Molecular Basis for Membrane Selectivity of Antimicrobial Peptide

Article Cite This: J. Chem. Inf. Model. XXXX, XXX, XXX−XXX

pubs.acs.org/jcim

Molecular Basis for Membrane Selectivity of Antimicrobial Peptide Pleurocidin in the Presence of Different Eukaryotic and Prokaryotic Model Membranes Reza Talandashti,† Hamid Mahdiuni,‡ Majid Jafari,† and Faramarz Mehrnejad*,† †

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Department of Life Science Engineering, Faculty of New Sciences and Technologies, University of Tehran, P.O. Box 14395-1561, Tehran, Iran ‡ Bioinformatics Lab., Department of Biology, School of Sciences, Razi University, P.O. Box 67149-67346, Kermanshah, Iran S Supporting Information *

ABSTRACT: Pleurocidin, a 25-residue cationic peptide, has antimicrobial activity against bacteria and fungi but exhibits very low hemolytic activity against human red blood cells (RBC). The peptide inserts into the bacterial membrane and causes the membrane to become permeable by either toroidal or carpet mechanism. Herein, to investigate the molecular basis for membrane selectivity of Pleurocidin, the interaction of the peptide with the different membrane models including the RBC, DOPC, DOPC/DOPG (3:1), POPE/ POPG (3:1), and POPE/POPG (1:3) bilayers were studied by performing allatom molecular dynamics (MD) simulation. The MD results indicated that the peptide interacted weakly with the neutral phospholipid bilayers (DOPC), whereas it made strong interactions with the negatively charged phospholipids. Pleurocidin maintained its α-helical structure during interactions with the anionic model membranes, but the peptide lost its secondary structure adjacent to the neutral model membranes. The results also revealed that the Trp-2, Phe-5, and Phe-6 residues, located in the Nterminal region of the peptide, played major roles in the insertion of the peptide into the model membranes. In addition, the peptide deeply inserted into the DOPC/DOPG membrane. The order analysis showed that Pleurocidin affected the order of anionic phospholipids more than zwitterionic phospholipids. The cholesterol molecules help the RBC membrane conserve integrity in response to Pleurocidin. This research has provided data on the Pleurocidin−membrane interactions and the reasons of resistance of eukaryotic membrane to the Pleurocidin at atomic details that are useful to develop potent AMPs targeting multidrug-resistant bacteria.

1. INTRODUCTION During the past decades, many antimicrobial peptides (AMPs) have been isolated and characterized from a wide range of organisms.1 They are involved in the immune response to the bacterial aggression and act as the first-line defense against invading microorganisms. AMPs usually cause rapid death of a wide range of targets, such as Gram-positive and -negative bacteria, viruses, parasites, and even cancer cells. They act on the target cells by different mechanisms.2,3 Most AMPs act directly on the plasma membrane of the target cell and disrupt it, whereas the others disturb cellular metabolisms by targeting the cell signaling pathways.4,5 Therefore, the ability of AMPs to interact and penetrate into the target cell membrane is the main point in the field of antimicrobial peptides research. Considering functions of AMPs, the peptides can be used as novel antibiotics instead of current antibiotics, particularly when there is resistance to antibiotic treatments.6 Pleurocidin is a cationic and yet amphiphilic antimicrobial peptide with 25 amino acids (GWGSFFKKAAHVGKHVGKAALTHYL) that was isolated from the skin mucous secretion of winter flounder (Pseudopleuronectes americanus).7 Previous © XXXX American Chemical Society

circular dichroism (CD) studies have shown that in the presence of trifluoroethanol (TFE), sodium dodecyl sulfate (SDS), and various types of membrane models Pleurocidin adopts an α helical structure with hydrophilic and hydrophobic residues on opposing sides of the helical structure.8−10 Previous NMR investigations have confirmed that Pleurocidin has 95% α helix conformation between residues Trp-2 and Lue-25 in the presence of 140 mM dodecylphosphocholine [DPC]. Furthermore, the analysis of NOESY spectra indicated that Pleurocidin at low peptide-to-lipid ratios bound to the membrane surface in the parallel orientation with respect to the membrane normal direction and at higher peptide-to-lipid ratios the peptide penetrated into the membrane and formed the toroidal pores.11,12 Pleurocidin also could affect various intracellular processes representing different activity mechanisms of the peptide, suggesting that Pleurocidin has a high potential for passing through the bacterial membrane.9,13 Experimental studies have also demonstrated that the peptide Received: March 22, 2019 Published: June 25, 2019 A

DOI: 10.1021/acs.jcim.9b00245 J. Chem. Inf. Model. XXXX, XXX, XXX−XXX

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Journal of Chemical Information and Modeling Table 1. Summary of the MD Simulation’s Details simulation systems peptide bilayer

bilayer

DOPC DOPC/DOPG (3:1) POPE/POPG (3:1) POPE/POPG (1:3) RBC DOPC DOPC/DOPG (3:1) POPE/POPG (3:1) POPE/POPG (1:3) RBC

acronym

no. lipid molecules

no. water molecule

temp (K)

SDP1 SDP2 SDP3 SDP4 SDP5 SD1 SD2 SD3 SD4 SD5

190 PC 142PC, 48PG 142PE, 48PG 142PG, 48PE a 190 PC 142PC, 48PG 142PE, 48PG 142PG, 48PE a

14 586 14 578 12 560 12 903 10 997 14 820 14 718 12 706 13 041 11 133

303 303 323 323 310 303 303 323 323 310

length (ns) 2× 2× 2× 2× 2× 100 100 100 100 100

500 500 500 500 500

a The RBC membrane lipid is composed of Chol, POPC, POPE, SM16, and DOPS with a ratio of 45:23:6:21:0 in the upper leaflet and 45:7:22:5:16 in the lower leaflet.

above the surface of the bilayer models with parallel orientation and each peptide−bilayer system was simulated for 500 ns. On the basis of previous studies, it seems that the length of the simulation is sufficient to study peptide−bilayer interactions and early insertion of Pleurocidin on the bilayer membrane models surface.25−28 Further simulation details are given in Table 1. 2.2. MD Systems Setup. All MD simulations were performed with the GROMACS package, version 5.1.4.29 The CHARMM force field was used for the peptide and all bilayer membrane models.30,31 The systems were neutralized by adding the appropriate number of sodium and chloride ions to each simulation box. The periodic boundary conditions (PBC) were applied in all simulation box axes and the TIP3P water model was used to solvate the systems.32 To remove bad contacts and steric hindrances in structures, all simulation systems were energy-minimized by using the steepest descent algorithm. The temperature was maintained at melting temperature (Tm) for each bilayer membrane using the Nosé−Hoover algorithm temperature (Table 1).33,34 To reach the target temperature for each system, all simulation systems were equilibrated under the NVT ensemble for 500 ps. The pressure for all the simulation systems was maintained 1 bar using the Parrinello−Rahman barostat under the NPT ensemble.35 The LINCS algorithm was used for constraining all covalent bonds.36 The particle mesh Ewald (PME) algorithm was used for calculation the long-range electrostatic interactions.37 van der Waals interactions and short-range electrostatic interactions were computed with 1.2 nm distance cutoff. Two independent trajectories were produced for each peptide−bilayer system. 2.3. Analyses. All analyses were performed over the last 100 ns of the simulations times. The APL value and membrane thickness were calculated using FATSLiM and GridMAT, respectively.38,39 The secondary structure of the peptide was investigated by a dictionary of secondary structure of proteins (DSSP) algorithm40 and the helicity of the peptide was calculated using “gmx helix” tools. The molecular mechanics Poisson−Boltzmann surface area (MM-PBSA) method is widely used for calculation of the binding free energy in biological systems such as protein− ligand and peptide−membrane interactions.26,28,41,42 The g_mmpbsa tool calculates the binding free energy using the molecular mechanics-Poisson−Boltzmann surface area (MMPBSA) method.43,44 The total binding free energy is obtained by the following equation:

has strong interactions with anionic lipids, while it weakly interacts with zwitterionic lipids.8−14 In addition, it has been shown that helical content and flexibility of the peptide structure directly affects its mechanism of action and its antibacterial activity.8,9,11,12,15,16 Pleurocidin has a broad antimicrobial activity against Gram-positive and Gram-negative bacteria while it has very low hemolytic activity against red blood cells [RBCs].8 Since the peptide has selective lipid membrane-perturbation activities,17 it is a suitable candidate for use in the treatment of bacterial infections such as sepsis,18 as well as cancer treatments.19,20 Previous studies have also indicated that the peptide has low toxicity on gingival fibroblasts, a cytolytic effect on breast cancer cells, antifungal activity, and growth prevention of tumor xenografts.17,19−21 Taking into account the mentioned features of Pleurocidin, our main goal in the study is to find out how the peptide can recognize the bacterial cell membranes from the eukaryotic cell. Therefore, all-atom MD simulations were used to study the interactions of the peptide with different lipid bilayer models. For this purpose, five lipid bilayer models were selected: 1,2-dioleoyl-sn-glycero-3-phosphocholine [DOPC]; a zwitterionic membrane model, DOPC/1,2-dioleoyl-sn-glycero3-phosphoglycerol [DOPG] (3:1); a model for anionic lipid bilayer; and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine [POPE]/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol [POPG] (3:1) and POPE/POPG (1:3) as models of Gram-negative and Gram-positive bacterial membranes, respectively. In addition, a model of a RBC membrane consisting of cholesterol, 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine [POPC], POPE, 1,2-dioleoyl-sn-glycero-3phospho-L-serine [DOPS], and N-palmitoyl-D-erythro-sphingosylphosphorylcholine [SM16] were used on the basis of a previous simulation study.22 The results of this study could provide useful information on the understanding of antimicrobial effects of Pleurocidin against different model membranes as well as the responses of those membranes to the antimicrobial peptide.

2. COMPUTATIONAL METHODS 2.1. Preparation of MD Simulation. The starting structure of the peptide was taken from RCSB Protein Data Bank (PDB ID: 1Z64).12 The initial lipid bilayer structures and their topologies were created using the CHARMM-GUI web tool.23,24 To obtain equilibrated systems, all pure bilayers were simulated for 100 ns and the last snapshots of each system were used as the initial structure for simulating each peptide− bilayer complex. The peptide was placed approximately 3 nm B


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Journal of Chemical Information and Modeling

Figure 1. Average distance of COM of each residue from the COM of the lipid bilayer models and helicity of the peptide during the last 100 ns in the (A) SDP1, (B) SDP2, (C) SDP3, (D) SDP4, and (E) SDP5 simulations. Radial distribution function (RDF) of water molecules around the peptide in all simulations (F). The term “Distance_Residue” refers to the average distance between COM of each residue and the COM of the membrane. The average distance between COM of phosphorus atoms and COM of the membrane is referred to as “Distance_P”, and the average distance between COM of the membrane and COM of O3 atoms is referred to as “Distance_O3”. The standard deviation was calculated in two replications.

ΔG binding = Gcomplex − G bilayer − Gpeptide

where the total binding free energy is obtained by summing up ΔGpb and ΔGnpb that are the polar binding free energy and nonpolar binding free energy, respectively. ΔGpb and ΔGnpb can be expressed by the following equations:

(1)

where Gcomplex, Gbilayer, and Gpeptide are the total free energy of the Pleurocidin−bilayer, bilayer, and Pleurocidin, respectively. Furthermore, this value is equal to the total free energy binding that can be expressed by the following equation: ΔG binding = ΔGpb + ΔGnpb

(2) C

ΔGpb = ΔGelec + ΔGpols

(3)

ΔGnpb = ΔGvdW + ΔGnpols

(4) DOI: 10.1021/acs.jcim.9b00245 J. Chem. Inf. Model. XXXX, XXX, XXX−XXX

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Journal of Chemical Information and Modeling where ΔGpols, ΔGnpols, ΔGelec, and ΔGvdW are the polar solvation energy, nonpolar solvation energy, electrostatic energy, and van der Waals energy, respectively. To calculate total binding free energy, 1000 snapshots were extracted from the last 100 ns of individual trajectories of each peptide− bilayer system.

those are in the C-terminal residues.21 These experimental results are in good agreement with our MD simulation results. As a result, Pleurocidin has a better penetration in the anionic membrane relative to zwitterionic membrane. In a similar study of MD simulations of human antimicrobial peptide Ll-37 in model bilayers, Zhao and colleagues illustrated that the peptide has a deeper penetration into the anionic membrane rather than the zwitterionic membrane.50 Previous studies have represented that the lipid head groups have a key role in the Pleurocidin interactions with a membrane.8,10 Mason et al. illustrated that the intrinsic fluorescence intensity of Trp-2 in Pleurocidin was significantly reduced when the lipid head groups were changed from PC to PE.10 These results suggested that the peptide in the POPE/ POPG (3:1) lipid bilayer was quite accessible to the aqueous environment although still associated with the lipid bilayer. Our simulations analysis demonstrated that, in SDP3 (the POPE/POPG (3:1) and Pleurocidin simulation system) and SDP4 (the POPE/POPG (1:3) and Pleurocidin simulation system), the peptide was attached to the bilayer surface after 10 ns and no penetration was observed. In both systems, the side chains of the aromatic residues were not able to cross the phosphorus atoms (Figure S1C,D). In the SDP4 (Grampositive model) system, the most Pleurocidin residues were adsorbed on the membrane surface and remained there and only four residues at the C-terminal region were unable to adsorb on the membrane surface (Figure 1D). In addition, the most residues in SDP3 (Gram-negative model) were not able to contact closely with the bilayer (Figure 1C). The plausible explanation for these findings can be assigned to the presence of more phospholipids with the negative PG head groups in the SDP4 membrane, which increases the binding affinity between the peptide and the membrane. Although we deal with the anionic lipid bilayers in both SDP3 and SDP4, the peptide was unable to cross the phosphate head groups and could not penetrate into the hydrophobic core of the membrane. By comparing the results of DOPC/DOPG (3:1) with the Gram-negative (SDP3) and Gram-positive (SDP4) lipid membranes, it could be concluded that in the anionic lipid mixture, Pleurocidin has a better penetration in the membrane when the zwitterionic lipid head groups are PC, while the peptide does not show any penetration when the zwitterionic lipid head groups are PE. This finding is in excellent agreement with the previous experimental studies and illustrates the role of lipid head groups in the peptide− membrane interactions.8,9,11 In a recent study, Mason and colleagues examined the effect of cholesterol on the Pleurocidin interactions with the lipid bilayers. Their results showed that the peptide was increasingly excluded from the hydrophobic core of the membrane and exposed to the aqueous environment when the concentration of cholesterol was increased in the anionic lipid bilayers.10 In addition, a recent molecular dynamics study has indicated that cholesterol conserves the membrane integrity during interactions with the proteins or peptides.25 Therefore, in this research, we used the RBC membrane model having a high percentage of the cholesterol in its constituent lipids (the SDP5 simulation system). In this way, the z-coordinates of the O3 atoms at the apex of cholesterol molecules were measured (Figure 1E and Figure S2). As depicted in Figure 1E, the residues Trp-2, Phe-5, and Phe-6 crossed the phosphorus atoms of the phospholipids and stuck on the O3 atoms of cholesterol at 60 and 90 ns, respectively (Figure S1E). The

3. RESULTS AND DISCUSSION 3.1. Behavior of Peptide toward the Lipid Bilayer Models. 3.1.1. Insertion Depth and Structural Change of Pleurocidin. In order to measure the proximity of Pleurocidin to the bilayer models, the z-coordinates of the center of mass (COM) of the peptide backbone and the COM of the phosphorus atoms (P) were calculated as a function of time (Figure S1). In addition, to provide more detailed information, the average distance between the COM of each residue of Pleurocidin and the COM of the bilayers was computed during the last 100 ns of simulations (Figure 1). Previous studies have shown that the aromatic residues have a key role in the early insertion of the peptide onto the membrane surface and they have a tendency to be placed in the membrane hydrophobic core.26,28 In addition, it has been shown that the Trp side chain acts as an anchor for peptides and could locate between the phospholipid acyl chains.45−48 It has also been proven that the cationic residues of AMPs have a major role in the peptide adsorption on the anionic membrane surfaces by electrostatic interactions.49 As shown in Figure 1A, in SDP1 (the DOPC and Pleurocidin simulation system), the peptide was not able to adsorb onto the DOPC membrane surface. The analyses showed that the approximate distance of the N-terminal residues of the peptide (residues 1−6) from the bilayer surface was 0.5−1.0 nm, while the central residues of the peptide were located at distances of 1.5−2.0 nm from the bilayer surface. The larger distances between the central region of the peptide and the membrane surface are possibly due to the strong electrostatic repulsions between the NH3+ groups of the Lys residues and the partially positive charge of the choline groups of the DOPC molecules; the deduction is confirmed by the binding energy results, which will be discussed in the following parts of this paper. As a result, the side chains of aromatic residues (Trp-2, Phe-5, and Phe-6) could not be inserted into the hydrophobic core of the zwitterionic membrane (Figure S1A). In contrast to SDP1, in SDP2 (the DOPC/DOPG and Pleurocidin simulation system) most of the peptide residues crossed the phosphorus atoms of the phospholipids so that the N-terminal residues inserted into the membrane deeper than the C-terminal residues (Figure 1B). In terms of penetration time, the whole peptide crossed the membrane surface after 110 ns, while the Trp-2, Phe-5, and Phe-6 residues passed from the phosphate headgroup of the bilayer only after 20 and 60 ns, respectively (Figure S1B). This finding demonstrates that Pleurocidin inserted into the lipid bilayers through its Nterminus residues. The results were confirmed by previous experimental study indicating Pleurocidin embedded further into the hydrophobic core of the anionic lipid membranes.8,11 Fluorescence spectroscopy studies have indicated that the fluorescence intensities of Pleurocidin are increased in the presence of anionic lipids such as POPG compared to the zwitterionic lipids.8 Intrinsic fluorescence results have confirmed that the N-terminus residues of Pleurocidin penetrate further into the hydrophobic core of the anionic membranes.11 In addition, the N-terminal residues have generally a more crucial role in the antimicrobial activity of Pleurocidin than D


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nearly its helical structure in the hydrophobic core of the membrane. The results of helicity analysis indicated that the peptide entirely loses the helical structure adjacent to the zwitterionic membrane. In contrast, almost all the Pleurocidin residues conserve their helical structure in contact with anionic membranes such as the DOPC/DOPG (3:1) lipid bilayer. In the Gram-negative, Gram-positive, and RBC membranes, the N-terminal residues of the peptide had great potential to conserve the helical structure compared with the C-terminal residues (Figure 1A−E). 3.1.2. Pleurocidin−Lipid Bilayer Interactions. To elucidate the affinity of Pleurocidin to lipid bilayer models, the average number of contacts between the peptide and the membrane models was measured (Table 3). Furthermore, to determine the crucial residues, the contributions of each amino acid in the total number of contacts between Pleurocidin and the membranes were also calculated (Figure 2). The highest and lowest number of contacts between Pleurocidin and the membranes were allocated to the SDP2 and SDP1 systems, respectively. The maximum number of contacts in the SDP2 system is due to the entire penetration of Pleurocidin into the lipid bilayer and, accordingly, its having more interactions with the acyl chains of the phospholipids. In the SDP2 and the SDP5 systems, the Trp-2, Phe-5, and Phe-6 residues have the maximum contacts with the lipid bilayers (Figure 2B). The average number of contacts for each residue in all simulations (Figure 2A) demonstrated that Trp-2 had maximum contacts with the membranes among the other residues, which is probably related to the anchor-like role of the Trp residues in proteins and peptides.45 The contact analysis illustrated that the Lys residues in SDP2 and SDP4 could establish the highest number of contacts with the corresponding membranes (Figure S3). An exception in this regard was the Lys-7 residue, which had many more contacts in SDP3 than in SDP4. The Lys residues in SDP1 had the fewest contacts with the corresponding membrane, which is due to the absence of the anionic lipids within the membrane model. In general, the N-terminal region of the peptide had more contacts with the lipid bilayer models than the C-terminal region. In conclusion, on the basis of the contact analysis results, it can be suggested that Pleurocidin has more affinity to the DOPC/DOPG (3:1) membrane than the others. This is possibly due to the presence of the anionic lipids and PC head groups, which causes the peptide has appropriate interactions with the membrane. 3.1.3. Interactions of Pleurocidin with Na+ and Cl− Ions. To indicate whether the peptide has direct interactions with the NA+ and Cl− ions, we calculated the average number of contacts between the peptide and the ions using a cutoff distance of 0.6 nm (Figure 3A). In the anionic membranes (SDP2, SDP3, and SDP4), the peptide had very low interactions with the Cl− ions. Due to electrostatic repulsions between negative charged membranes and the chloride ions, the Cl− ions were not able to be close enough to the peptide to create lasting interactions with Pleurocidin. However, in the

COM of Pleurocidin reached to the membrane surface after 180 ns (Figure S1E). Similar to the other simulations, the Nterminal residues of the peptide had higher contributions to interact with the RBC membrane and penetrated deeper than C-terminus ones. The distribution of water molecules around Pleurocidin was decreased after penetration of the peptide into the membrane. To examine the distribution of water molecules around the peptide, the radial distribution functions (RDFs) between the water molecules and the peptide were calculated in all MD simulations. According to the RDF graphs, the first peaks (range of 0.14−0.22 nm that separately shown) in all simulations are related to the hydration shell of water molecules around the peptide (Figure 1F). The results illustrated that in the DOPC/DOPG (3:1) membrane the water molecules are excluded from the Pleurocidin surface and the peptide is sat in the hydrophobic environment. In order to gain insight into the effect of lipid bilayer models on the secondary structure content of Pleurocidin, the dictionary of secondary structure of proteins (DSSP) algorithm40 and the helicity analysis were measured for the peptide over the last 100 ns of trajectories (Figure 1 and Table 2). On the basis of the secondary structure, AMPs are Table 2. Percent of the Pleurocidin Helix Content at the Different Bilayer Models during the Last 100 ns of the MD Trajectoriesa helix content

SDP1

SDP2

SDP3

SDP4

SDP5

3±2

81 ± 8

40 ± 12

50 ± 16

47 ± 15

a

Standard deviation was calculated in two replications.

categorized into four families: α-AMPs, β-AMPs, αβ-AMPs, and non-αβ AMPs.51 Most of the α-AMPs maintain the helical structures in the hydrophobic environment and lose their secondary structure contents in an aqueous ambient environment.52 Point mutation experiments have revealed that the antimicrobial activity of Pleurocidin is more dependent on the helical contents of the peptide structure, rather than net charge and hydrophobicity of the peptide.8,15,21,53,54 As shown in Table 2, Pleurocidin in the SDP2 system kept a higher percent of its helical contents among others and maintained approximately 81% of its helicity. Surprisingly, in SDP1, the peptide lost almost entirely the helical structure and turned to the coil structure. In the other simulation systems, Pleurocidin retained 50% of its helical contents on average. Circular dichroism (CD) studies on the Pleurocidin structure in the different lipid bilayer models have indicated that the peptide in the DOPC bilayer loses its helical structure, while it can keep the helicity in DOPC/DOPG (3:1).8 Further experimental studies have demonstrated that Pleurocidin has partially lost its helix structure in the POPE/POPG (3:1) lipid bilayers.9−11,16 Evidently, the CD data are in good agreement with our MD results, indicating the peptide lost its structure in the aqueous environment and zwitterionic membrane, while sustained

Table 3. Number of Contacts (0.6). (B) Average number of ions around the peptide at distances less than 1 nm.

zwitterionic membranes (SDP1 and SDP5) the Cl− ions make more interactions with the peptide compared with the Na+ ions. In the SDP4 system, considering the membrane is a highly negative charged bilayer, the Na+ ions were adsorbed on the membrane surface, which can make more interactions with the peptide. In addition, we calculated the average number of ions surrounding the peptide at a distance of less than 1 nm (Figure 3B). As illustrated in Figure 3B, in SDP2 and SDP5 the average number of ions around the peptide was very low since the peptide was significantly penetrated into the membrane. In SDP3 and SDP4, there were more Na+ ions around the peptide than the Cl− ions. While in the SDP1 system, there were more Cl− ions around the peptide than the Na+ ions.

3.1.4. Peptide Location during the Interactions with Lipid Bilayers. As mentioned, in all MD simulations the peptide was located at 3 nm distance above the lipid bilayers. The hydrophilic side of the peptide was toward the membrane surface, and the hydrophobic side of the peptide was toward the aqueous environment. In Figure 4, the structure and location of Pleurocidin at the end of peptide−membrane trajectories were illustrated. In SDP1, the peptide completely lost its helical structure and the side chains of basic residues were oriented on the opposite side of the membrane surface (Figure 4A). The aromatic residues were unable to penetrate into the membrane core because the peptide could not get enough close to the membrane surface, which is a required condition for the penetration. However, in the SDP2 system, F


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Figure 4. Structure and orientation toward the membrane of Pleurocidin in all simulations after 500 ns. (A), (B), (C), (D), and (F) represent the last snapshot of trajectory in the SDP1, SDP2, SDP3, SDP4, and SDP5 systems, respectively. The peptide structure is shown as a cartoon model and the lipid acyl chains are not displayed. The P atoms are shown as spheres and cholesterol molecules in (F) are shown in yellow. The Lys and aromatic residues are shown in magenta and cyanine, respectively.

head groups was calculated and the findings revealed that the SDP3 and SDP4 systems had the highest number of H-bonds (Figure S4). The POPE phospholipids have an amine (NH3+) group, which can act as either donor or receptor for hydrogen bonding. In membranes, the amine groups can organize a network of H-bonds, keeping the lipid head groups together and raising the high-energy barrier against the peptide penetration.56 Therefore, the inability of Pleurocidin to insert into the Gram-negative and Gram-positive membranes can be attributed to the existence of a large number H-bonds between the lipid head groups. To provide a better insight into the peptide behavior during interactions with the membrane, a series of snapshots through the trajectories were extracted and shown in Figures S5−S9. 3.2. Structural and Dynamical Changes of Membranes in Response to Pleurocidin. 3.2.1. Order and Area per Lipid Parameters. Bacterial membranes have been identified as the major targets for the activity of most AMPs.57 The majority of investigations on the AMP interactions with lipid bilayers have focused on the membrane integrity and structural changes of the peptides.9−11,26,28,55,58,59 It has been shown that the lipid composition of membranes can control the mechanisms of action and the activities of AMPs.60 Actually, the evolution of AMP-resistant bacteria has been linked to the modification of lipid composition since

the peptide thoroughly penetrated into the membrane and the side chains of the aromatic residues were oriented toward the hydrophobic core of the membrane (Figure 4B). However, the penetration of the peptide into the membrane was stopped on the interface of head groups and hydrophobic tails. This anchoring fashion is due to hydrogen bindings and electrostatic interactions between the Lys residues and the phosphate head groups.26,55 A similar phenomenon occurred in SDP5 with the difference that only the N-terminal region of the peptide was able to penetrate into the membrane. The cholesterol molecules of the RBC’s membranes act as a barrier for the peptide insertion and restricted the peptide’s penetration much more than the DOPC/DOPG (3:1) system (Figure 4E).25 In both SDP3 (the Gram-negative model) and SDP4 (the Gram-positive model), the peptide binds to the membrane surface via hydrogen bonds (H-bonds) and electrostatic interactions between the basic amino acids of Pleurocidin and the phosphate head groups of the membranes.26 These interactions are altogether driving forces for the peptide adsorption from its hydrophilic side to the membrane surfaces. During the simulation of SDP3 and SDP4, the aromatic residues are unable to pass from the phosphate head groups, possibly due to the interconnected network of H-bonds forming by the PE head groups of the POPE components. For this purpose, the total number of H-bonds between the lipid G


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Figure 5. Order parameters of the lipid acyl chains in the pure bilayer and the peptide−membrane complex: (A), (C), (E) Sn1 acyl chain; (B), (D), (F) Sn2 acyl chain.

bilayers increases the lipid chains order.10−12,16 As depicted in Figure 5, in all the peptide−membrane systems the order of acyl chains was decreased by Pleurocidin. Although the peptide equally affected the sn-1 lipid chains in both SDP1 and SDP2, it changed the order of the sn-2 chains in SDP2 more than in SDP1. These results indicated that Pleurocidin can induce further disordering in the anionic phospholipids relative to the zwitterionic phospholipids, which is in line with experimental results.16 The SCD for both acyl chains of SD3 and SDP3 was more than that of SD4 and SDP4, which shows the lipid acyl chain is disordered by increasing the level of the anionic lipids. The lipid acyl chains in the RBC membrane had highest degree of order among the other membrane models, which is due to the high level of cholesterol in the RBC membrane. Deuterium NMR investigations have shown that the cholesterol molecules

changing the lipid components can affect the membrane fluidity and integrity.61,62 The previous computational studies have shown that AMPs such as Melittin and Pardaxin induced a disordering effect on the lipid acyl chains.50,55,63 To understand the mechanisms of action of AMPs, determining the dynamical changes of the membrane in the presence of AMPs is of great importance. Therefore, the deuterium order parameters (SCD) of the acyl chains were calculated in both pure lipid bilayers and the Pleurocidin−membrane complexes (Figure 5). Experimentally, it has been proved that Pleurocidin has disordering effect on the hydrophobic acyl chains.11 In addition, the peptide has a greater effect on the ordering of the anionic lipids relative to the zwitterionic lipids.16 It has also been shown that the presence of cholesterol in the lipid H


a

I

CHL

0.40 ± 0.008

SDP5 0.48 ± 0.02

SD5

POPC 0.50 ± 0.02

SDP5

0.62 ± 0.024

0.62 ± 0.02

Standard deviation was calculated in two replications.

0.38 ± 0.009

SD5

SDP4

SD4

POPE

SDP3 0.61 ± 0.014

SD3

0.59 ± 0.013

0.70 ± 0.015

0.68 ± 0.011 POPE

SDP2

SD2

DOPC

SD5

SD2

0.46 ± 0.017

POPE

RBC

0.49 ± 0.022

SDP5

POPE/POPG(1:3)

POPE/POPG(3:1)

0.43 ± 0.019

SD5

0.69 ± 0.011

SD4

0.67 ± 0.021

SD3

0.69 ± 0.018

SM16 0.49 ± 0.033

SDP5

POPG

POPG

DOPG

0.72 ± 0.003

0.68 ± 0.011 DOPC/DOPG(3:1)

SDP1

DOPC SD1

Table 4. Area per Lipids (APL) of Each Lipid in All MD Simulationsa

SDP2

0.46 ± 0.022

SD5

DOPS

0.69 ± 0.014

SDP4

0.69 ± 0.023

SDP3

0.71 ± 0.021

SDP5 0.51 ± 0.033

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Figure 6. Average membrane thickness during the last 100 ns. (A), (B), (C), (D), and (E) show the local membrane thinning in SDP1, SDP2, SDP3, SDP4, and SDP5, respectively. The average membrane thickness in the pure lipid bilayer and the Pleurocidin−membrane complex is shown in (F).

make the lipid acyl chain more ordered.64,65 Further, Urbina et al. showed that the addition of cholesterol to the phosphatidylcholine membrane increased the lipid chain order.59 Previous molecular dynamics studies have also shown that cholesterol makes the lipid acyl chains more ordered.66,67 The order of lipid acyl chains of the RBC membrane was affected by the peptide (Figure 5E,F); however, in comparison with the other cholesterol-free membranes, the hydrophobic acyl chains were less disordered. The results of the order analysis suggest that Pleurocidin behaves similarly to other AMPs such as Melittin that disturb membranes using the carpet mechanism.68,69 Therefore, by scrutiny of the obtaining results, it can be concluded that the Pleurocidin activity is dependent on both lipid head groups and hydrophobic acyl chain types in the target lipid bilayer.

The data for the effect of the Pleurocidin insertion on the area per lipid (APL) for each lipid type in the lipid bilayer models have been presented in Table 4. The results showed that APL is increased in the presence of the peptide for all of the investigated systems except POPE/POPG (1:3). However, the interactions of the peptide with different lipid bilayer models make the lipid tails more fluid in movement. The values obtained for APL are in good agreement with previous simulation and experimental studies.70−72 Previous NMR spectroscopy investigations and MD studies have shown that the presence of cholesterol in the membrane causes the membrane to become horizontally packed, resulting in lower APL.73,74 The values calculated for APL in the RBC membrane indicated that APL declines due to the presence of cholesterol in the membrane, which is in a good agreement with the pervious study.22 J


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Figure 7. Binding free energy between Pleurocidin and the lipid bilayer models. (A) represents the nonpolar binding energy, (B) represents the polar binding energy, and (C) represents the total binding free energy between Pleurocidin and the membrane models. Standard deviation was calculated in two replications.

3.2.2. Membranes Thickness. Membrane thickness is defined as the distance between the lipid head groups of the upper and lower leaflets.75 To compare the bilayers thickness, fluctuations of the membrane thickness were calculated during the whole trajectories in the pure lipid bilayers and the peptide−membrane complexes (Figure S10). In this way, to evaluate local membrane thinning, the average membrane thickness was also computed during the last 100 ns of MD simulation (note: to better compare membranes, the position of the peptide was fixed in the center of simulation box) (Figure 6). Jafari et al. illustrated that Pardaxin causes a decrease in membrane thickness.34 As shown in Figure 6F, the Pleurocidin insertion in the lipid bilayers caused the membrane thinning in all of the systems, except POPE/POPG (1:3) that there was no significant difference. The greatest decrease in the membrane thickness was related to SDP1, SDP2, and SDP5. In SDP2 and SDP5, the lipid acyl chains were disordered by the Pleurocidin insertion into the membranes (Figure 5A,B and Figure 5E,F) and, hence, the two leaflets of the membrane could get closer to each other. In SDP4, the lipid acyl chains were disordered less than the other membrane models (Figure 5C,D) and, consequently, the membrane thickness was not decreased tangibly in response to the peptide. In the pure lipid bilayer models, the order of membrane thickness was SD5 > SD3 > SD1 > SD2 > SD4 (Figure S10A). It can be interpreted that the existence of a higher amount of the anionic lipids in SD4 makes the membrane thinner than in SD3. The cholesterol molecules increase the ordering of lipid acyl chains, causing the RBC membrane thicker. The values of the

membrane thickness are in good agreement with previous experimental and MD simulation studies.76,77 Interactions of Pleurocidin with the lipid bilayers cause a reduction in the average thickness of membranes (Figure 6F and Figure S10B). The membrane thickness was changed in the range 3.3−4 nm in SDP1, SDP2, SDP3, and SDP4 and in SDP5 changed in the range 3.3−4.4 nm (Figure 6A−E). The membrane was thinned in the location of peptide penetration, which is specially observed in anionic and RBC membranes (Figure 6B,E). In Gram-positive membrane and to a lesser extent in Gramnegative membrane, Pleurocidin also caused the local membrane thinning (Figure 6D,C). In the zwitterionic lipid bilayer, local membrane thinning was not observed. 3.3. Binding Free Energy. As indicated in previous studies, Pleurocidin has a strong interaction with the anionic membranes and can penetrate into the membranes.8−11,16,17,21,54 On the contrary, the peptide has weak interactions with the zwitterionic membranes. Previous computational studies on different AMPs have indicated that the basic residues have strong electrostatic interactions with the anionic lipids. In addition, the aromatic amino acids were anchored within the membrane models by hydrophobic interactions.26,28,55,78−81 To get more information about the interaction of Pleurocidin with the membrane models, we calculated the total binding free energy between the peptide and the lipid bilayer models using the g_mmpbsa tool (Figure 7). As expected, the major contributor of the nonpolar energy was van der Waals (vdW) interactions. The vdW interactions play a key role in the Pleurocidin−membrane interactions, K


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Figure 8. Contribution energy of each amino acid in the Pleurocidin−membrane interactions. The contribution of those amino acids that have a low energy are shown for (A) SDP1, (B) SDP2, (C) SDP3, (D) SDP4, and (E) SDP5. The contribution energy of the residues with the largest energy is shown in (F). Standard deviation was calculated in two replications.

bilayers. The electrostatic energy plays a major role in the Pleurocidin−membrane interactions as well (Figure 7B). The highest contribution of electrostatic energy was related to the Gram-positive membrane because of the high number of anionic lipids in the membrane, and the lowest contribution of electrostatic energy was related to the zwitterionic membrane due to the lack of the anionic lipids in the lipid bilayer. The polar solvation energy in all systems had positive values (Figure 7B). Collectively, considering the total binding energy

particularly in SDP2 and SDP5 (Figure 7A). This is possibly due to the favorable hydrophobic interaction of the peptide with the membrane, causing all atoms of the peptide to have a chance to interact with the hydrophobic lipid tails by vdW forces. By contrast, in the SDP1, SDP3, and SDP4 systems, the contribution of vdW energy to the Pleurocidin−membrane interactions was low because in these models the peptide was located far away from the hydrophobic core of the lipid L


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interactions of the peptide with the membrane models. Pleurocidin, due to the presence of the anionic lipids and the PC head groups, has more proper interactions with the DOPC/DOPG (3:1) membrane than with the other bilayers. The MD results indicated that the peptide can induce further disordering in the anionic lipid bilayer relative to the zwitterionic phospholipids, which is in line with experimental results.16 The research also demonstrated that cholesterol molecules help the RBC membrane to conserve its integrity during interactions with Pleurocidin. Therefore, it can be concluded that very low hemolytic activity of Pleurocidin is due to the presence of cholesterol molecules in the eukaryotic membranes. It may be suggested that the Pleurocidin activity is dependent on both lipid head groups and hydrophobic acyl chain types in the target lipid bilayer. The binding free calculation showed that electrostatic interactions of the Lys amino acids play a major role in the interactions of the peptide with lipid bilayers, specially the anionic, Gram-negative, and Gram-positive membranes. The findings obtained here at the molecular level might shed light on a better understanding of the actual mechanism(s) by which the novel AMPs interact with the bacterial membranes. In addition, the present research can help to understand the role of different lipid constituent’s in the evolution of antibiotic-resistant bacteria.

as strength criterion for a complex, the SDP4 (Gram-positive membrane) system constitutes the strongest complex among the others and SDP1 (zwitterionic membrane) makes the weakest one (Figure 7C). In addition, Pleurocidin makes weak interactions with the RBC membrane because the outer leaflet of the RBC membrane does not have negatively charged lipids. The total binding free energy in both SDP2 and SDP3 were equal. In Figure 8, we have also illustrated the contributions of each residue to the total binding free energies for the peptide− membrane systems. To better clarify the results, the residues with higher values of binding energy have been shown in a separate graph (Figure 8F). As depicted in Figure 8, the residues Gly-1, Lys-7, Lys-8, Lys-14, Lys-18, and Lue-25 were hotspot residues for the peptide−membrane interactions. The Gly-1 (due to contain free N-terminal NH3+ group) and Lys residues (due to have an NH3+ group in their side chains) had favorable electrostatic interactions with the lipid bilayers except for DOPC (membrane component of SDP1), and their binding energies were highly negative. In SDP1, the Lys residues made electrostatic repulsions with the membrane and, hence, the binding energy values were positive. The Lue-25 residue is the C-terminal residue containing the free carboxyl (COO−) group, and consequently, it established electrostatic repulsions with the negative phospholipids within the membranes. The aromatic residues contributed largely to the membrane interactions due to hydrophobic forces. For example, the Trp-2 residue in SDP1 and SDP2 had a contribution energy of approximately −60 kJ/mol (Figure 8A,B). It can be interpreted that bulky hydrophobic residues such as Trp would have a more opportunity to interact with the membrane containing the phosphatidylcholine (PC) head groups. In the all of systems except SDP1, the value of contribution energy for the His residues was negative especially in SDP4 in which His-15 had the largest contribution energy among the other residues (Figure 8D). By comparing SDP2 and SDP4 (Figure 7A,B), it can be suggested that the higher values of the total binding free energy for SDP4 (Figure 7C) is due to the stronger electrostatic interactions of the Lys residues. However, the contributions of the other residues to the binding energy in SDP2 were higher than those in SDP4. It should be noted that the residues do not have the same roles in the Pleurocidin− membrane interactions. Some residues such as Lys and His may contribute largely to membrane adsorption via electrostatic interactions while certain residues such as Trp and Phe play a dual role; on one hand, they participate in the membrane adsorption, and on the other hand, they play an anchor role for maintaining the peptide in a certain location within the membranes.28,45 After SDP2, the peptide had the highest vdW energy in SDP5 (Figure 7A) and those residues that were passed from the phosphate head groups had a favorable contribution energy (Figure 8E). Overall, these findings indicate that the N-terminal region plays an important role in the Pleurocidin−membrane interactions and the antimicrobial activity of Pleurocidin.

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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.jcim.9b00245.

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Further information related to the distance between peptide and lipid bilayers as a function of time, the 2D and 3D structures of the cholesterol molecule and its distribution in the RBC membrane, the number of contacts between Lys residues and membrane models, the average of the hydrogen bonds between lipid head groups, the snapshots during trajectories for each peptide−membrane complex, and membrane thickness for the pure membrane and peptide−membrane systems as a function of time (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Faramarz Mehrnejad: 0000-0002-4444-6990 Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Hancock, R. E.; Lehrer, R. Cationic Peptides: A New Source of Antibiotics. Trends Biotechnol. 1998, 16, 82−88. (2) Guilhelmelli, F.; Vilela, N.; Albuquerque, P.; Derengowski Lda, S.; Silva-Pereira, I.; Kyaw, C. M. Antibiotic Development Challenges: The Various Mechanisms of Action of Antimicrobial Peptides and of Bacterial Resistance. Front. Microbiol. 2013, 4, 353. (3) Schwierz, N.; Krysiak, S.; Hugel, T.; Zacharias, M. Mechanism of Reversible Peptide-Bilayer Attachment: Combined Simulation and Experimental Single-Molecule Study. Langmuir 2016, 32, 810−821. (4) Prenner, E. J.; Lewis, R. N.; McElhaney, R. N. The Interaction of the Antimicrobial Peptide Gramicidin S with Lipid Bilayer Model and Biological Membranes. Biochim. Biophys. Acta, Biomembr. 1999, 1462, 201−221.

4. CONCLUSIONS In the present study, we investigated the molecular basis for membrane selectivity of Pleurocidin in the presence of different membrane models and we also explored the role of different phospholipids and cholesterol molecules in the membrane−Pleurocidin interactions. It was observed that the N-terminal region of Pleurocidin has a crucial role in the M


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Realistic Biological Membrane Simulations. J. Comput. Chem. 2014, 35, 1997−2004. (25) Xiang, N.; Lyu, Y.; Zhu, X.; Narsimhan, G. Investigation of the Interaction of Amyloid Beta Peptide (11−42) Oligomers with a 1Palmitoyl-2-Oleoyl-Sn-Glycero-3-Phosphocholine (Popc) Membrane Using Molecular Dynamics Simulation. Phys. Chem. Chem. Phys. 2018, 20, 6817−6829. (26) Jafari, M.; Mehrnejad, F.; Aghdami, R.; Chaparzadeh, N.; Razaghi Moghadam Kashani, Z.; Doustdar, F. Identification of the Crucial Residues in the Early Insertion of Pardaxin into Different Phospholipid Bilayers. J. Chem. Inf. Model. 2017, 57, 929−941. (27) Qian, Z.; Zou, Y.; Zhang, Q.; Chen, P.; Ma, B.; Wei, G.; Nussinov, R. Atomistic-Level Study of the Interactions between Hiapp Protofibrils and Membranes: Influence of Ph and Lipid Composition. Biochim. Biophys. Acta, Biomembr. 2018, 1860, 1818. (28) Jahangiri, S.; Jafari, M.; Arjomand, M.; Mehrnejad, F. Molecular Insights into the Interactions of Gf-17 with the Gram-Negative and Gram-Positive Bacterial Lipid Bilayers. J. Cell. Biochem. 2018, 119, 9205. (29) Van Der Spoel, D.; Lindahl, E.; Hess, B.; Groenhof, G.; Mark, A. E.; Berendsen, H. J. Gromacs: Fast, Flexible, and Free. J. Comput. Chem. 2005, 26, 1701−1718. (30) Best, R. B.; Zhu, X.; Shim, J.; Lopes, P. E.; Mittal, J.; Feig, M.; Mackerell, A. D., Jr. Optimization of the Additive Charmm All-Atom Protein Force Field Targeting Improved Sampling of the Backbone Phi, Psi and Side-Chain Chi(1) and Chi(2) Dihedral Angles. J. Chem. Theory Comput. 2012, 8, 3257−3273. (31) Klauda, J. B.; Venable, R. M.; Freites, J. A.; O’Connor, J. W.; Tobias, D. J.; Mondragon-Ramirez, C.; Vorobyov, I.; MacKerell, A. D., Jr.; Pastor, R. W. Update of the Charmm All-Atom Additive Force Field for Lipids: Validation on Six Lipid Types. J. Phys. Chem. B 2010, 114, 7830−7843. (32) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L. Comparison of Simple Potential Functions for Simulating Liquid Water. J. Chem. Phys. 1983, 79, 926−935. (33) Hoover, W. G. Canonical Dynamics: Equilibrium Phase-Space Distributions. Phys. Rev. A: At., Mol., Opt. Phys. 1985, 31, 1695−1697. (34) Nose, S. A Molecular-Dynamics Method for Simulations in the Canonical Ensemble. Mol. Phys. 1984, 52, 255−268. (35) Parrinello, M.; Rahman, A. Polymorphic Transitions in SingleCrystals - a New Molecular-Dynamics Method. J. Appl. Phys. 1981, 52, 7182−7190. (36) Hess, B. P-Lincs: A Parallel Linear Constraint Solver for Molecular Simulation. J. Chem. Theory Comput. 2008, 4, 116−122. (37) Darden, T.; York, D.; Pedersen, L. Particle Mesh Ewald - an N. Log(N) Method for Ewald Sums in Large Systems. J. Chem. Phys. 1993, 98, 10089−10092. (38) Allen, W. J.; Lemkul, J. A.; Bevan, D. R. Gridmat-Md: A GridBased Membrane Analysis Tool for Use with Molecular Dynamics. J. Comput. Chem. 2009, 30, 1952−1958. (39) Buchoux, S. Fatslim: A Fast and Robust Software to Analyze Md Simulations of Membranes. Bioinformatics 2017, 33, 133−134. (40) Kabsch, W.; Sander, C. Dictionary of Protein Secondary Structure: Pattern Recognition of Hydrogen-Bonded and Geometrical Features. Biopolymers 1983, 22, 2577−2637. (41) Yahyaei, M.; Mehrnejad, F.; Naderi-Manesh, H.; Rezayan, A. H. Protein Adsorption onto Polysaccharides: Comparison of Chitosan and Chitin Polymers. Carbohydr. Polym. 2018, 191, 191−197. (42) Jafari, M.; Mehrnejad, F.; Rahimi, F.; Asghari, S. M. The Molecular Basis of the Sodium Dodecyl Sulfate Effect on Human Ubiquitin Structure: A Molecular Dynamics Simulation Study. Sci. Rep. 2018, 8, 2150. (43) Kumari, R.; Kumar, R.; Open Source Drug Discovery, C.; Lynn, A. G_Mmpbsa–a Gromacs Tool for High-Throughput Mm-Pbsa Calculations. J. Chem. Inf. Model. 2014, 54, 1951−1962. (44) Baker, N. A.; Sept, D.; Joseph, S.; Holst, M. J.; McCammon, J. A. Electrostatics of Nanosystems: Application to Microtubules and the Ribosome. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 10037−10041.

(5) Brogden, K. A. Antimicrobial Peptides: Pore Formers or Metabolic Inhibitors in Bacteria? Nat. Rev. Microbiol. 2005, 3, 238− 250. (6) Hancock, R. E. Host Defence (Cationic) Peptides: What Is Their Future Clinical Potential? Drugs 1999, 57, 469−473. (7) Cole, A. M.; Weis, P.; Diamond, G. Isolation and Characterization of Pleurocidin, an Antimicrobial Peptide in the Skin Secretions of Winter Flounder. J. Biol. Chem. 1997, 272, 12008−12013. (8) Yoshida, K.; Mukai, Y.; Niidome, T.; Takashi, C.; Tokunaga, Y.; Hatakeyama, T.; Aoyagi, H. Interaction of Pleurocidin and Its Analogs with Phospholipid Membrane and Their Antibacterial Activity. J. Pept. Res. 2001, 57, 119. (9) Lan, Y.; Ye, Y.; Kozlowska, J.; Lam, J. K.; Drake, A. F.; Mason, A. J. Structural Contributions to the Intracellular Targeting Strategies of Antimicrobial Peptides. Biochim. Biophys. Acta, Biomembr. 2010, 1798, 1934−1943. (10) Mason, A. J.; Marquette, A.; Bechinger, B. Zwitterionic Phospholipids and Sterols Modulate Antimicrobial Peptide-Induced Membrane Destabilization. Biophys. J. 2007, 93, 4289−4299. (11) Mason, A. J.; Chotimah, I. N.; Bertani, P.; Bechinger, B. A Spectroscopic Study of the Membrane Interaction of the Antimicrobial Peptide Pleurocidin. Mol. Membr. Biol. 2006, 23, 185−194. (12) Syvitski, R. T.; Burton, I.; Mattatall, N. R.; Douglas, S. E.; Jakeman, D. L. Structural Characterization of the Antimicrobial Peptide Pleurocidin from Winter Flounder. Biochemistry 2005, 44, 7282−7293. (13) Pundir, P.; Catalli, A.; Leggiadro, C.; Douglas, S. E.; Kulka, M. Pleurocidin, a Novel Antimicrobial Peptide, Induces Human Mast Cell Activation through the Fprl1 Receptor. Mucosal Immunol. 2014, 7, 177−187. (14) Saint, N.; Cadiou, H.; Bessin, Y.; Molle, G. r. Antibacterial Peptide Pleurocidin Forms Ion Channels in Planar Lipid Bilayers. Biochim. Biophys. Acta, Biomembr. 2002, 1564, 359−364. (15) Lee, J.; Lee, D. G. Structure-Antimicrobial Activity Relationship between Pleurocidin and Its Enantiomer. Exp. Mol. Med. 2008, 40, 370−376. (16) Amos, S. T.; Vermeer, L. S.; Ferguson, P. M.; Kozlowska, J.; Davy, M.; Bui, T. T.; Drake, A. F.; Lorenz, C. D.; Mason, A. J. Antimicrobial Peptide Potency Is Facilitated by Greater Conformational Flexibility When Binding to Gram-Negative Bacterial Inner Membranes. Sci. Rep. 2016, 6, 37639. (17) Sung, W. S.; Lee, D. G. Pleurocidin-Derived Antifungal Peptides with Selective Membrane-Disruption Effect. Biochem. Biophys. Res. Commun. 2008, 369, 858−861. (18) Hotchkiss, R. S.; Moldawer, L. L.; Opal, S. M.; Reinhart, K.; Turnbull, I. R.; Vincent, J. L. Sepsis and Septic Shock. Nat. Rev. Dis Primers 2016, 2, 16045. (19) Hilchie, A. L.; Doucette, C. D.; Pinto, D. M.; Patrzykat, A.; Douglas, S.; Hoskin, D. W. Pleurocidin-Family Cationic Antimicrobial Peptides Are Cytolytic for Breast Carcinoma Cells and Prevent Growth of Tumor Xenografts. Breast Cancer Res. 2011, 13, R102. (20) Hilchie, A. L.; Haney, E. F.; Pinto, D. M.; Hancock, R. E.; Hoskin, D. W. Enhanced Killing of Breast Cancer Cells by a D-Amino Acid Analog of the Winter Flounder-Derived Pleurocidin Nrc-03. Exp. Mol. Pathol. 2015, 99, 426−434. (21) Zhang, M.; Wei, W.; Sun, Y.; Jiang, X.; Ying, X.; Tao, R.; Ni, L. Pleurocidin Congeners Demonstrate Activity against Streptococcus and Low Toxicity on Gingival Fibroblasts. Arch. Oral Biol. 2016, 70, 79−87. (22) Gorai, B.; Sivaraman, T. Delineating Residues for Haemolytic Activities of Snake Venom Cardiotoxin 1 from Naja Naja as Probed by Molecular Dynamics Simulations and in Vitro Validations. Int. J. Biol. Macromol. 2017, 95, 1022−1036. (23) Jo, S.; Kim, T.; Iyer, V. G.; Im, W. Charmm-Gui: A Web-Based Graphical User Interface for Charmm. J. Comput. Chem. 2008, 29, 1859−1865. (24) Wu, E. L.; Cheng, X.; Jo, S.; Rui, H.; Song, K. C.; DavilaContreras, E. M.; Qi, Y.; Lee, J.; Monje-Galvan, V.; Venable, R. M.; Klauda, J. B.; Im, W. Charmm-Gui Membrane Builder toward N


Article

Journal of Chemical Information and Modeling (45) Situ, A. J.; Kang, S. M.; Frey, B. B.; An, W.; Kim, C.; Ulmer, T. S. Membrane Anchoring of Alpha-Helical Proteins: Role of Tryptophan. J. Phys. Chem. B 2018, 122, 1185−1194. (46) Bi, X.; Wang, C.; Ma, L.; Sun, Y.; Shang, D. Investigation of the Role of Tryptophan Residues in Cationic Antimicrobial Peptides to Determine the Mechanism of Antimicrobial Action. J. Appl. Microbiol. 2013, 115, 663−672. (47) Chan, D. I.; Prenner, E. J.; Vogel, H. J. Tryptophan- and Arginine-Rich Antimicrobial Peptides: Structures and Mechanisms of Action. Biochim. Biophys. Acta, Biomembr. 2006, 1758, 1184−1202. (48) Datta, A.; Bhattacharyya, D.; Singh, S.; Ghosh, A.; Schmidtchen, A.; Malmsten, M.; Bhunia, A. Role of Aromatic Amino Acids in Lipopolysaccharide and Membrane Interactions of Antimicrobial Peptides for Use in Plant Disease Control. J. Biol. Chem. 2016, 291, 13301−13317. (49) Malanovic, N.; Lohner, K. Antimicrobial Peptides Targeting Gram-Positive Bacteria. Pharmaceuticals 2016, 9, 59. (50) Zhao, L.; Cao, Z.; Bian, Y.; Hu, G.; Wang, J.; Zhou, Y. Molecular Dynamics Simulations of Human Antimicrobial Peptide Ll37 in Model Popc and Popg Lipid Bilayers. Int. J. Mol. Sci. 2018, 19, 1186. (51) Jenssen, H.; Hamill, P.; Hancock, R. E. Peptide Antimicrobial Agents. Clin. Microbiol. Rev. 2006, 19, 491−511. (52) Gennaro, R.; Zanetti, M. Structural Features and Biological Activities of the Cathelicidin-Derived Antimicrobial Peptides. Biopolymers 2000, 55, 31−49. (53) Lim, S.; et al. Effects of Two Glycine Residues in Positions 13 and 17 of Pleurocidin on Structure and Bacterial Cell Selectivity. Protein Pept. Lett. 2004, 11, 35−40. (54) Souza, A. L.; Diaz-Dellavalle, P.; Cabrera, A.; Larranaga, P.; Dalla-Rizza, M.; De-Simone, S. G. Antimicrobial Activity of Pleurocidin Is Retained in Plc-2, a C-Terminal 12-Amino Acid Fragment. Peptides 2013, 45, 78−84. (55) Jafari, M.; Mehrnejad, F.; Doustdar, F. Insight into the Interactions, Residue Snorkeling, and Membrane Disordering Potency of a Single Antimicrobial Peptide into Different Lipid Bilayers. PLoS One 2017, 12, No. e0187216. (56) Boggs, J. M. Lipid Intermolecular Hydrogen Bonding: Influence on Structural Organization and Membrane Function. Biochim. Biophys. Acta, Rev. Biomembr. 1987, 906, 353−404. (57) Sato, H.; Feix, J. B. Peptide-Membrane Interactions and Mechanisms of Membrane Destruction by Amphipathic Alpha-Helical Antimicrobial Peptides. Biochim. Biophys. Acta, Biomembr. 2006, 1758, 1245−1256. (58) Galdiero, S.; Falanga, A.; Cantisani, M.; Vitiello, M.; Morelli, G.; Galdiero, M. Peptide-Lipid Interactions: Experiments and Applications. Int. J. Mol. Sci. 2013, 14, 18758−18789. (59) Urbina, J. A.; Pekerar, S.; Le, H.-b.; Patterson, J.; Montez, B.; Oldfield, E. Molecular Order and Dynamics of Phosphatidylcholine Bilayer Membranes in the Presence of Cholesterol, Ergosterol and Lanosterol: A Comparative Study Using 2h-, 13c- and 31p-Nmr Spectroscopy. Biochim. Biophys. Acta, Biomembr. 1995, 1238, 163− 176. (60) Lee, T. H.; Sani, M. A.; Overall, S.; Separovic, F.; Aguilar, M. I. Effect of Phosphatidylcholine Bilayer Thickness and Molecular Order on the Binding of the Antimicrobial Peptide Maculatin 1.1. Biochim. Biophys. Acta, Biomembr. 2018, 1860, 300−309. (61) Omardien, S.; Brul, S.; Zaat, S. A. Antimicrobial Activity of Cationic Antimicrobial Peptides against Gram-Positives: Current Progress Made in Understanding the Mode of Action and the Response of Bacteria. Front. Cell Dev. Biol. 2016, 4, 111. (62) Mishra, N. N.; Bayer, A. S. Correlation of Cell Membrane Lipid Profiles with Daptomycin Resistance in Methicillin-Resistant Staphylococcus Aureus. Antimicrob. Agents Chemother. 2013, 57, 1082− 1085. (63) Sengupta, D.; Leontiadou, H.; Mark, A. E.; Marrink, S.-J. Toroidal Pores Formed by Antimicrobial Peptides Show Significant Disorder. Biochim. Biophys. Acta, Biomembr. 2008, 1778, 2308−2317.

(64) Thewalt, J. L.; Bloom, M. Phosphatidylcholine: Cholesterol Phase Diagrams. Biophys. J. 1992, 63, 1176−1181. (65) Henriksen, J.; Rowat, A. C.; Brief, E.; Hsueh, Y. W.; Thewalt, J. L.; Zuckermann, M. J.; Ipsen, J. H. Universal Behavior of Membranes with Sterols. Biophys. J. 2006, 90, 1639−1649. (66) Ferreira, T. M.; Coreta-Gomes, F.; Ollila, O. H.; Moreno, M. J.; Vaz, W. L.; Topgaard, D. Cholesterol and Popc Segmental Order Parameters in Lipid Membranes: Solid State 1h-13c Nmr and Md Simulation Studies. Phys. Chem. Chem. Phys. 2013, 15, 1976−1989. (67) Vermeer, L. S.; de Groot, B. L.; Reat, V.; Milon, A.; Czaplicki, J. Acyl Chain Order Parameter Profiles in Phospholipid Bilayers: Computation from Molecular Dynamics Simulations and Comparison with 2h Nmr Experiments. Eur. Biophys. J. 2007, 36, 919−931. (68) Wimley, W. C. Describing the Mechanism of Antimicrobial Peptide Action with the Interfacial Activity Model. ACS Chem. Biol. 2010, 5, 905−917. (69) Hristova, K.; Dempsey, C. E.; White, S. H. Structure, Location, and Lipid Perturbations of Melittin at the Membrane Interface. Biophys. J. 2001, 80, 801−811. (70) Polyansky, A. A.; Volynsky, P. E.; Nolde, D. E.; Arseniev, A. S.; Efremov, R. G. Role of Lipid Charge in Organization of Water/Lipid Bilayer Interface: Insights Via Computer Simulations. J. Phys. Chem. B 2005, 109, 15052−15059. (71) Petrache, H. I.; Tristram-Nagle, S.; Gawrisch, K.; Harries, D.; Parsegian, V. A.; Nagle, J. F. Structure and Fluctuations of Charged Phosphatidylserine Bilayers in the Absence of Salt. Biophys. J. 2004, 86, 1574−1586. (72) Pan, J.; Heberle, F. A.; Tristram-Nagle, S.; Szymanski, M.; Koepfinger, M.; Katsaras, J.; Kucerka, N. Molecular Structures of Fluid Phase Phosphatidylglycerol Bilayers as Determined by Small Angle Neutron and X-Ray Scattering. Biochim. Biophys. Acta, Biomembr. 2012, 1818, 2135−2148. (73) Leftin, A.; Molugu, T. R.; Job, C.; Beyer, K.; Brown, M. F. Area Per Lipid and Cholesterol Interactions in Membranes from Separated Local-Field (13)C Nmr Spectroscopy. Biophys. J. 2014, 107, 2274− 2286. (74) Adams, M.; Wang, E.; Zhuang, X.; Klauda, J. B. Simulations of Simple Bovine and Homo Sapiens Outer Cortex Ocular Lens Membrane Models with a Majority Concentration of Cholesterol. Biochim. Biophys. Acta, Biomembr. 2018, 1860, 2134. (75) Engelman, D. M. Membranes Are More Mosaic Than Fluid. Nature 2005, 438, 578−580. (76) Venable, R. M.; Brown, F. L. H.; Pastor, R. W. Mechanical Properties of Lipid Bilayers from Molecular Dynamics Simulation. Chem. Phys. Lipids 2015, 192, 60−74. (77) Pluhackova, K.; Kirsch, S. A.; Han, J.; Sun, L.; Jiang, Z.; Unruh, T.; Bockmann, R. A. A Critical Comparison of Biomembrane Force Fields: Structure and Dynamics of Model Dmpc, Popc, and Pope Bilayers. J. Phys. Chem. B 2016, 120, 3888−3903. (78) Bortolus, M.; Dalzini, A.; Maniero, A. L.; Panighel, G.; Siano, A.; Toniolo, C.; De Zotti, M.; Formaggio, F. Insights into PeptideMembrane Interactions of Newly Synthesized, Nitroxide-Containing Analogs of the Peptaibiotic Trichogin Ga Iv Using Epr. Biopolymers 2017, 108, e22913. (79) Yin, L. M.; Edwards, M. A.; Li, J.; Yip, C. M.; Deber, C. M. Roles of Hydrophobicity and Charge Distribution of Cationic Antimicrobial Peptides in Peptide-Membrane Interactions. J. Biol. Chem. 2012, 287, 7738−7745. (80) Wang, G.; Hanke, M. L.; Mishra, B.; Lushnikova, T.; Heim, C. E.; Chittezham Thomas, V.; Bayles, K. W.; Kielian, T. Transformation of Human Cathelicidin Ll-37 into Selective, Stable, and Potent Antimicrobial Compounds. ACS Chem. Biol. 2014, 9, 1997−2002. (81) Mishra, B.; Golla, R. M.; Lau, K.; Lushnikova, T.; Wang, G. Anti-Staphylococcal Biofilm Effects of Human Cathelicidin Peptides. ACS Med. Chem. Lett. 2016, 7, 117−121.

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Molecular Basis for Membrane Selectivity of Antimicrobial Peptide

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