2 2

9 9 9 9 9 9

200 200 200 200 200 200

ns ns ns ns ns ns

POPC, palmitoyl–oleoyl–phosphatidylcholine; POPG, palmitoyl–oleoyl–phosphatidylglycerol.

Chem Biol Drug Des 2014

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Results and Discussion A fundamental understanding the selectivity of different AMPs such as LAH4 for mammalian and bacterial membranes would not only of fundamental biophysical interest but also be crucial for the development of these peptides as novel antibiotic agents. LAH4, a histidine-rich amphipathic alpha-helical peptide, exhibits antimicrobial activity over a wide range of bacteria including Escherichia coli or Bacillus megaterium (11). Results of NMR (8,10,19), circular dichroism (9,10,18), and FTIR spectroscopies (18,37) have confirmed that LAH4 adopts a helical conformation in membrane environments and indicated that the topology of LAH4 alters in a pH-dependent manner. These experimental studies have also shown that LAH4 kills bacteria both at neutral and acidic pH, although it is highly active at acidic pH (9,11). Based on the experimental data, we have performed over 2000 ns simulations on LAH4 to systematically study its interactions with two model lipid bilayers, POPC and POPG, which mimic the mammalian and bacterial membranes, respectively. Two copies of the peptide (A and B) were initially placed 5.0 nm away from the membranes, in an alpha-helical conformation (Figure 2). To better realize the position of the peptide in the various bilayers, we have considered penetration and orientation of LAH4 at pH 5.0 and pH 7.0 by calculating membrane properties such as order parameter of the acyl chains, the density profile of different groups, and the peptide-membrane distance.

Position and orientation of LAH4 Density profiles in the presence of two copies of LAH4 on both sides of the lipid bilayers have shown in Figure 3. These data compare mass density distributions of lipid head groups, lipid tails, peptide, ions, and water in the z-direction normal to the lipid bilayer plane. The distribu-

tions show stable bilayers for all four systems, and no water or lipid head group is observed in the hydrophobic interior of membranes. The head groups (including choline, glycerol, and phosphate) are located between water and the lipid tails. Compared with head group for POPC, the head groups of POPG are clearly shifted toward the water phase, showing a stronger hydration. During the MD simulations, the peptide has reoriented and permanently attached with the hydrophobic face to the POPC bilayer. For comparison, LAH4 can preferentially interact with the lipids via their N-terminal hydrophilic residues in anionic membrane. It is interesting to note that the peptide has penetrated into the POPC bilayer more than the POPG bilayer, whereas for POPG system, the peptide is clearly shifted toward the water phase and demonstrating a stronger hydration. In comparison between diverse systems, these observations have also indicated that LAH4 inserted more deeply in zwitterionic membrane especially at pH 7.0 and has at least penetrated when inserted in POPG bilayer at pH 5.0 (Figure 4). Indeed, protonation of histidines at pH 5.0 can lead to more polar interactions for LAH4 with the water molecules and may prevent from the arrival of the peptide into the bilayer in comparison with the neutral pH that the histidines are discharged. It is possible that there is a relation between deprotonation of the histidines and charge of the membrane with a penetration depth of into the membrane and ultimately an alignment of the peptide. These results are also consistent with previous studies that the alpha-helical structure of the peptide adopts a transmembrane alignment at pH 7.0, but flips to lie parallel to the membrane surface at acidic pH, when the histidines carry positive charges (19). In recognition of this issue at pH 7.0 in the presence of zwitterionic bilayer and discharged histidines, LAH4 inserts in most depth (Figure 4). On the contrary, at pH 5.0 in the presence of the anionic bilayer because of existence positive and negative charge between cationic peptide and

Figure 3: The density profiles for LAH4, lipid head groups, lipid tails, ions, and water along the z-axis of the simulation box.

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Chem Biol Drug Des 2014

Study of LAH4–Membrane Interactions

Figure 4: Final snapshots (t = 200 ns) of LAH4 peptide– bilayer simulations.

anionic membrane, LAH4 is trapped among lipid head groups and cannot deeply penetrate into the hydrophobic core of the anionic membranes (Figure 4). This work indicates that most parts of the peptide interact with the lipid head group and tail regions of zwitterionic membrane and thus allow for the anchoring of the peptide to the POPC bilayer. In contrast, some regions of LAH4 are still located within the water phase in the POPG system, and in agreement with the experimental studies (10,38), the peptide adopts a parallel alignment to the bilayer surface at pH 5.0 (Figure 4). Our results can also demonstrate the significant role of electrostatic and polar interactions to the distribution and penetration of LAH4 into the different bilayers that leads to the different density profile for the peptide. The previous experimental studies have shown that the peptide has excellent killing ability toward the tumor and bacterial cell, while maintaining low toxicity against mammalian cells (8,9,11,12,18). To understand this selectivity, we have also determined the distance between the center of mass of LAH4 backbone and the center of mass of the phosphate groups in the lipid bilayer for each of the systems by MD simulations (Figure 5). It is interesting to note that the peptide has penetrated into a greater depth in POPC than POPG bilayer. At pH 5.0, LAH4 was rapidly adsorbed onto the anionic membrane (within 5.0 ns), Chem Biol Drug Des 2014

driven by strong long-range electrostatic attractions between the positively charged peptide and the negatively charged bilayer. It seems that LAH4 attaches more slowly but more deeply into the POPC bilayer than into the POPG bilayer. Upon adsorption, the affinity of LAH4 for the anionic bilayer is further enhanced as a result of the formation of many hydrogen bonds between the peptide and the head groups of bilayer (Table 2).

Peptide–lipid interactions The previous studies have demonstrated that hydrogen bonds and electrostatic interactions are the major driving forces to associate the AMPs with the cell membrane (39,40). To investigate the detailed mechanism, we have performed a hydrogen-bonding analysis on the results of all the simulations, focusing on the interactions between the peptide and the lipid head groups (Table 2). The results have shown that the total number of hydrogen bonds between the following pairs at early and late stages of the MD simulations. These results are to be expected, because the peptide forms hydrogen bonds with bilayers during the MD simulations. In almost all the simulations, abundant hydrogen bonding is observed between the lipid head groups and the peptide. Peptide–water bonds have decreased early in the simulation process, while peptide–lipid hydrogen bonds have increased. The MD 5

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Figure 5: Peptide positions relative to the center of mass of the adjacent lipid leaflet. Table 2: Average number of hydrogen bonds formed at the early and the last stages of the simulations for peptide–lipid pairs and peptide–water pairs pH = 5.0

LAH4-POPC LAH4-water (POPC) LAH4-POPG LAH4-water (POPG)

pH = 7.0

5–10 ns

195–200 ns

5–10 ns

195–200 ns

7 66

50 27

6 71

37 19

27 77

65 46

17 71

50 58

POPC, palmitoyl–oleoyl–phosphatidylcholine; POPG, palmitoyl– oleoyl–phosphatidylglycerol.

simulations have indicated that the peptide moves from water environment to the bilayer environment, and in the process, forms persistent hydrogen bonds with lipid head group regions (Table 2). This effectively reduces the number of hydrogen bonds between the peptide and water. As shown in Table 2, LAH4 strongly binds to the anionic lipid at acidic pH. To demonstrate the structural features of the peptide–lipid mixed system, we have also calculated various radial distribution functions between the histidines side chains and the head groups of lipids in during the last 20 ns of the simulations. These data have informed us of the detailed location and orientation of the peptide with respect to the 6

bilayers as well as give information on hydrogen-bondformation patterns and electrostatics interactions. As we discussed earlier, the side-chain imidazole group of histidine is of great importance in the chemistry of LAH4. This means that in the acidic pH, histidines can afford donor and acceptor atoms for hydrogen-bond formation. LAH4 contains 4 histidines that are capable of forming hydrogen bonds, the experimental studies have shown that it interacts preferentially with anionic lipids, and this interaction is enhanced at acidic pH when the four histidine residues become positively charged (14,16,17). Although histidines have been suggested to play an important role, experimental evidence for such a role is not still lacking. Therefore, our MD results guided by experimental data help us to better understand the events involved in the molecular recognition of an interface. For this purpose, we have calculated the RDFs between the side chains of histidines and the phosphorus atoms of the lipids. These results have revealed that histidines contribute significantly to the hydrogen-bonding interactions to the peptide (Figure 6). As can be seen, the side chain of His11 can interact with lipid bilayers in all simulations. At acidic condition, the imidazole ring of His11 formed strong hydrogen bond with POPG molecules (Figure 7). These data suggest then that the imidazole functional group contributes most significantly to the hydrogen bonding potential of the histidine, which in turn contributes to the hydrogen bonds formed between LAH4 and membranes. The side chain of His11 has peaks at 0.29 nm and 0.4 nm, indicating hydrogenbonding/electrostatic interactions with POPC and POPG. All of the RDFs in the Figure 7 have a first sharp and narrow peak at about 0.29 nm, indicating the formation of stable hydrogen bonds between the proton on the residue and the oxygen atom in the phosphate group for each of the amino acid–lipid pairs (Figure 7). The magnitude of the second peak of the RDF between His11 and phosphor atoms shows a further of probability to the presence of LAH4 within lipid phosphate head groups under acidic conditions and demonstrates more preferential binding of this positively charged residue to POPG bilayer at pH 5.0 so that can result strongly interacting with anionic membrane (Figure 8). In combination of previous experimental study (19) and our MD results, we can demonstrate that electrostatic interactions between the side chain of His11 and the phosphor atoms of bilayers should have a significant impact on the activity of LAH4. Thus, it can be responsible for its favorable function of LAH4 faced with the anionic membrane and improve anticancer activity of LAH4 whereby this cationic antimicrobial peptide can destabilize membranes of microbial and cancerous cells.

Effect of peptide on membranes Lipid tail ordering Order parameter, |SCD|, compares the order of the lipid tails between the peptide-free bilayer and the peptideChem Biol Drug Des 2014

Study of LAH4–Membrane Interactions

Figure 6: The radial distribution functions between imidazole ring of histidines and the phosphor atoms of the lipids during the last 20 ns of the simulation time.

Figure 7: The radial distribution functions between imidazole ring of His11 and the phosphor atoms of the lipids during the last 20 ns of the simulation time.

bound bilayer (41,42). The lipid tail order parameters for both sn1 and sn2 acyl chains of the peptide-free bilayer and peptide-associated bilayer have been shown in Figure 9. In agreement with the previous experimental study (8), our results have shown that the |SCD| values of sn1 and sn2 acyl chains in the LAH4-POPC system slightly are lower than those in the pure POPC simulation at both the acidic and neutral pH (Figure 9). Interestingly, this study has also demonstrated that the disruption of POPC by LAH4 is very similar at pH 5.0 and 7.0 (Figure 9). At pH 5.0, our analyses have shown that binding of LAH4 significantly reduces the lipid acyl chain order for POPG bilayer. Remarkably, in agreement with the previous experimental study (8), our data have also indicated that POPG acyl chains are disordered close to the lipid head group more than center of the bilayer (Figure 9). LAH4 was shown to be able to interact in a stronger manner with POPG model membranes; both its Lennard-Jones and Coulomb interaction energies (LAH4-POPG) are higher than LAH4-POPC. This result proposes a potential mechanism whereby LAH4 penetrates into the bilayer and preferentially inserts in around the head group interface (8). At pH 7.0, the presence of the peptide causes a slight Chem Biol Drug Des 2014

increase in the order parameter of two fatty acyl chains of the PG molecules with respect to the pure POPG bilayer (Figure 9). Consistent with experimental studies (7,8,11), these results have strongly reflected that the membrane models in these simulations have responded differently to the LAH4 binding. The POPG lipids tend to be more disordered in the presence of LAH4. Interestingly, this reduction in the order of POPG fatty acyl chains followed by inserting LAH4 in bilayer has revealed a selective disruption for the anionic membrane. Previous experimental investigations (6,8,9,11,12,18) have shown that the peptide has indicated an anionic membrane-destabilizing activity, and its antibiotic ability has become enhanced at pH 5.0 when it has oriented parallel to the membrane surface and histidines have charge. Due to the different lipid components in membrane of cancer cells and normal cell surfaces, these results can provide useful insights about how the presence of different lipids can alter LAH4 structure–function relationships. Because a high selectivity and killing capability are basic requirements for clinical use of AMPs, these data can prove this claim that LAH4 may be considered as an anticancer and antimicrobial drug. Therefore, understanding the mechanism of action such an amphipathic peptide might be a major advance in cancer therapy.

Energetics of LAH4 binding These findings have revealed that LAH4 is able to interact with anionic PG membranes more effectively than zwitterionic PC membranes. Experimental studies suggest that the peptide binds to POPG bilayers strongly (9–11). To compare the interaction between LAH4 and the model lipid bilayers, we have calculated the one-dimensional PMF, W (z), for all systems, as a function of the reaction co-ordinate z (Figure 10). The profiles constructed along the normal bilayer show that the binding of LAH4 to POPC bilayer is weaker than POPG bilayer. As is evident from Figure 10, the placement of the peptide at the membrane 7

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Figure 8: Representative snapshots of histidines, interacting with palmitoyl–oleoyl– phosphatidylglycerol lipid bilayer.

Figure 9: Deuterium order parameter of lipid acyl chains at pH 7.0 and pH 5.0.

surface was associated with a minimum PMF
0.7 kcal/ mol at 0.39 nm for POPC and
0.90 kcal/mol at 0.44 nm for POPG at pH 7.0, compared with -0.95 kcal/mol at 0.43 nm for POPC and
1.2 kcal/mol at 0.45 nm for POPG at pH 5.0. This difference in the PMF profiles of LAH4 at pH 5.0 and pH 7.0 has indicated more 8

tendency and favorable interactions of LAH4 for lipid head groups under acidic conditions. By analyzing the PMF profile at acidic pH, we have found that the negatively charged lipid forms a relatively strong binding complex with LAH4 (compared with zwitterionic membranes). Chem Biol Drug Des 2014

Study of LAH4–Membrane Interactions

Figure 10: Potential of mean force (PMF) the Cb of hydrophobic residues obtained from palmitoyl–oleoyl–phosphatidylcholine and palmitoyl–oleoyl–phosphatidylglycerol simulations of LAH4.

diffusive compared with the POPG bilayer at pH 5.0 (Figure 11). This suggests that the interactions between disordered POPG acyl chains probably limited POPG molecules to move throughout the membrane. Our results have demonstrated the lateral diffusion coefficient, D, for POPG bilayers (at acidic pH) is significantly lower compared with POPC bilayer (Table 3). Therefore, we can conclude LAH4 have stronger interactions with anionic bilayer head than zwitterionic one, and it has resulted in the reduced rate of lateral diffusions for POPG bilayer.

Conclusion Lipid diffusion We have obtained the lateral diffusion coefficient from the mean square displacements (MSD) for the lipid molecules versus time (Figure 11). The lateral diffusive rate of lipid molecules on a membrane surface is considered as function of the membrane composition, the concentration of an obstacle, temperature, the hydration level, and the observed timescale (43–45). Kim et al. (45) have shown that in the presence of antimicrobial peptides, the rate of lateral diffusions of lipids in bilayers would be affected due to the electrostatic, hydrophilic, and/or hydrophobic peptide–lipid interactions. In agreement with this study(45), our findings have indicated that the POPC membrane is more

B

A

Figure 11: (A) Mean square displacement (MSD) curves of palmitoyl– oleoyl–phosphatidylcholine and palmitoyl–oleoyl–phos-phatidylglycerol (solid) calculated from the last 20 ns of the simulation time, with colors corresponding to different simulations. (B) Averaged MSD (dashed).

Table 3: Lateral diffusion coefficients for membrane lipids Dlat (10
8 cm2/s)

System LAH4 + LAH4 + LAH4 + LAH4 + POPC POPG

POPG (pH POPC (pH POPG (pH POPC (pH

= = = =

5.0) 5.0) 7.0) 7.0)

3.71 5.22 4.61 6.41 7.12 2.73

     

0.01 0.01 0.02 0.01 0.04 0.01

POPC, palmitoyl–oleoyl–phosphatidylcholine; POPG, palmitoyl– oleoyl–phosphatidylglycerol.

Chem Biol Drug Des 2014

In conclusion, the results of the current study have shown that the depth of LAH4 penetration depends on membrane composition and change in pH (19,46). Accordingly, this cationic peptide penetrates more deeply into the zwitterionic POPC bilayer compared with the anionic POPG membrane model. In agreement with experimental studies (8,11,16,26), these simulation results have also shown that PG acyl chains were disordered near the lipid head group more than center of the bilayer when the peptide was incorporated into the lipid bilayer that such effect could drive membrane disruption. We suggest occurrence of electrostatic interaction between the cationic histidine residues (especially His-11), and the anionic head groups enhances LAH4 affinity to negatively charged membrane. Therefore, it can be an important factor in determining activity of this peptide (11,19). Indeed, change in pH and its effect on protonation of imidazole ring can influence LAH4-induced membrane disrupting. We have inferred that electrostatic interactions are responsible for initial peptide–membrane binding (18,47,48) and subsequently establish van der Waals contacts and hydrophobic interactions through its non-polar residues (18,49). It can be concluded; at acidic pH, LAH4 preferentially interacts with anionic lipid bilayers more than zwitterionic lipid membrane (8,18). Consequently, surface exposure of anionic lipids such as phosphatidylserine and phosphatidylglycerol, respectively, at plasma membrane of tumor cells and bacterial membranes can be resulted selective toxicity of LAH4 toward for these two cell types compared with normal cells. Previous experimental studies have indicated the presence of anionic lipid can lead to preferential interaction between tumor cells and alpha-helical cationic AMPs. After entering into membrane bilayer, AMPs induce acyl chains disordering and subsequent membrane disrupting that could cause death of cancer cell (6,49–51). In fact, the differences in the head groups of lipids, such as PG head groups versus PC, can be considered as crucial factor affecting the membrane structure and the preference of LAH4 for bacterial membranes. Therefore, LAH4 can undoubtedly be considered as an excellent candidate for the development of a new class of antibiotics. To obtain the mechanism of pore formation by LAH4, multiple peptides in bilayers of varying compositions need to be investigated. 9

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Acknowledgments The support of the Iran National Science Foundation (INSF) is gratefully acknowledged. We thank all of our colleagues for help and advice.

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