Melittin Creates Transient Pores in a Lipid Bilayer: Results from

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Melittin Creates Transient Pores in a Lipid Bilayer: Results from Computer Simulations Kolattukudy P. Santo, Sheeba Jem Irudayam, and Max L Berkowitz J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/jp312328n • Publication Date (Web): 27 Mar 2013 Downloaded from http://pubs.acs.org on April 15, 2013

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The Journal of Physical Chemistry

Melittin Creates Transient Pores in a Lipid Bilayer: Results from Computer Simulations.

Kolattukudy P. Santo, Sheeba J. Irudayam and Max L. Berkowitz* Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599

Abstract: To study the interaction between melittin peptides and lipid bilayer we performed coarsegrained simulations on systems containing zwitterionic dipalmitoylphosphatidylcholine (DPPC) and anionic palmitoyloleoylphosphatidylglycerol (POPG) phospholipids in a 7:3 ratio. Eight different systems were considered: four at low and four at high peptide to lipid (P/L) ratios. In case of low P/L ratio we did not observe any pore creation in the bilayer. In two out of four of the simulations with the high P/L ratio, appearance of transient pores in the bilayer was observed. These pores were created due to an assembly of 3-5 melittin peptides. Not all of the peptides in the pores were in a transmembrane conformation; many of them had their termini residues anchored to the same leaflet and these peptides assumed bent, U-shaped, conformations. We propose that when an assembly of melittin peptides creates pores, such an assembly acts as a “wedge” that splits the bilayer. To get a more detailed description of melittin on the bilayer surface and in transient pores we performed coarse-grained to united-atom scale transformations and after that performed 50 ns molecular dynamics simulations using the united atom description of the systems. While these simulations did not show much of the change in the pore structure during the 50 ns time interval, they clearly showed presence of water in the transient pores. Appearance of transient pores together with the translocation of peptides across the membranes is consistent with the mechanism proposed to explain graded dye leakage from large vesicles in the presence of melittin.

*e-mail: [email protected]; tel: 919-962-1218 Keywords: antimicrobial peptides, molecular dynamics, multiscale simulations, peptide/membrane interaction

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Introduction. Antimicrobial peptides (AMP) were discovered around thirty years ago, but we still do not understand the principles of their action1,2. AMPs are usually cationic, amphipathic peptides that have a broad spectrum of antimicrobial activity. The adsorption of cationic AMPs on anionic bacterial membrane surfaces is facilitated by the electrostatic interaction. While adsorbed on a membrane surface, a large group of AMPs, that have a coil structure in aqueous solutions, undergo transformation to a helical structure orienting themselves with their helical axis parallel to the surface. What happens next is not very clear, in spite that a large number of experiments using different techniques were performed to study the behaviour of AMPs interacting with membranes1,2. It was suggested that in some cases, when peptide concentration reaches a critical peptide to lipid ratio (P/L), peptides reorient to a transmembrane (TM) conformation and the TM peptides assemble to create pores3,4. The architecture of the pore walls can be different: If the walls of the pores are lined up by peptides only, the pores are called barrel-stave. If pore walls are lined up by peptides and lipid headgroups, creating a toroidal surface, the pores are called toroidal. It is believed that alamethicin, a 20-residue fungal peptide, creates barrel-stave pores, while melittin, a peptide from a honey bee venom, is engaged in creation of toroidal pores4. To explain action of some other AMPs such as cecropin P1, a “carpet model” was proposed5. In this model, AMPs cover the membrane surface as a carpet; when their concentration is large they tear apart the membrane causing cell destruction. Recently, another model explaining action of AMPs was suggested: the interfacial activity model1. This model explains the AMPs action as due to a strong perturbation of the interface between hydrophobic and hydrophilic regions of the lipid by adsorbed peptides and therefore as a disruption of membrane functionality. The study of AMP properties is often done using giant unilamellar vesicles (GUV) made up of a single type of phospholipid molecules or mixture of different types of phospholipid molecules, including some anionic lipids6. To detect the lytic activity of peptides, dyes are placed inside the GUVs cores and their


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leakage due to AMP action is monitored. It was observed that leakage process occurs in two different ways: continuous or graded and all-or none. Melittin peptide causes graded leakage, while magainin produces all-or-none. It was suggested that in graded case transient pores are created and peptides translocate across the membrane7. Creation of transient pores facilitates gradual leakage of the vesicle content. In the all–or-none case, peptides adsorbed on the membrane surface induce high tension that can open a large channel in the membrane8. In both graded and all-or-none mechanisms peptide action has a stochastic character, i.e. channel opening or membrane rupture is a random process. Computer simulations can often clarify the molecular picture behind the mechanism of biological phenomena. Simulations were also performed to study the action of AMPs9. Since the opening of a pore in the membrane by a peptide happens on a time scale that is long compared to time scales that routinely can be reached in detailed atomic simulations of systems containing AMPs and bilayers, often the pores in such simulations are prepared and their evolution is monitored10-12. To observe a spontaneous pore creation, computer simulations that use coarse-grained (CG) description of a system are performed. We also recently performed such a CG simulation of melittin interacting with DPPC bilayer using MARTINI force field and non-polar water13. In our case, for melittin to create pores, we had to place the peptides deep into the interfacial region between lipid headgroups and the hydrophobic core, so that melittins were able to overcome the barrier to enter the core region. Recent simulations that use MARTINI force field consider more sophisticated water model, the polarizable water model14, and it is observed that its application improves the agreement between CG and detailed atomic simulations when pores are formed in lipid bilayers15. In this work we report results from our CG and also united atom (UA) simulations performed on systems containing melittin peptides interacting with a bilayer consisting of zwitterionic dipalmitoylphosphatidylcholine (DPPC) and anionic palmitoyloleoylphosphatidylglycerol (POPG)


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phospholipids in a 7:3 ratio at low and high peptide to lipid (P/L) ratio. A mixture containing PC and PG type lipids is often used to model a bacterial membrane in both experiments and in simulations 16,17.. Our simulations show that melittin peptides adsorb on membrane surfaces and at a high peptide to lipid ratio some of the peptides penetrate the membrane creating transient pores in it. The stochastic character of the pore creation and its decay, architecture and structure of the pores are discussed below.

Methods Coarse-Grained Simulations: We performed 8 CG simulations of systems containing melittin peptides (26 residues: GIGAVLKVLTTGLPALISWIKRKRQQ) and a phospholipid bilayer consisting of a mixture of two types of phospholipids. In four of the simulations, the peptide to lipid ratio was relatively small (P/L=6/608~1/100) and in other four it was relatively large (P/L=28/608~1/21; see Table 1). The simulations were done starting with different initial distribution of peptides, and also with different initial velocity distributions. The initial peptides structure was obtained from their crystal structure 18,19 with the 88% helicity. During our simulations using the CG MARTINI force field20-22 this helicity value remained fixed and it was imposed by including a dihedral potential (with force constant 400 kJ /mol) between the backbone atoms and also by changing the polarity of the backbone beads22. Melittin dissolved in water assumes a coil conformation, when located on a membrane surface it is in a helical conformation. Therefore, by choosing the helicity of melittin to be 88% it is appropriate to place the peptides initially on a membrane surface. When adsorbed on surfaces of one component lipid bilayers melittin peptides have a lower helicity than 88%; indeed we observed in our previous united-atoms simulations of melittin adsorbed on the surface of a pure POPC bilayer that peptide helicity was ~73% 23. Experiments indicate that the presence of PG type lipids increases the peptide helicity24, thus justifiying


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the choice of 88% value. The crystal structure of melittin shows that it actually consists of two helices separated by a hinge at the PRO14 residue. In our simulations the peptide had a net charge of +6, arising from a protonated N-terminus (NH3+), 3 lysine (LYS) and 2 arginine (ARG) residues25 . As we already mentioned it, the bilayer in our simulations was represented by a mixture of zwitterionic (DPPC) and anionic (POPG) phospholipids taken in a 7:3 ratio, specifically, the bilayer contained a total of 608 lipids with 432 DPPC and 176 POPG molecules. This bilayer was constructed through self-assembly from a mixture of randomly placed DPPC and POPG molecules into standard CG water. Firstly a 152 lipid (DPPC-108, POPG-44) mixed bilayer was constructed and after 40 ns simulation run this bilayer was replicated twice in x and y directions to get a 608-lipid bilayer, which was then equilibrated in CG water for 60 ns. In all simulations melittin peptides were placed on one side of the bilayer, with their helical axes parallel to the membrane surface and with their centers of mass located at a distance around 3.0 nm from the bilayer center. The membrane surface was placed in the xy-plane and different initial structures (see Table 1) were set-up by varying peptide orientations within the xy plane. Such a placement was chosen in order for the peptides to get attached initially only on one side of the membrane (asymmetric attachment). The side of the bilayer the peptides were initially placed on was called side 1 or leaflet 1, the opposite side was side 2. Every system was solvated with the CG MARTINI polarizable water (PW)14 and to make the systems electroneutral a proper number of Na+ and Cl- ions were added. Initially Na+ ions were placed ~2 nm away from the membrane surface and Cl- ions were placed closer to the peptides. The systems were energy-minimized and then simulated in the NPT ensemble. CG simulations were performed using the Gromacs 4.5.5 software package with the MARTINI force field. Energy minimizations were done with the steepest-descent method. The non-bonded LJ interactions were cut off at 1.2 nm. For peptide-lipid systems, electrostatics was treated using Particle Mesh Ewald


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(PME) scheme26,27 in accordance with the protocols for MARTINI polarizable water. The use of PME is appropriate with the polarizable water, although it is non-standard with the non-polarizable CG water. Nevertheless, the use of PME was found to be important in pore formation studies and has been used even with non-polarizable water earlier 9. All production runs were simulated in the NPT ensemble using Berendsen coupling scheme with the temperature maintained at 323 K and pressure kept at 1. 0 bar with semi-isotropic coupling28. We chose the temperature of 323 K to make sure that the bilayer remains in the liquid crystal phase. The time constants for the pressure and temperature couplings were 3.0 ps and 0.3 ps respectively, and the compressibility value was 3×10-4 bar-1 . All simulation runs were performed utilizing periodic boundary conditions with a time step of 0.020 ps. Initial size of all systems was taken to be 14.2 nm, 14.2nm and 15.0 nm in x, y and z directions respectively. System configurations were visualized using VMD software29 . To compare the results of our simulations with the results of the simulations when no peptides were present in the system we also performed a CG simulation for the bilayer containing a 7:3 mixture of DPPC with POPG. This simulation was performed for a time period of 1 µs. Finally, we need to mention here that a time period of the run in the CG MARTINI simulations roughly corresponds to a period four times longer in all atoms or united atoms simulations. All the times we mention in this paper when describing CG simulation results are the CG run times, which should be multiplied by four, if comparison with a run time from an atomic simulation is required. CG particles to UA transformation procedure: Configurations of our systems obtained from the CG simulations, four from the simulations with a low P/L (~1/100) ratio and four from the simulations with a high P/L (~1/21) ratio were chosen and transformed to configurations with united atom (UA) description, following the procedure developed by Rzepiela et al30.


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The initial positions of the atomic particles were built using the g_fg2cg tool in the modified version of Gromacs 3.3.130. For melittin, 262 atoms were mapped from 55 CG beads. The resulting atomistic melittin had a net charge of +6 with a protonated N-terminus and an amidated C-terminus. For the POPG lipid, 53 atoms were mapped from the 13 CG beads and for the DPPC, 50 atoms were mapped from the 12 CG beads. Melittin, DPPC and POPG were described by GROMOS96 53a6 united atom force field31,32. Following the initial mapping, restrained simulated annealing was performed for 60 ps to reduce the temperature from 1300 K to 323 K using a restraining force constant of 12000 kJ/(mol nm2). The restraints were then smoothly released over 30 ps. The transformed atomistic structures were simulated for 50 ns with a time step of 2 fs using Gromacs 4.5.5. A similar procedure was used to transform a lipid bilayer without any peptides from a CG to an atomistic scale by starting with a configuration obtained from a CG snapshot made at 1000 ns. SPC water model was used and Na+ and Clcounterions were added by replacing in the transformed atomistic structure water molecules that were closest to the position of the ions in the CG snapshot. Berendsen thermostat and barostat were used to maintain the temperature at 323 K and the pressure was maintained at 1 bar with semi-isotropic pressure coupling. The temperature and pressure coupling constants were 1.0 ps. Periodic boundary conditions, PME for electrostatics and LINCS algorithm33 to constrain covalent bonds were also used. To avoid loss of information during the CG to AA transformation, for each CG snapshot the resolution transformation was repeated to get two atomistic structures, which were simulated using the parameters listed above. Gromacs analysis tools were used to calculate the densities; 2D distributions were visualised using MATLAB®(2010a) and the snapshots were plotted using VMD.

Results and Discussion. CG simulations with a low P/L ratio


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The four CG systems with the low P/L ratio were simulated up to a time of 5400 ns. None of them displayed pore formation in the bilayer or peptide translocation across the bilayer. Snapshots at different times from one of the runs, run 4, are shown in Figure 1. As indicated by these snapshots, peptides remained at the interface until the end of the run. This was expected, since a critical P/L ratio is generally required to observe some AMP action 3,4. Molecular dynamics simulations from Marrink’s group34 reported that melittin formed pores only when the value of P/L ratio was larger than 1/64. The experimental findings are somewhat controversial; based on some data melittin translocation across the membrane is estimated to occur even at a P/L ratio ~ 1/100 or smaller35, although some more recent experiments do not indicate that melittin translocates at these small P/L ratios16. In Figure 2a we present density profiles of water, membrane phosphates and peptide residues for run 4 (the densities were calculated for a time period of 100 ns at the 5000 ns point of the run) and compare these profiles with the ones from the run without peptides. Water and phosphate density curves nearly overlap with the corresponding curves obtained from the simulations of the bilayer without peptides. We observed that due to the incorporation of melittin the increase in the area per lipid from run 4 was less than 3 %, and the corresponding membrane thinning was practically absent (less than 1%). Figure 2a also shows the distribution of the hydrophobic and hydrophilic residues of the peptides. The hydrophobic residues (ALA, ILE, LEU, TRP and VAL) of the peptides were found located deeper in the bilayer, at distances nearly 1 nm from the bilayer center, while hydrophilic residues (GLN, LYS, ARG, THR and SER) were mostly located in the headgroup region. This separation of residues can be achieved by creating conformations where melittin peptide assumes a bent shape, anchoring residues located next to termini in the bilayer headgroup region. The presence of PRO14 residue in melittin facilitates creation of such bent conformations; we already observed them in our recent CG simulations of melittin in pure DPPC bilayers and called them “U-shaped”13. Bent conformations of melittin called “banana-shaped”


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were also observed previously in a study where combined pulse EPR and Monte Carlo simulation methods were used36. CG simulations with a high P/L ratio We also performed 4 CG simulations with high peptide to lipid ratio of P/L~1/21. To see the effect of initial configuration we performed three of the simulations (runs 5,6 and 7) starting from the same configuration, but with different initial velocities. Run 8 started from a different configuration. Density distributions from run 5, similar to the ones obtained from simulations at low P/L, are shown in Figure 2b (these distributions were calculated from the data obtained from the time interval of 100 ns at the end of the run). In case of high P/L the presence of peptides increased the area per lipid by nearly 12% and caused a membrane thinning by about 13%. The distribution of the peptide residues from Figure 2b shows that some of the peptides translocated across the bilayer. The change in the shape of the phosphate group density indicates a reorientation of this group needed to create a toroidal pore. The separation of hydrophobic and hydrophilic residues was found to be similar to the one observed in the low P/L case, thus also indicating the presence of U-shaped peptides. In all four simulations with the high P/L, all peptides quickly got strongly attached to one side (side 1) of the membrane. In two of the simulations some of the peptides were found to penetrate into the bilayer and even translocate to the other leaflet of the bilayer. In other two simulations peptides remained attached to one side of the membrane for around 5000 ns and no pore formation was observed. Although peptide concentration was high, all of them remained in the monomeric state without aggregating into large clusters while adsorbed on the membrane surface, in agreement with the experiments indicating that melittin remains mostly in the monomeric form when attached to the membrane surface6,16. (Figure S1 of the supporting information shows that melittin on a membrane surface remained mostly un-aggregated over a time span of 10000 ns). Furthermore, while being


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monomeric on the surface, melittin associates with the charged POPG lipids, causing them to aggregate around the peptides, in agreement with the results from recent simulation of Polyansky et al.37, who studied the AMP action on a PE/PG mixed bilayer. The observed behaviour of melittin, when located on a PC/PG membrane surface, can be directly linked to the high positive charge (+6) of the peptide. While the high charge on the peptide tends to prevent peptide aggregation into clusters or oligomers, it also causes the association of peptides with the negatively charged POPG headgroups, thus promoting this lipid aggregation around the peptide. In two of our simulations, designated as runs 5 and 6, peptides slowly penetrated into the bilayer interior. During run 5 one of the peptides started to penetrate the membrane at ~255 ns of the run (Figure S2) by first inserting its N-terminus into the bilayer and then assuming a trans-membrane (TM) orientation (see the snapshot at 260 ns in Figure S2). After that, assisted by the reorientation of the neighboring lipid headgroups, peptides started to aggregate in the bilayer interior (see snapshots at 1000 ns and 2000 ns from Figure S2). This process occurred on a relatively slow, microsecond time scale, until the aggregate contained 3 peptides. Notice that not all of the peptides in the aggregate were in a TM conformation; two of the three peptides from the aggregate in the snapshot at 2000 ns were in the U-shaped conformation with both of their termini attached to the same leaflet. The slowness in the peptide aggregation can be related to the slow diffusion of the peptides on the membrane surface, probably due to their association with the PG lipids. The same association resulted in a slow melittin reorientation from the surface state to a TM conformation. For example, in run 6 peptide penetration into the bilayer interior was observed at even longer times, after 1600 ns of the run. In that case melittin penetrated deeply into the membrane, although still remaining in the U-shaped conformation (Figure S3 of the supporting information). This was followed up by an aggregation with a second Ushaped peptide. The aggregates of melittin containing peptides with U-shaped conformation due to charge distribution on melittin and also peptide bending around the kink at PRO14 residue were also


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observed in our previous simulations13, where we suggested that the presence of U-shaped peptides may obstruct the water-flow in pores formed by melittin. Such U-shaped conformations were also found to aid partitioning of the antimicrobial peptide maculatin 1.1 into the membrane interior38,39. To understand if the presence of peptide aggregates is equivalent to the presence of pores filled with water, we monitored the amount of water molecules in the middle of the bilayer as a function of time. For this purpose we calculated the running average (taken over time intervals of 50 ns) of the number of CG water molecules in the slab of 1 nm width around the middle of the bilayer. This running average is depicted in Figure 3a. It shows that for run 5 pores may be present during the time intervals around 3560 ns and around 12500 ns. More detailed investigation of the run 5 results, by. inspecting the running average calculated over 10 ns time intervals for the number of waters in the middle slab, as shown in Figure 3b, and also by directly observing the snapshots from the run, showed that the pore in the bilayer opened up only during a relatively short time period around the 3560 ns time point of the run. The structure of this pore and its decay can be seen from the snapshots depicted in Figure 4. The snapshot at 3540 ns shows an aggregate formed by 3 peptides, but by 3560 ns the aggregate had changed into a pore through which water was flowing. As snapshot at 3560 ns shows, the phosphate groups were present in the pore, giving the pore a toroidal character. Further 30 ns of the time evolution saw the beginning of the peptide assembly disintegration along with the translocation of two peptides in the aggregate to the other leaflet of the membrane. By 3570 ns four peptides were present in the pore region; one of them detached from leaflet 1 and moved to the leaflet 2, starting the peptide assembly disintegration process. As can be seen from the Figure, at 3570 ns the pore increased in size, causing a large efflux of water. However, the snapshot at 3590 ns shows that as peptides fully translocated, the pore became less toroidal and tended to close, thus reducing the water flux. The snapshots at 3710 ns and finally at 4130 ns show that the translocated peptides moved away from each other and the assembly region. Also we observed that the pore fully disintegrated, and only one


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peptide remained in the TM orientation. The aggregate in run 6 became a pore at the time point ~3200 ns, that also disintegrated (Figure S4 of the supporting information) in a similar fashion. Thus, in both simulations the peptides translocation opened a transient toroidal pore causing the flow of water through the pore for a short time. The number of peptides in the pore region did not remain stable, and was changing from 3 to 4 in run 5 and from 3 to 5 in run 6. In Figure 5 we display density profiles similar to profiles from Figure 2, but densities were calculated as averages over a relatively short time period of 10 ns. These profiles clearly illustrate the presence of water in the pore and the transient character of such pores. The lifetime of the pores that appeared in our systems in both runs 5 and 6 was in the range of ~30-40 ns in CG simulations, equivalent to ~120-160 ns of real time. After we observed in simulations 5 and 6 the disintegration of the peptide aggregates that caused transient pore formation in the bilayer and translocation of the peptides, these simulations were continued for several more microseconds. Thus, in run 5 we observed that after some of the peptides translocated one peptide still remained in its TM orientation. The 5000 ns snapshot from Figure S5 shows one peptide in TM orientation, while two translocated peptides oriented parallel to the membrane surface in the other leaflet of the bilayer had moved away from the TM peptide. At this point, in some respect, the conformation of the system became similar to the one observed at 260 ns, however the imbalance in the number of peptides on the two leaflets became smaller. Again, as during the initial stage of the simulation, the presence of peptide in TM region probably caused subsequent aggregation and translocation of peptides, although due to the smaller peptide imbalance on membrane surfaces, longer time scales for aggregation may likely be involved. Indeed, for the run 5 the reformation of the aggregates in the membrane was not observed until the time point of about 6500 ns of the simulation (see Figure S5). By 8000 ns of the run, an aggregate containing 4 peptides was formed. Two of the additional peptides in this aggregate came from leaflet 1, with one more peptide from leaflet 2, where it appeared after initial translocation. This structure of the aggregate persisted for the


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remaining time of the simulation, until the time point of 16180 ns was reached (Figure S6a). In run 6 only one peptide translocated during the transient pore formation (compared to 2 peptides in run 5). The translocated peptide reunited with the aggregate after 6000 ns of the simulation time, forming again a 4-peptide aggregate, which remained intact until the end of the simulation at 10920 ns (Figure S6 b). The structures of these final aggregates are given in Figures S6c and S6d. As we can see, the aggregate in run 5 contained two peptides in TM orientation and two peptides in the U-shaped conformation, while in run 6 the aggregate had three U-shaped and one TM peptides. It is interesting that in both runs 5 and 6 these aggregates did not transform into pores. It was observed that AMPs induce lipid flip-flops in membranes40. We also found that in our simulation runs 5 and 6, melittin translocation caused rearrangement of lipids between the leaflets. Such lipid flipflops that occurred in run 5 are illustrated in Figure 6. In this figure all lipids initially present in the leaflet 1 are shown in green. We can see from the snapshot at 3540 ns that none of the lipids in leaflet 1 had flipped to the other leaflet at this point of the simulation. However, at 3590 ns lipids started to flip, as peptides started translocating. The snapshot at 10800 ns illustrates that a number of lipids from leaflet 1 have flipped to leaflet 2. Lipid flip-flops also occurred from leaflet 2 to leaflet 1 in run 5. Flipflops were observed in run 6 as well. In run 5, nine lipids flipped from leaflet 1 to leaflet 2; four of them were DPPC and five of them were POPG. Four lipids flipped from leaflet 2 to leaflet 1, all them were POPG. Thus a total of 13 lipids flipped between both leaflets, 9 of them were POPG, while 4 of them were DPPC molecules. In run 6 nine lipids (three of them DPPC and six of them POPG) flipped from leaflet 1 to leaflet 2, while eight lipids (all POPG) flipped from leaflet 2 to leaflet 1. Thus, the total number of lipids flipped between the leaflets in this case was 17; 14 of them being POPG and 3 of them being DPPC. As we can see, the lipids that flipped were mostly POPG (~77%). Since melittin tends to associate with the charged POPG lipids, it is the reason why a larger number of POPG lipids flip-flopped. POPG lipids being negatively charged and located in the neighborhood of the positively charged N-


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terminus follow the translocation of this terminus. While the total number of lipids that undergo a flipflop is ~3%, the total net balance of flipped lipids indicates that the flip-flop process does not produce a large compositional change in the membrane: for run 5 the net balance shows that only 4 DPPC and 1 POPG lipids moved from side 1 to side 2, while in run 6 only 3 DPPC lipids flipped from side 1 to side 2, while 2 POPG flipped from side 2 to side 1. UA simulations with a low P/L ratio: To get a more detailed picture about conformations of the peptides and penetration of water into the bilayer, we performed transformations from the CG description to the UA and consequently ran 50 ns molecular dynamics simulations of our systems in the united atom representation, starting from the configurations obtained after transformation. For the low P/L case (P/L ~ 1/100), conformations at 3500 ns from runs 1 and 3 and conformations at 5400 ns from runs 2 and 4 were transformed to the united atom description. These runs performed using the UA description were designated as runs 1a, 2a, 3a and 4a. The transformation was repeated and the corresponding runs were designated as 1b, 2b, 3b and 4b. Snapshots from the four runs, 1a - 4a at 0 and 50 ns are shown in Figure 7 along with the density distributions of the bilayer groups, water and the peptides. Density distributions indicate that peptides are positioned in the lipid headgroup-tail interface region. This is consistent with the previous observations on the peptide location in the bilayers41. A few peptides are observed to adopt a U-shaped conformation described in our earlier work13,23 and observed in our present CG simulations. The CG simulations had the peptide helicity fixed at 88%, but after the transformation to the united atom representation we observed that most of the peptides started to loose their helicity. We observe from Figure 7 that the density distributions for the bilayers with low P/L ratio deviate very slightly from the distributions of the bilayer without peptides. The main reason we performed the resolution transformations was to see what are the properties of water defects in the bilayer and also if water


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permeation across the bilayer takes place due to the presence of melittin peptides. Therefore, we calculated water densities and the time averaged water density plots from the simulations are shown in Figure 8. Interestingly, from this Figure we observe that water defects for the systems with the low P/L ratio (runs 1a, 2a, 3a and 4a) are similar to the water defects observed in the pure bilayer system, indicating that low peptide concentration does not cause significant perturbations of the lipid bilayer. The same conclusions can be reached based on the results from simulations 1b, 2b, 3b and 4b. However, for the simulation 4b, one of the peptides was observed to penetrate deeper into the bilayer. To see if this can produce larger defects that can grow and eventually result in a pore, we extended the simulation 4b to a longer time of 150 ns, but did not observe large changes happening to the system in the time interval 50 to 150 ns. UA simulations with a high P/L ratio: Four configurations from a run with high P/L ratio (~1/21) were transformed from the CG to the atomistic scale to study pores in more detail. Since a pore was observed for run 5, snapshots at 3540ns, 3570 ns, 3590 ns and 4130 ns were chosen from this run to transform to united atom representation. We designated our runs as runs 5a, 5b, 5c and 5d respectively. The first three snapshots describe the pore and the translocation of melittin. The final snapshot at 4130 ns depicts the state with a closed pore and two melittin peptides translocated, although one melittin still remained in a transmembrane orientation. During the 50 ns of atomistic simulations, no significant changes occurred in the studied systems. A few of the peptides were observed to insert a little deeper into the bilayer core and new water defects were formed, as the initial pore began to close. Figure 9 shows the snapshots at the beginning of the run (at 0 ns) and at the end (50 ns), the peptides in the pore are colored in a consistent way, so that the dynamics of a particular peptide could be followed. In the run 5a which started from a snapshot made at


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3540 ns of the CG simulation, the pore is lined up by the lipid headgroup atoms and three peptides, one of which is in a transmembrane orientation (magenta), two others (orange and ochre) are in a bent Ushaped conformation. Figure 9 does not represent a pre-arranged toroidal pore described for melittin in earlier works,10-12 but looks more like a disordered toroidal pore34. In the run 5b that started at 3570 ns of the CG, a fourth peptide shown in red also lines the pore. The structure of the four peptides in the pore is shown more clearly in Figure S7 where we observe that bent conformations do not remain helical; one or both helices of the peptide unwind. The peptide in the transmembrane orientation is observed to remain in this orientation and the peptides that translocate are those with bent conformation. Once the translocation is complete, the pore closes and new water defects begin to form in different regions. The creation of temporary pores is consistent with the picture of the graded dye release, proposed to explain melittin action on vesicles containing an embedded dye6. The density distributions of the peptides from Figure 9 clearly show the translocation of peptides when going from simulation 5a to 5d. The peptide in the transmembrane orientation has its N-terminus attached to the lower leaflet, suggesting that during the reorientation of melittin it is more likely that the N-terminus with a charge of +1 traverses the bilayer core rather than the C-terminus with a higher charge of +4. This supports the choice made in our earlier work on the reorientation of melittin to pull the N-terminus from the upper to the lower leaflet23. The top view of the bilayer displayed in Figure 9 shows that charged POPG lipids line-up at the edges of the pore where the concentration of the peptides is higher. We also observed that in all four systems both leaflets of the bilayer are perturbed in the presence of the peptides, although the peptides are added only to the upper leaflet. The observed decrease in the bilayer thickness when the P/L ratio is large resulted in the increase in area of the bilayer.


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The distribution of the positions of water molecules in Figure 8 gives a clear picture of the undulations in the bilayer, water defects and presence of water in the pore. In the run 5a, although it appears that there are two pores, the pore at x = 8 nm is an artefact of the visualization and represents water defects in the upper and lower leaflet that have different y positions. Consistent with the snapshots from Figure 4, the water density for run 5d displayed on Figure 8 does not show existence of a water-permeated pore. It should also be noted, that since we present average positions of the water molecules, the distribution in the pore region broadens from the pore diffusion and hence cannot be accurately correlated to the actual pore size.

Conclusions. We performed CG simulations to study the interaction of melittin peptide with a phospholipid membrane consisting of a 7:3 mixture of PC/PG molecules. Our CG simulations performed at a low P/L ratio ( ~1/100) showed that melittin slightly perturbs the PC/PG bilayer and does not create pores during a few microseconds of simulation time. Nevertheless the simulations show that peptides move into the interface between the headgroups and tail regions of membranes. In two of the four simulations performed at a high P/L ratio (~1/21), few melittin peptides aggregated around a peptide in a transmembrane conformation. These aggregates caused an opening of a transient toroidal pore; eventually some of the peptides were observed to translocate to the other side of the membrane. Our observations are consistent with the conclusions reached by Matsuzaki et al.35 who observed a graded efflux of calcein from egg PC/egg PG (9:1) large unilamellar vesicles (LUVs) and who also proposed that pore formation is coupled with translocation of the peptides. Our observations are also consistent with more recent conclusions made in the work by Gordon-Grossman et al. about peptide translocation at higher P/L16.


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The fact that we observed transient pores in only two of the 4 simulations with a high P/L ratio is perhaps not surprising. The initial penetration of melittin into the membrane interior is determined by many factors; the peptide helicity is probably an important one. Our CG simulations keep the helicity constant at 88%, but more detailed UA simulations show that the helicity of peptides is reduced. In our previous simulations of pre-arranged pore containing four melittin peptides12 we also observed that helicity is reduced. This indicates that it is important to perform CG simulations where peptides dihedral angles can be flexible. Another important factor that may play a role in the AMP activity is the membrane curvature. Thus Hallock et al. 42 have observed that melittin-like AMPs imprint a positive curvature on the membrane. It is also known that proteins containing domains with amphipatic motifs such as BAR domains sense membrane curvature.43,44 Due to the periodic boundary conditions the membrane in the simulations cannot assume large curvatures. In addition, one should also remember, that experiments indicate that even at high P/L ratio, melittin peptides do not cause 100% of vesicle rupture16. In conclusion, our simulations where we study the interaction of melittin with a bilayer containing a DPPC/POPG 7:3 mixture of lipids show that at a low P/L ratio melittin weakly perturbs a bilayer, although appearance of defects in a bilayer structure caused by peptide deeper penetration into the bilayer can be seen in the united atom simulations. In simulations with a high P/L ratio, we observed an assembly of peptides in the membrane hydrophobic region. Not all the peptides in the assembly were in the transmembrane conformation; many had a bent U-shape conformation with both termini anchored to the same bilayer leaflet. From our simulations we observed that some of the peptide aggregates became pores and some did not. The aggregates with peptides containing U-shaped melittins with their termini anchored to the same leaflet created pores. In this cases the aggregate acted as a “wedge” that effectively ruptured the membrane. UA simulations performed after transformation from the CG scale to a more detailed scale confirmed the presence of large amount of water in the pores. The number of


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peptides in the pores was not stationary, it was mostly fluctuating between 3 and 4, but even 5 peptides were present in a pore for short time duration in run 6. When some of the U-shaped peptides were anchored to leaflet 1 and other to leaflet 2, the assemblies did not become pores, probably loosing their “wedge” character and also blocking water flux. We observed this kind of peptide assemblies over relatively long time periods in runs 5 and 6 towards the end of the runs. The pores we observed had a transient character. The existence of transient pores and translocation of AMPs across the bilayer was proposed to explain the mechanism of graded dye efflux from vesicles. Our simulations confirm the existence of such a mechanism and they also provide a more detailed picture of the transient pore structure. Based on the observations from our simulations we propose a “wedge” mechanism model for pore creation that is a combination of both pore creation and peptide interfacial activity models.

Acknowledgement This work was supported by the National Science Foundation under grant MCB-0950280.

Supplementary Information Available A supplementary file displays the snapshots of the top view of the system in run 5, peptides insertion and aggregation in runs 5 and 6, transient pore formation in run6, post translocational behaviour of the system in run 5, final structure of the peptide aggregates at the end of the simulations in runs 5 and 6 and snapshots of the peptides in the pore in UA simulations. This material is available free of charge via the Internet at http://pubs.acs.org


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REFERENCES

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(18) Terwilliger, T. C.; Eisenberg, D. The Structure of Melittin .1. Structure Determination and Partial Refinement. J. Biol. Chem. 1982, 257, 6010-6015. (19) Terwilliger, T. C.; Eisenberg, D. The Structure of Melittin .2. Interpretation of the Structure. J. Biol. Chem. 1982, 257, 6016-6022. (20) Marrink, S. J.; de Vries, A. H.; Mark, A. E. Coarse Grained Model for Semiquantitative Lipid Simulations. J. Phys. Chem. B 2004, 108, 750-760. (21) Marrink, S. J.; Risselada, H. J.; Yefimov, S.; Tieleman, D. P.; de Vries, A. H. The Martini Force Field: Coarse Grained Model for Biomolecular Simulations. J. Phys. Chem. B 2007, 111, 7812-7824. (22) Monticelli, L.; Kandasamy, S. K.; Periole, X.; Larson, R. G.; Tieleman, D. P.; Marrink, S. J. The Martini Coarse-Grained Force Field: Extension to Proteins. J. Chem. Theory Comput. 2008, 4, 819834. (23) Irudayam, S. J.; Berkowitz, M. L. Binding and Reorientation of Melittin in a Popc Bilayer: Computer Simulations. Biochim. Biophys. Acta-Biomembr. 2012, 1818, 2975-2981. (24) Asthana, N.; Yadav, S. P.; Ghosh, J. K. Dissection of Antibacterial and Toxic Activity of Melittin - a Leucine Zipper Motif Plays a Crucial Role in Determining Its Hemolytic Activity but Not Antibacterial Activity. J. Biol. Chem. 2004, 279, 55042-55050. (25) Raghuraman, H.; Chattopadhyay, A. Melittin: A Membrane-Active Peptide with Diverse Functions. Biosci. Rep. 2007, 27, 189-223. (26) Essmann, U.; Perera, L.; Berkowitz, M. L.; Darden, T.; Lee, H.; Pedersen, L. G. A Smooth Particle Mesh Ewald Method. J. Chem. Phys. 1995, 103, 8577-8593. (27) 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. (28) Berendsen, H. J. C.; Postma, J. P. M.; Vangunsteren, W. F.; Dinola, A.; Haak, J. R. Molecular-Dynamics with Coupling to an External Bath. J. Chem. Phys. 1984, 81, 3684-3690. (29) Humphrey, W.; Dalke, A.; Schulten, K. Vmd: Visual Molecular Dynamics. J. Mol. Graphics Modell. 1996, 14, 33-&. (30) Rzepiela, A. J.; Schafer, L. V.; Goga, N.; Risselada, H. J.; De Vries, A. H.; Marrink, S. J. Reconstruction of Atomistic Details from Coarse-Grained Structures. J. Comput. Chem. 2010, 31, 13331343. (31) Kukol, A. Lipid Models for United-Atom Molecular Dynamics Simulations of Proteins. J. Chem. Theory Comput. 2009, 5, 615-626. (32) Oostenbrink, C.; Villa, A.; Mark, A. E.; Van Gunsteren, W. F. A Biomolecular Force Field Based on the Free Enthalpy of Hydration and Solvation: The Gromos Force-Field Parameter Sets 53a5 and 53a6. J. Comput. Chem. 2004, 25, 1656-1676. (33) Hess, B.; Bekker, H.; Berendsen, H. J. C.; Fraaije, J. G. E. M. Lincs: A Linear Constraint Solver for Molecular Simulations. J. Comput. Chem. 1997, 18, 1463-1472. (34) 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, 23082317. (35) Matsuzaki, K.; Yoneyama, S.; Miyajima, K. Pore Formation and Translocation of Melittin. Biophys. J. 1997, 73, 831-838. (36) Gordon-Grossman, M.; Gofman, Y.; Zimmermann, H.; Frydman, V.; Shai, Y.; Ben-Tal, N.; Goldfarb, D. A Combined Pulse Epr and Monte Carlo Simulation Study Provides Molecular Insight on Peptide-Membrane Interactions. J. Phys. Chem. B 2009, 113, 12687-12695. (37) Polyansky, A. A.; Ramaswamy, R.; Volynsky, P. E.; Sbalzarini, I. F.; Marrink, S. J.; Efremov, R. G. Antimicrobial Peptides Induce Growth of Phosphatidylglycerol Domains in a Model Bacterial Membrane. J. Phys. Chem. Lett. 2010, 1, 3108-3111.


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(38) Bond, P. J.; Parton, D. L.; Clark, J. F.; Sansom, M. S. P. Coarse-Grained Simulations of the Membrane-Active Antimicrobial Peptide Maculatin 1.1. Biophys. J. 2008, 95, 3802-3815. (39) Parton, D. L.; Akhmatskaya, E. V.; Sansom, M. S. P. Multiscale Simulations of the Antimicrobial Peptide Maculatin 1.1: Water Permeation through Disordered Aggregates. J. Phys. Chem. B 2012, 116, 8485-8493. (40) Fattal, E.; Nir, S.; Parente, R. A.; Szoka, F. C. Pore-Forming Peptides Induce Rapid Phospholipid Flip-Flop in Membranes. Biochemistry 1994, 33, 6721-6731. (41) 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. (42) Hallock, K. J.; Lee, D. K.; Ramamoorthy, A. Msi-78, an Analogue of the Magainin Antimicrobial Peptides, Disrupts Lipid Bilayer Structure Via Positive Curvature Strain. Biophys. J. 2003, 84, 3052-3060. (43) Bhatia, V. K.; Madsen, K. L.; Bolinger, P. Y.; Kunding, A.; Hedegard, P.; Gether, U.; Stamou, D. Amphipathic Motifs in Bar Domains Are Essential for Membrane Curvature Sensing. EMBO J. 2009, 28, 3303-3314. (44) Cui, H. S.; Lyman, E.; Voth, G. A. Mechanism of Membrane Curvature Sensing by Amphipathic Helix Containing Proteins. Biophys. J. 2011, 100, 1271-1279.


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Tables Table 1. Details of the coarse-grained simulations: melittins were initially placed on one side of the membrane consisting of 432 DPPC and 176 POPG lipids.


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Figure Captions Figure 1: Snapshots from simulations with low peptide density, P/L~1/100 (run 4 in Table 1). Initially peptides were placed on the membrane surface and as the simulations progressed they tended to partition into the bilayer. Color codes; lime: DPPC, grey: POPG, pink: PO4. Peptides are shown in different colors but the color scheme is the same in all snapshots. Figure 2. Partial density profiles of the components in simulations with (a) low peptide density, P/L ~1/100 (run 4 in Table 1) and with (b) high peptide density, P/L ~1/21 ( run 5 in Table 1) . Color codes: black –water; red-PO4; green- hydrophilic residues (ARG, LYS, GLN, THR and SER); blue- hydrophobic residues (ILE, LEU, ALA, TRP and VAL). Black and red dashed lines indicate respectively water and PO4 densities obtained from simulations of pure bilayer. Figure 3. (a) The running average of the number of water molecules NAve present in the middle of the bilayer in the simulations run 5 (red) and run 6 (blue) as a function of the simulation time. Only those water molecules are counted that are present in the region between two planes parallel to membrane surface. The planes are placed 0.5 nm above and below the xy plane in the middle of the bilayer. The running average is taken over 50 ns of the simulation time. (b) The same as in (a), only the time period considered corresponds to the time when the pore is present in the system and the running average NAve is taken over a time interval of 10 ns. Figure 4. Transient pore formation and translocation by melittins in run 5 (P/L ~1/21). In (a) peptides are shown in yellow color, except the two peptides that translocate, which are shown in purple and cyan respectively. In (b) peptides are shown in transparent red so that water flux can be visible. Other colors: green-polarizable water; pink spheres: PO4 groups Figure 5. Partial densities of water (black) and lipid phosphates (red) averaged over 10 ns time intervals centred at different time points (a) 3540 ns, (b) 3570 ns, (c) 3590 ns and (d) 3690 ns during the transient pore formation in the CG simulation run 5. While densities at 3540 ns (plot a) show presence of little water or phosphate groups inside the bilayer, plot (b) shows a considerable amount of water and PO4 in the middle of the bilayer, indicating the presence a toroidal pore. In (c) these densities are substantially diminished and in (d) the densities again look like in (a). Figure 6. Snapshots from run 5 showing lipid flip-flops during melittin insertion and translocation. Lipids in the leaflet 1 at the start of the simulation are shown in green, PO4 groups in pink, while peptides are not shown for clarity. The peptides in other snapshots are shown in red. Figure 7. Initial and final snapshots of the four transformed atomistic systems at a P/L ratio ~ 1/21. The phosphorous atoms of DPPC and POPG lipids are represented by green spheres, peptides by blue ribbons and water by red dots. Water is not shown for the snapshot at 0 ns for clarity. The density distributions of the peptides (blue), lipid bilayer (green), water (red) and lipid bilayer without peptides (black) are also given for each system.


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Figure 8. The average position of the water molecules in the xz plane for the UA systems. Top row: pure bilayer without any peptides; Middle row: four systems with low P/L (~1/100), (1a to 4a); Bottom row: four systems with high P/L (~1/21)(5a to 5d). The positions were averaged over the last 10 ns of the simulation. The color scheme (red to blue) represents the fraction of water molecules (high to low) at a given xz position. Figure 9. Initial and final snapshots of the four UA systems at P/L ratio ~ 1/21 along with a top view and density distributions. The colors in the snapshots and the density distributions are the same as described in Figure 7, with the exception that the four peptides involved in the pore are colored in red, orange, ochre and magenta. In the top view, the DPPC lipids are shown in blue and POPG lipids in red.


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run #

Na+/ClNo. of melittins

Simulation length (ns)

Remarks

176/36 176/36 176/36 176/36 176/168

No of polarizable water molecules 16444 16429 16430 16443 15640

1 2 3 4 5

6 6 6 6 28

5400 5400 5400 5400 16180

28

176/168

15640

10920

28 28

176/168 176/168

15640 15696

5480 4970

No pore No pore No pore No pore Transient pore, peptide translocation and aggregates Transient pore, peptide translocation and aggregates No pore No pore

6

7 8

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Table 1.


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Figure 1.


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Figure 2.


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Figure 3


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Figure 4.


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Figure 5


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Figure 6


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Figure 7.


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Figure 8.


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Figure 9.


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TOC Figure.


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Melittin Creates Transient Pores in a Lipid Bilayer: Results from

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