Cause and Effect of Melittin-Induced Pore Formation: A Computational

Sep 16, 2009 - The simulation results indicate the incipience of an ion-permeable water pore through collective membrane perturbation by bound peptide...
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Cause and Effect of Melittin-Induced Pore Formation: A Computational Approach Moutusi Manna and Chaitali Mukhopadhyay* Department of Chemistry, University of Calcutta, 92, A. P. C. Road, Kolkata-700 009, India Received February 4, 2009 Melittin embedded in a palmitoyl oleyl phosphatidylcholine bilayer at a high peptide/lipid ratio (1:30) was simulated in the presence of explicit water and ions. The simulation results indicate the incipience of an ion-permeable water pore through collective membrane perturbation by bound peptides. The positively charged residues of melittin not only act as “anchors” but also disrupt the membrane, leading to cell lysis. A detailed analysis of the lipid tail order parameter profile depicts localized membrane perturbation. The lipids in the vicinity of the aqueous cavity adopt a tilted conformation, which allows local bilayer thinning. The prepore thus formed can be considered as the melittin-induced structural defects in the bilayer membrane. Because of the strong cationic nature, the melittin-induced prepore exhibits selectivity toward anions over cations. As Cl- ions entered into the prepore, they are electrostatically entrapped by positively charged residues located at its wall. The confined motion of the Cl- ions in the membrane interior is obvious from calculated diffusion coefficients. Moreover, reorientation of the local lipids occurs in such a way that few lipid heads along with peptide helices can line the surface of the penetrating aqueous phase. The flipping of lipids argued in favor of melittininduced toroidal pore over a barrel-stave mechanism. Thus, our result provides atomistic level details of the mechanism of membrane disruption by antimicrobial peptide melittin.

Introduction

*To whom correspondence should be addressed. Tel: 91-33-2350-8386. Fax: 91-33-2351-9755. E-mail: [email protected] or cmchem@caluniv. ac.in.

surface binding of peptides (carpet mechanism) in a detergent-like manner.5 In the barrel-stave model, the peptides span a nearly flat bilayer and aggregate to line the pore surface,10-12 whereas, in the toroidal pore model, peptides perturb the local bilayer structure. The lipids in each leaflet sharply bend so that the lipid head groups along with peptide helices line the aqueous channel.8,12,13 On the other hand, in the carpet mechanism, the peptides form a carpetlike monolayer on the bilayer surface, driven predominantly by electrostatic interactions.8,14 Melittin is a naturally occurring AMP with pronounced cytolytic potency. It is the principal toxic component of the European honeybee venom, Apis mellifera.15 It is a highly basic, amphipathic hexacosa peptide (GIGAVLKVLTTGLPALISWIKRKRQQ-CONH2), with a large hydrophobic region (residues 1-20) and a stretch of predominantly hydrophilic amino acids (residues 21-26) at the carboxy terminal. Because of poor cell selectivity, it exhibits strong hemolytic activity against both bacterial and mammalian cells.6,13,16-19 At a moderately high concentration, melittin is known to cause micellization as well as membrane fusion,20,21 in addition to voltage-dependent ion channel formation across the planar lipid bilayer.22-24

(1) Chromek, M.; Slamova, Z.; Bergman, P.; Kovacs, L.; Podracka, L; Ehren, I.; H€okfelt, T.; Gudmundsson, G. H.; Gallo, R. L.; Agerberth, B.; Brauner, A. Nat. Med. 2006, 12, 636–641. (2) Hancock, R. E.; Scott, M. G. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 8856– 8861. (3) Li, M.; Lai, Y.; Villaruz, A. E.; Cha, D. J.; Sturdevant, D. E.; Otto, M. Proc. Natl. Acad. Sci. 2007, 104, 9469–9474. (4) Zasloff, M. Nature 2002, 415, 389–395. (5) Brogden, K. A. Nat. Rev. Microbiol. 2005, 3, 238–250. (6) Lee, M.-T.; Hung, W.-C.; Chen, F.-Y.; Huang, H. W. Proc. Natl. Acad. Sci. 2008, 105, 5087–5092. (7) Dubovskii, P. V.; Volynsky, P. E.; Polyansky, A. A.; Karpunin, D. V.; Chupin, V. V.; Efremov, R. G.; Arseniev, A. S. Biochemistry 2008, 47, 3525–3533. (8) Bond, P. J.; Parton, D. L.; Clark, J. F.; Sansom, M. S. P. Biophys. J. 2008, 95, 3802–3815. (9) Bringezu, F.; Wen, S.; Dante, S.; Hauss, T.; Majerowicz, M.; Waring, A. Biochemistry 2007, 46, 5678–5686. (10) Langham, A. A.; Ahmad, A. S.; Kaznessis, Y. N. J. Am. Chem. Soc. 2008, 130, 4338–4346. (11) Sanchez-Martı´ nez, S.; Huarte, N.; Maeso, R.; Madan, V.; Carrasco, L.; Nieva, J. L. Biochemistry 2008, 47, 10731–10739.

(12) Yang, L.; Harroun, T. A.; Weiss, T. M.; Ding, L.; Huang, H. W. Biophys. J. 2001, 81, 1475–1485. (13) Allende, D.; Simon, S. A.; McIntosh, T. J. Biophys. J. 2005, 88, 1828–1837. (14) Papo, N.; Shai, Y. Biochemistry 2003, 42, 458–466. (15) Habermann, E. Science 1972, 177, 314–332. (16) Asthana, N.; Yadav, S. P.; Ghosh, J. K. J. Biol. Chem. 2004, 279, 55042– 55050. (17) Sharon, M.; Oren, Z.; Shai, Y.; Anglister, J. Biochemistry 1999, 38, 15305– 15316. (18) Lee, M.-T.; Chen, F.-Y.; Huang, H. W. Biochemistry 2004, 43, 3590–3599. (19) Matsuzaki, K.; Yoneyama, S.; Miyajima, K. Biophys. J. 1997, 73, 831–838. (20) Toraya, S.; Nagao, T.; Norisada, K.; Tuzi, S.; Sait^o, H.; Izumi, S.; Naito, A. Biophys. J. 2005, 89, 3214–3222. (21) Naito, A.; Nagao, T.; Norisada, K.; Mizuno, T.; Tuzi, S.; Saito, H. Biophys. J. 2000, 78, 2405–2417. (22) Tosteson, M. T.; Tosteson, D. C. Biophys. J. 1981, 36, 109–116. (23) Stankowski, S.; Pawlak, M.; Kaisheva, E.; Robert, C. H.; Schwarz, G. Biochim. Biophys. Acta 1991, 1069, 77–86. (24) Becucci, L.; Guidelli, R. Langmuir 2007, 23, 5601–5608.

A large group of membrane-active peptides, such as antimicrobial peptides and toxins, are known to cause membrane damage, which galvanizes cell death. Such peptides are often used by nature in the defensive and offensive systems all across the plant and animal kingdoms. Antimicrobial peptides (AMPs), which are considered as “the native line of defense throughout nature”, show a high toxicity against both Gram-positive and Gramnegative bacteria as well as fungi, viruses, and mycobacteria, etc.1-4 Research in this field is of growing interest, as AMPs can act as potential alternatives to conventional antibiotics. AMPs are typically small (∼10-50 residues), cationic, amphipathic peptides, known to permeate microbial cell walls, thus inducing leakage of the cellular components across the bilayer.5-11 Among the different mechanisms proposed so far, the mode of action of AMPs can be explained either by the formation of transmembrane (TM) ion permeable pores (barrel-stave or toroidal pore model) or by the

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DOI: 10.1021/la902660q

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Melittin is mostly disordered as free monomer in solution; however, at high ionic strength, pH, or peptide concentration, monomers self-associate to form an R-helical tetrameric aggregate.25-27 The tetramer is too stable, with a buried hydrophobic and exposed hydrophilic face, to be inserted into the bilayer.28 In a lipidic environment, melittin adopts a bent R-helical conformation (small hinge at Pro14) with segregated hydrophobic and hydrophilic faces.29,30 The association of melittin with phospholipid bilayer initiates the lytic mechanism. Melittin is thought to lyse the membrane by disrupting the bilayer barrier property.31 Several studies reveal that the interaction is sensitive to peptide concentration,6 lipid composition,13,32-34 ionic strength,35 hydration level,36 and membrane potential.24 The orientation of melittin helix inside the lipid bilayer also plays a crucial role in melittin-induced cell lysis.6 Depending upon the “physicochemical” conditions, melittin can either lie laterally across the membrane (“Wedge” model) or insert itself parallel to lipid normal.12 The two binding states can be interconverted under equilibrium conditions, which is directly related to lytic activity. The previous experimental studies proposed that at a peptide concentration higher than a certain threshold concentration, melittin changes from a surface-associated state to an inserted state.18 Again, with protonation of the N-terminus, melittin adopts a transbilayer orientation.37 However, a recent article deduced a more complicated oriental distribution of melittin.38 According to them, about three-fourths of melittin molecules orient parallel to the bilayer surface with a slight tilt, while the rest orient almost parallel to surface normal. Although there is no consensus regarding its orientation, it is well-established that melittin can form a TM pore only in an inserted state, and in parallel orientations, peptides cause bilayer thinning in proportion to peptide concentration.6 Although a large number of studies have been undertaken to determine the architecture of the pore induced by AMP, the poreforming mechanism of melittin still remains controversial. Some studies suggest the formation of a barrel-stave or toroidal-shaped pore, while others envisage a carpet mechanism.12-14,39,40 Despite the ambiguity, the melittin pore is thought to be due to the defects in the bilayer, produced by the collective perturbation of a membrane structure by bound peptides.6,13 To gain better insight into the relevant lytic mechanism, we need atomistic level resolution of specific lipid-peptide interactions, details of perturbations produced in the membrane bilayer, and also

environmental responses of surrounding ions and the aqueous medium. The objective of the present work is to investigate the molecular details of the pore-forming mechanism by venom toxin melittin, which is still in chaos. Baumgaertner et al.39 earlier investigated the stability of a hypothetical melittin pore consisting of a melittin tetramer in a membrane bilayer. They found that as the pore expanded, the initial tetrameric configuration decayed into a stable trimer and one monomer. Now, instead of immersing a pore structure, here, we have added four melittin monomers in a hydrated palmitoyl oleyl phosphatidylcholine (POPC) bilayer. In our system, the peptide to lipid (P/L) ratio, 1:30, is well above the critical peptide to lipid ratio of 1:62.18 Melittins are inserted in a transbilayer orientation with their hydrophilic C-terminal protruding out of the membrane. As revealed by previous experiments, the conditions that we chose are necessary for the pore formation by cytolytic peptide melittin. Within the simulation time scale, we observe the formation of a prepore in the host bilayer accomplished by a severe membrane perturbation. The evidence that we have from the simulation shows preference for the toroidal pore model over the barrel-stave mechanism. We have also noticed the movement of the chloride ion inside the prepore, which is complementary to the experimental finding of anion selectivity of the melittin channel.22 To the best of our knowledge, this is probably the first computational approach that reports such spontaneous initiation of ion-permeable toroidalshaped pore by hemolytic melittin, at a high peptide concentration. Few additional simulations were run to study the influence of the initial peptide position and the effect of the peptide charge state in cell lysis (Supporting Information). When melittins are more deeply inserted in the membrane, so their N-terminals reach nearly the lower interfacial region, we again observe the formation of a water defect in the inner bilayer leaflet. The highly basic peptide causes serious bilayer deformation, opening up a region through which water and ions can penetrate the bilayer. However, when they were placed further away from each other, each isolated melittin individually exhibited cell lysis. Again, if Lys-7, which is exposed in the hydrophobic interior is made uncharged, we observe a drastic drop in water penetration, which indicates the importance of this specific amino acid residue or, broadly, the peptide charge state in melittin-induced lysis. These results point toward the fact that the presence of highly basic peptides perturbs the host bilayer, which ultimately leads to bilayer disruption.

(25) Terwilliger, T. C.; Eisenberg, D. J. Biol. Chem. 1982, 257, 6016–6022. (26) Hartings, M. R.; Gray, H. B.; Winkler, J. R. J. Phys. Chem. B 2008, 112, 3202–3207. (27) Qiu, W.; Zhang, L.; Kao, Y.-T.; Lu, W.; Li, T.; Kim, J.; Sollenberger, G. M.; Wang, L.; Zhong, D. J. Phys. Chem. B 2005, 109, 16901–16910. (28) Hua, L.; Huang, X.; Liu, P.; Zhou, R.; Berne, B. J. J. Phys. Chem. B 2007, 111, 9069–9077. (29) Chatterjee, C.; Mukhopadhyay, C. Biochem. Biophys. Res. Commun. 2002, 292, 579–585. (30) Smith, R.; Separovic, F.; Milne, T. J.; Whittaker, A.; Bennett, F. M.; Cornell, B. A.; Makriyannis, A. J. Mol. Biol. 1994, 241, 456–466. (31) Epand, R. M.; Vogel, H. J. Biochim. Biophys. Acta 1999, 1462, 11–28. (32) Raghuraman, H.; Chattopadhyay, A. Eur. Biophys. J. 2004, 33, 611–622. (33) Alakoskela, J.-M.; Sabatini, K.; Jiang, X.; Laitala, V.; Covey, D. F.; Kinnunen, P. K. J. Langmuir 2008, 24, 830–836. (34) Wessman, P.; Str€omstedt, A. A.; Malmsten, M.; Edwards, K. Biophys. J. 2008, 95, 4324–4336. (35) Raghuraman, H.; Ganguly, S.; Chattopadhyay, A. Biophys. Chem. 2006, 124, 115–124. (36) Raghuraman, H.; Chattopadhyay, A. Langmuir 2003, 19, 10332–10341. (37) Bradshaw, J. P.; Dempsey, C. E.; Watts, A. Mol. Membr. Biol. 1994, 11, 79–86. (38) Chen, X.; Wang, J.; Boughton, A. P.; Kristalyn, C. B.; Chen, Z. J. Am. Chem. Soc. 2007, 129, 1420–1427. (39) Lin, J.-H.; Baumgaertner, A. Biophys. J. 2000, 78, 1714–1724. (40) Ladokhin, A. S.; White, S. H. Biochim. Biophys. Acta, Biomembr. 2001, 1514, 253–260.

Simulation Details

12236 DOI: 10.1021/la902660q

All molecular dynamics simulations were performed with the program CHARMM (Chemistry at Harvard Macromolecular Mechanics)41 using the CHARMM27 parameter set,42 including dihedral cross-term corrections (CMAP)43 for peptides and modified TIP3 water models.44 The long-range electrostatic interactions were treated via a Particle Mesh Ewald (PME) method using a Gaussian distribution width of k = 0.34 A˚-1, a real space cutoff of 12 A˚, and fft grid points of 1 A˚ in all directions, and a fifth order β-spline was used for the interpolation. The Lennard-Jones (LJ) potential was smoothly switched off between 10 and 12 A˚. The temperature and pressure of the system were kept constant at 300 K and 1 atm, respectively. The (41) Brooks, B. R.; Bruccoleri, R. E.; Olafson, B. D.; States, D. J.; Swaminathan, S.; Karplus, M. J. Comput. Chem. 1983, 4, 187–217. (42) MacKerell, A. D. J. Phys. Chem. B 1998, 102, 3586–3616. (43) MacKerell, A. D.; Feig, M.; Brooks, C. L. J. Comput. Chem. 2004, 25, 1400– 1415. (44) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L. J. Chem. Phys. 1983, 79, 926–935.

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temperature was controlled by a Hoover thermostat with a coupling constant of 20000 kcal mol-1 ps-2 and pressure with a pressure piston mass of 2000 amu. Periodic boundary conditions (PBC) were applied in all three directions using a tetragonal lattice. The leapfrog Verlet integrator with a time step of 1 fs was used to solve Newton’s equations of motion. Nonbonded and image lists were updated every 20 integration steps. The overall rotational and translational motions of the system were removed every 500 integration steps. All covalent bonds involving hydrogen were constrained with the SHAKE algorithm. Pure POPC Bilayer. The model bilayer containing 128 POPC lipids (64 lipids in each leaflet) was constructed following the method as reported in refs 45 and 46. The starting configuration for a phospholipid system was prepared from random selection of lipids from a pre-equilibrated, prehydrated set and then placing them in a bilayer. The short contacts between heavy atoms were reduced through systematic rotations (around the z-axis) and translations (in the xy plane) of the lipids. The bilayer was then solvated, and the overlapping waters were deleted within 2.6 A˚ from the lipid molecules. The simulation cell dimension was initially set to allow 64 A˚2 area per lipid. Short steepest descent (SD) minimizations, followed by long adapted basis NewtonRaphson (ABNR) minimization, were performed to remove bad atom contacts. The pure bilayer was then simulated with NPT ensemble, as it is the most natural choice of an ensemble in membrane simulation. As the POPC lipid has a low gel-to-fluid transition temperature (Tm ≈ -5 C or 268 K),47,48 simulations were performed at 300 K to maintain bilayer fluidity. After 10 ns of NPT simulation, the bilayer shrinks considerably along the X-Y plane, resulting in an area per lipid of ∼56 A˚2, which is significantly smaller than the experimentally reported value of 68.3 A˚2 (for an area per lipid vs time plot, see the Supporting Information, Figure S1).49 As a consequence, the lipid tails became highly ordered (Figure S2 of the Supporting Information), indicating a liquid-to-gel phase transition of lipid bilayer. A similar trend of the decreasing area per lipid, using CHARMM NPT simulation protocol, had been reported previously in the literature.50 To alleviate this problem of CHARMM bilayer simulation, NPAT (constant number of atoms, pressure, cross-sectional area, and temperature) or NγPT (constant number of atoms, pressure, surface tension, and temperature) ensembles are typically recommended, although NγPT is preferred for simulating membrane embedded proteins/peptides.51 In the present simulation, to expand the area per lipid to its desired value, we then switched to the NγPT ensemble,45,52 with γ=50 dyn/cm. After the desired area per lipid was reached, the final coordinate set was then taken to initiate the additional NγPT simulation, at γ = 20 dyn/cm. The choice of the optimal γ value was based on recent simulation studies of the liquid DPPC membrane, where the surface tensions of 20 and 24.5 dyn/cm gave the best agreement with the known area per lipid.52,53 After a 10 ns production run, the simulation box dimensions became 65.0265.0181 A˚3, (45) Skibinsky, A.; Venable, R. M.; Pastor, R. W. Biophys. J. 2005, 89, 4111– 4121. (46) Mondal, S.; Mukhopadhyay, C. Langmuir 2008, 24, 10298–10305. (47) Litman, B. J.; Lewis, E. N.; Levin, I. W. Biochemistry 1991, 30, 313–319. (48) Leekumjorn, S.; Sum, A. K. J. Phys. Chem. B 2007, 111, 6026–6033. (49) Kucerka, N.; Tristram-Nagle, S.; Nagle, J. F. J. Membr. Biol. 2005, 208, 193–202. (50) Jensen, M. Ø.; Mouritsen, O. G.; Peters, G. H. Biophys. J. 2004, 86, 3556– 3575. (51) Feller, S. E.; Zhang, Y.; Pastor, R. W. J. Chem. Phys. 1995, 103, 10267– 10276. (52) Dolan, E. A.; Venable, R. M.; Pastor, R. W.; Brooks, B. R. Biophys. J. 2002, 82, 2317–2325. (53) Cournia, Z.; Ullmann, G. M.; Smith, J. C. J. Phys. Chem. B 2007, 111, 1786–1801.

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with an area per lipid (Figure S3 of the Supporting Information) of ÆAæ = 66 ( 0.5 A˚2. The result agrees well with the area per POPC, 63.5 A˚2 at 310 K and 66.5 A˚2 at 303 K, obtained from recent simulation studies (using AMBER and GROMACS, respectively).54,55 Combined System. The melittin atomic coordinate was retrieved from the Protein Data Bank (PDB code: 2mlt). Four melittin monomers (each adjacent pair is separated by two lipid molecules) were inserted vertically into the pre-equilibrated (final coordinate of 10 ns NγPT run) bilayer (Figure 1a), following the procedure as described in refs 56 and 57. In the present simulation, melittin spans only ∼2/3 of the complete bilayer width; thus, the N-terminal remains free into the low-density tail region of the lower layer. For each melittin, two lipid molecules were removed only from the upper leaflet, resulting in a P/L ratio of 1:30. All overlapping water molecules within 2.6 A˚ of the peptide were removed. On the basis of the theoretical study56 and experimental observation,58 Trp19 was placed near the carbonyl group, which is considered to be its highly energetically favorable interaction site. As a result, the charged C-terminal moiety was nicely positioned into the lipid/water interface, inserting the predominately hydrophobic N-terminal segment into the bilayer hydrophobic core. Two charged residues of the TM segment, that is, Lys7 and the protonated N-terminus of all the four melittins, were so oriented that they face each other and also to the center of bilayer. To maintain electrical neutrality of the system, 4  6 Clcounterions were added and additional sodium chloride was added to produce the physical salt concentration of 150 mM. Then, a series of minimizations were performed to relax the system.52 First, the system was subjected to SD algorithm with fixed constraints on the peptide followed by ABNR minimization with harmonic constraints applied on the peptide backbone. Finally, peptides were released, and the fully unconstrained system was then subjected to energy minimization (SD), followed by a production run, 15 ns long, using the NγPT ensemble, with γ = 20 dyn/cm.59 A few control simulations were performed to study the effects of the initial peptide position and the peptide charge state on cell lysis (see the Supporting Information), using the same simulation protocol. A list of total simulations performed is given in the Supporting Information (Table S1.)

Results and Discussion Binding of melittin causes sizable disruption of the host POPC bilayer, leading to the initiation of an ion-permeable water pore. In this section, we focus on peptide conformation, details of peptide-membrane interaction, differential effects of the peptide on the membrane structure and dynamics, membrane permeability for water and ions, and the architecture of the prepore formed. Peptide Conformation. Peptide conformational studies are necessary to gauge the influence of the lipid matrix on the structure and dynamics of the bound peptide. In the present simulation, the conformation of melittin remained stable over the entire simulation time scale. Thus, in a bilayer, melittin retains its R-helical structure (Figure S4 of the Supporting Information), with a time average bend angle of ÆΩæ ≈ 145 ( 10 (Figure S5 of (54) Rog, T.; Murzyn, K.; Pasenkiewicz-Gierula, M. Acta Biochim. Pol. 2003, 50, 789–798. (55) Pandit, S. A.; Chiu, S.-W.; Jakobsson, E.; Grama, A.; Scott, H. L. Biophys. J. 2007, 92, 920–927. (56) Bachar, M.; Becker, O. M. Biophys. J. 2000, 78, 1359–1375. (57) Kandt, C.; Ash, W. L.; Tieleman, D. P. Methods 2007, 41, 475–488. (58) Vogel, H.; J€ahnig, F. Biophys. J. 1986, 50, 573–582. (59) Gullingsrud, J.; Babakhani, A.; McCammon, J. A. Mol. Simul. 2006, 32, 831–838.

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Figure 1. (a) Initial and (b) final snapshot of the system, with melittin in magenta, POPC in silver, and water in green. The headgroup phosphate atoms and chloride ions are highlighted as silver and red van der Waals spheres, respectively. The image rendering was done with VMD.87 The figure represents considerable water penetration followed by reorientation of lipids, especially from the lower leaflet. Few chloride ions are shown to enter into the membrane core, along with water.

the Supporting Information). A very similar bend angle of ÆΩæ ≈ 134 ( 20 was reported by Baumgaertner et al. for a melittin pore immersed in the POPC bilayer.39 On the basis of a solid-state NMR study, Naito et al.21 proposed that the kink angle between the N- and the C-terminal helical rods of melittin in the lipid bilayer is ∼140 or ∼160. The detail of the conformational drift was obtained by computing the root-mean-square deviation (rmsd) for CR atoms of the peptide backbone relative to the starting point (Figure S6 of the Supporting Information).60,61 For each peptide, the rmsd rises initially and closely approaches convergence after about 8 ns. The rmsd values range from 2.7 to 3.5 A˚ (last 7 ns), indicating a small structural fluctuation relative to the starting point, that is, stability of the helical structure in the bilayer environment. Solvation of Peptide: Peptide-Lipid and Peptide-Water Interactions. Lipid-protein interactions are crucial for many cellular processes, including membrane trafficking, transport, and signal transductions.62 Here, we have calculated the interaction energies of melittin with surrounding lipid and water molecules and also the partition of the total interaction energy into electrostatic and van der Waals (vdW) terms (Figure 2), as in refs 63 and 64. In the distribution curve for the peptide-lipid interaction (Figure 2a), a peak for the electrostatic contribution per melittin is located at ∼-190 kcal/mol, whereas that for the vdW contribution is located at ∼-160 kcal/mol. The result is in close proximity with results obtained for the interaction of single PG1 peptide with a mixed POPC/POPG bilayer.63 The source of the columbic interaction is hydrogen bonding between hydrophilic amino acid residues of melittin with negatively charged lipid head groups, whereas the vdW contribution is the outcome of a nonbonded (60) Psachoulia, E.; Sansom, M. S. P. Biochemistry 2008, 47, 4211–4220.  J. Phys. Chem. B 2008, (61) Jardon-Valadez, E,; Ulloa-Aguirre, A.; Pi~neiro, A. 112, 10704–10713. (62) Cho, W.; Stahelin, R. V. Annu. Rev. Biophys. Biomol. Struct. 2005, 34, 119– 151. (63) Jang, H.; Ma, B.; Woolf, T. B.; Nussinov, R. Biophys. J. 2006, 91, 2848– 2859. (64) Cordomı´ , A.; Perez, J. J. J. Phys. Chem. B 2007, 111, 7052–7063.

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Figure 2. Probability distribution of the interaction energy of melittin (per monomer) with surrounding (a) lipid and (b) water molecules. The total interaction energy is partitioned into the electrostatic and vdW terms.

interaction between the hydrophobic TM segment of melittin and the lipid acyl tails. These interactions lead to the formation of a supramolecular lipid-peptide complex. For a more focused examination of environment response to melittin, we further calculated the number of contacts and also the number of hydrogen bonds of peptides with surrounding lipid and solvent molecules, as described in the literature.60,65-68 First, we calculated the average number of water, phosphate, choline, ester, and acyl group heavy atoms within 4 A˚ of the peptide heavy atoms (Figure 3), to understand the microenvironment of melittin.65 The positively charged residues Lys21-Arg22-Lys23-Arg24 of the C-terminal region, nicely positioned at the energetically favorable water/lipid interface, are highly solvated by water and negatively (65) Gorfe, A. A.; Babakhani, A.; McCammon, J. A. J. Am. Chem. Soc. 2007, 129, 12280–12286. (66) Lorenz, C. D.; Faraudo, J.; Travesset, A. Langmuir 2008, 24, 1654–1658. (67) Wee, C. L.; Balali-Mood, K.; Gavaghan, D.; Sansom, M. S. P. Biophys. J. 2008, 95, 1649–1657. (68) Deol, S. S.; Bond, P. J.; Domene, C.; Sansom, M. S. P. Biophys. J. 2004, 87, 3737–3749.

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Figure 3. Contribution from the main components of the membrane system (water oxygens, choline, phosphate, and ester polar headgroups and acyl chain carbons) to the solvation of the individual residues of melittin (per monomer). The data in this figure are averaged over the last 7 ns.

charged lipid head groups. The polar Ser18-Trp19 residues exhibit a more complex solvation pattern. They are exposed to both nonpolar side chains and water by expelling their hydroxyl and indole side chains into the aqueous phase. This also indicates that the bound peptides enhance the water penetration into the hydrophilic-hydrophobic interfacial region of the phsopholipid bilayer. As expected, the hydrophobic TM segment exhibits a large number of contacts with the lipid tails. However, few N-terminal residues, especially Lys7 and protonated N-terminal Gly1, are shown to be highly solvated by water molecules. The apparent discrepancy arises due to the unfavorable partitioning of positively charged residues into the lipid tail region. The system then undergoes necessary adjustments and drags water by disrupting membranes. As explained by Becker,69 the dipole associated with Lys7 acts as an electrostatic “beacon”, steering water penetration from the extracellular side of the membrane. Roux et al. proposed a very similar solvation pattern of melittin.70 As compared to our result, they observed a lesser extent of water around Lys7, in accord with the parallel orientation of melittin in the plane of the outer leaflet. To elucidate the electrostatic interaction, we then calculated the number of hydrogen bonds (H-bond) shared by peptide residues. The criteria that we chose for H-bonding are as follows: The hydrogen-acceptor distance was e2.5 A˚, and the donorhydrogen-acceptor angle was e60.71 As depicted in Figure 4, the C-terminal charged residues tightly interact with the headgroup phosphate and “lock” the peptide into its trans-membrane orientation within the bilayer, whereas the membrane-buried Lys7 and charged N-terminal show a high H-bonding propensity for water oxygen. The N-terminal hydrophobic Ile2-Gly3-Ala4 residues exhibit dual contribution, H-bonding with water via a backbone amide nitrogen (resulting in the reduction in the helical content of these residues, as reflected from the secondary structure analysis, Figure S4 of the Supporting Information) and nonbonded interaction via hydrophobic side chains. For visual perception, Figure S7 of the Supporting Information represents the snapshots of few peptide-lipid supramolecular complexes. Figures 3 and 4 exhibit a large number of water penetration in the bilayer hydrophobic core, due to melittin-induced cell lysis,12,69 (69) Bachar, M.; Becker, O. M. J. Chem. Phys. 1999, 111, 8672–8685. (70) Berneche, S.; Nina, M.; Roux, B. Biophys. J. 1998, 75, 1603–1618. (71) Zhao, W.; Rog, T.; Gurtovenko, A. A.; Vattulainen, I.; Karttunen, M. Biophys. J. 2007, 92, 1114–1124.

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Figure 4. Time-averaged melittin-POPC and melittin-water H-bonds. In this figure, the last 7 ns of data has been used.

and also indicate the role of specific amino acid residues in membrane disruption. Membrane-Perturbing Effects of Melittin. Lipid Tail Order. The perturbation produced by melittin on the average structure of the bilayer interior can be characterized using the C-H bond orientational order parameter, SCH S CH ¼ 0:5Æ3 cos2 θ -1æ where θ is the angle between the C-H bond vector and the membrane normal and the angular brackets indicate averaging over time and over lipids. To investigate the effect of the prepore on local lipid perturbation, lipids are divided into two categories: local (those are within 10 A˚ of peptides) and bulk (all others) lipids. The marked asymmetry of the system is reflected in the order parameters profile of the acyl chains of the two bilayer leaflets (Figure 5). In the case of a lower layer, a drastic drop in SCH values of local lipids indicates a localized but strong bilayer perturbation by the loosely bound melittin N-terminals. As compared to the pure bilayer, bulk lipids in the lower layer exhibit a striking increase in order parameter values. The local bilayer disorder is also observed for the upper layer lipids but to a lesser extent. The SCH value of pure POPC bilayer agrees well with the previous findings.48,63 Another interesting observation is that the lipid tail ends become more ordered in the presence of peptide. The earlier theoretical studies indicate a very similar membrane perturbing effect by melittin56 and other membrane-active peptides.50,63,65 Thus, our result suggests that melittin strongly influences the structure and dynamics of the phospholipid membrane. In our case, the extracellular layer is less affected by the embedded peptide, whereas there is an increased level of disorder and structural deformation of lower-layer phospholipids in the immediate vicinity of the peptide. As a result, cell lysis starts from the inner side of the bilayer. In a recent article, Dufourc et al.72 have investigated the pore formation by an AMP cateslytin on the DMPC bilayer. The association of the charged peptide with the outer leaflet induces an electric field (ca. 0.1 V/nm, on the basis of electrophysiological measurement) inside the membrane, roughly perpendicular to the average plane of the bilayer. Because of the asymmetric charge distribution, the water defect initiates only from the inner bilayer leaflet. Membrane Thinning and Lipid Orientation. To study the impact of melittin on bilayer thickness, we plot the ensemble (72) Jean-Franc-ois, F.; Elezgaray, J.; Berson, P.; Vacher, P.; Dufourc, E. J. Biophys. J. 2008, 95, 5748–5756.

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Figure 5. Order parameters, SCH, for the palmitoyl (left column) and oleoyl (right column) chains for the different lipid categories, for the upper (upper panel) and the lower (lower panel) layer lipids and that of the pure POPC bilayer.

average (last 7 ns) position of 120 PO4- groups along the bilayer normal, as a function of radial distance from the center of the prepore (Figure 6) (which is defined as the center of the penetrating aqueous phase in the bilayer hydrophobic core, bound by four melittins). The peptides induce an asymmetry in the POPC headgroup’s distribution. In the case of the upper layer, no significant change in surface corrugation is observed. However, in the lower layer, the loosely bound N-terminals from melittin monomers perturb the regular lamellar structure of the lipid head groups. Membrane thinning is apparent from the thickness around ∼30 A˚ at the prepore, as compared to its original value of ∼38 A˚ away from the prepore (the bilayer thickness of the pure POPC bilayer is ∼37.5 A˚; Figure S8 of the Supporting Information). The local bilayer thinning by melittin6,56,70 and other membrane-active proteins/peptides10,63 have been reported previously by several groups. As depicted in Figure 7, lipids closer to the prepore adopted a more tilted conformation. A considerable reorientation takes place for lower-layer lipids. Few lipids in the lower layer changed the orientation of their acyl chains with respect to the membrane normal from parallel to perpendicular. The superposition of lipid conformations, Figure S9 of the Supporting Information, supports this fact. The reorientation allows the hydrophobic lipid tails to avoid being in contact with the penetrating aqueous phase. The flipped lipid heads are now translocated from the membrane surface to the surface of the aqueous channel. The rapid flip-flop of membrane lipids near melittin pore was demonstrated earlier.39,73 Such bending of bilayer leaflets is responsible for the localized membrane thinning. It has been earlier suggested that the presence of the peptide in a PC bilayer should alter the average orientation of the headgroup dipoles.74,75 Although in the present simulation, the PC headgroup of upper layer lipids remain (73) Fattal, E.; Nir, S.; Parente, R. A.; Szoka, F. C., Jr. Biochemistry 1994, 33, 6721–6731. (74) Shepherd, C. M.; Schaus, K. A.; Vogel, H. J.; Juffer, A. H. Biophys. J. 2001, 80, 579–596. (75) Kuchinka, E.; Seelig, J. Biochemistry 1989, 28, 4216–4221.

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Figure 6. Time average positions of PO4- head groups along the bilayer normal as a function of distance from the prepore.

roughly parallel to the membrane surface,48 some extent of perturbation of the polar lipid head in the lower leaflet is obvious from a broader headgroup distribution (Figure S10 of the Supporting Information). Membrane Permeability. Water Penetration. Because TM pore formation is central to many biological processes, it is an area of intense research.76-80 Recent theoretical studies have examined the water penetration through bilayer deformation by highly charged peptides.81 In the present simulation, we have already examined an enormous amount of water penetration into the membrane core. The extent of water penetration can be visualized from the atom density distribution along the bilayer normal.82 Figure S11 of the Supporting Information shows the (76) Thøgersen, L.; Schiøtt, B.; Vosegaard, T.; Nielsen, N. C.; Tajkhorshid, E. Biophys. J. 2008, 95, 4337–4347. (77) Illya, G.; Deserno, M. Biophys. J. 2008, 95, 4163–4173. (78) Mani, R.; Cady, S. D.; Tang, M.; Waring, A. J.; Lehrer, R. I.; Hong, M. Proc. Natl. Acad. Sci. 2006, 103, 16242–16247. (79) Gkeka, P.; Sarkisov, L. J. Phys. Chem. B 2009, 113, 6–8. (80) Notman, R.; Anwar, J.; Briels, W. J.; Noro, M. G.; Otter, W. K. d. Biophys. J. 2008, 95, 4763–4771. (81) Denning, E. J.; Woolf, T. B. Biophys. J. 2008, 95, 3161–3173. (82) Mondal, S.; Mukhopadhyay, C. Chem. Phys. Lett. 2007, 439, 166–170.

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Figure 7. Time average lipid tilt angle as a function of radial distance from the prepore (last 7 ns). The average tilt angle distribution of the pure POPC bilayer is shown in the inset. The lipid tilt angle is the angle between the saturated lipid tail (from carbon numbers 2 to 16) and the membrane normal.

average distribution (last 7 ns) of water, along with the distribution of water, carbonyl, and phosphate, at the starting point. The broadening of the water peak indicates water penetration from both leaflets of the membrane, although major lysis takes place from the lower layer. The prepore thus formed is of a well-defined size6,19 (∼25 A˚ in diameter, as reflected from the representation of the prepore in X-Y plane; Figure S12 of the Supporting Information), capable of ion permeation, rather than the hypothetical single-file water pore efficient for proton transfer, although the pore width is not the same all across the bilayer. In a study of melittin-induced hemolysis, Katsu et al.83 showed that the pores of 1.3 and 2.4 nm are formed at a melittin concentration of ∼0.2 and 0.8 μM, respectively. To get a quantitative estimation of water penetration, we then calculated the number of penetrating water molecules (NW) as a function of time. Figure S13 of the Supporting Information indicates a sharp rise in water penetration followed by a gradual increase. The result agrees well with the rapid water penetration observed by Roux et al.,70 immediately after the protonation of the N-terminus of melittin. Former experimental37 and theoretical studies69 estimated the number of penetrating water molecules in the hydrophobic region of the membrane for each melittin as ∼20-30. The pore size as well as the number of penetrating waters increase with an increase in the P/L ratio.19 Baumgaertner et al. investigated the stability of a hypothetical melittin pore constructed by a melittin tetramer in a POPC bilayer.39 According to their result, the number of water molecules inside the pore is ∼400 at the end of the simulation. A smaller number of penetrating water molecules, approximately ∼240, is observed in the present simulation (almost after 8 ns). Ion Penetration. Although the melittin-induced pore is welldocumented in the literature, only scarce information is available for ion conductance.22,23 The present study has explored the movement of chloride ion inside the prepore; however, no such evidence is observed for sodium ions. The selectivity of the prepore for anions over cations is presumably due to its strong cationic nature. To monitor the movement of chloride ions, we have plotted the Z-coordinates of Cl- ions as a function of time. Figure 8 shows that with the progress of time, few Cl- ions penetrate deep into the membrane and are entrapped by the positively charged N-terminus and Lys7 residues of melittin. Two events are observed where Cl- ions enter and exit the prepore from the same side. Our result is compatible with the movement of Cl- ions in an octameric PG-1 pore, as described in ref 10. Figure S14 of the Supporting Information shows the radial distribution (83) Katsu, T.; Ninomiya, C.; Kuroko, M.; Kobayashi, H.; Hirota, T.; Fujita, Y. Biochim. Biophys. Acta 1988, 939, 57–63.

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Figure 8. Time profiles of the z-coordinate of representative chloride ions. The blue and yellow curves correspond to the Z-position of Lys-7 and N-terminal Gly-1 respectively, as a function of time.

function (RDFs) of various nitrogen (both backbone and side chain) atoms of the TM segment of melittin with chloride ions, those that have entered into the aqueous cavity. As depicted in Figure S14 of the Supporting Information, mainly the positively charged Lys7 and N-terminus of melittin govern the electrostatic interaction. To evaluate the chloride occupancy of the prepore, we have calculated the number of penetrating Cl- ions as a function of time. Figure S15 of the Supporting Information reveals that on average ∼5-6 Cl- ions reside inside the prepore. The multiple ion occupancy is supportive to the formation of a prepore with welldefined size, in spite of a single-file water pore. Tosteson et al.22 earlier conjectured that four melittin monomers are needed to form the channel and at least four gating charges, that is, one charge per melittin monomer, is needed to compensate the positive charge on Lys7. Thus, Lys7 plays a crucial role in anion transport through the melittin pore. Moreover, the electrostatic repulsion between the positively charged residues protects the prepore from hydrophobic collapse. Furthermore, we also investigate the diffusive nature of chloride ions in bulk as well as in the interior of the prepore by calculating the diffusion coefficient of Cl- ions along the membrane normal as a function of the distance from and position along the axis of the prepore10,84 (Figure 9). The confined motion of Cl- ions near the center of the prepore is due to their strong electrostatic bonding with Lys7. As a result, they exhibit slower dynamics as compared to bulk Cl- ion. The calculated diffusion coefficient is comparable with the former theoretical finding.10 Altogether, our simulation data provide compelling evidence of the preference of melittin pore toward anions over cations and also present a qualitative scenario of the movement of chloride ions in the pore interior. Thus, our simulation results point toward the initiation of an ion-permeable water pore in the absence of TM potential19 (movies showing the initiation of cell lysis by melittin and the chloride ion penetration into membrane core are in the Supporting Information). Architecture of the Prepore. Despite intense research, there is an ongoing debate about the structure of the melittin pore. Few studies suggest the barrel-stave mechanism, while others invoke a toroidal (“worm-hole”) pore model. These two TM pores are actually differentiated on the basis of lipid orientation near the pore.12 In the barrel-stave model, peptides stand upright around a central lumen. However, in the toroidal pore model, both of the (84) Makarov, V. A.; Feig, M.; Andrews, B. K.; Pettitt, B. M. Biophys. J. 1998, 75, 150–158.

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Figure 9. Diffusion coefficient of Cl- ions along the z-axis, as a function of the distance from and the position along the axis of the prepore (r,z).

bilayer leaflets sharply bend in the fashion of a toroidal hole, so that the pore is lined by both peptide and lipid headgroups. Our simulation results exhibit the tilting of lipid near the aqueous cavity. Few lipids with their lipid acyl tails undergo orientational transition from parallel to perpendicular wrt lipid normal. The pore-lining lipid heads, which are originally on the membrane surface, are now flipped to line the prepore, in support of the toroidal pore model. Few theoretical as well as experimental studies examined the reorientation of the lipid during the formation of a toroidal-shaped pore previously.39,73,85 It has been suggested that toroidal pore formation would be favored by the presence of lipids with a positive curvature but opposed by lipids with a negative curvature.13 On the basis of oriented circular dichroism (OCD) and neutron scattering studies, Huang et al. made a case study on the melittin pore.12 They were able to crystallize the melittin pore at low temperature and low humidity. The properties of melittin pore are closely similar to those of magainin (toroidal pore) but unlike those of alamethicin (barrel stave pore).12 The single channel conductance of the melittin pore measured by Sansom et al. is similar to magainin, which supports the toroidal model.86 Again, any mismatch between the hydrophobic length of membrane and the peptide favors the toroidal pore over the barrel-stave pore,13 which is why in the latter case the bilayer remains almost flat. In our simulation, the N-terminus of melittin in the lower half of the leaflet causes serious local deformation and ultimately leads to membrane disruption. So, the evidence that we are able to gather from our simulation argued in favor of the melittin-induced toroidal pore over the barrel-stave mechanism.

Conclusion The present work addresses the mechanism of action of AMP melittin on the bilayer membrane. The cytolytic activity of melittin arises due to the defects introduced in the membrane by a highly cationic peptide. The present simulation indicates the (85) Matsuzaki, K.; Murase, O.; Tokuda, H.; Fujii, N.; Miyajima, K. Biochemistry 1996, 35, 11361–11368. (86) Sansom, M. P. Prog. Biophys. Mol. Biol. 1991, 55, 139–215. (87) Humphrey, W.; Dalke, A.; Schulten, K. J. Mol. Graphics 1996, 14, 33–38.

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formation of a prepore through collective membrane perturbation by bound peptide. The analysis of the “lipid-peptide” interaction energy and also the partition of total interaction energy into electrostatic and vdW terms will help to shed light on the factors that confer the stability and activity of melittin in the membrane. A detailed analysis of the average number of contacts and also the number of hydrogen bonds shared by the peptide with its surrounding environment enables us to explore the active role of specific amino acid residues in cell lysis. The charged residues of melittin not only act as “anchors” but also disrupt the membrane by dragging water from both sides, leading to the initiation of a water-filled pore, which is also supported by the surrounding lipid matrix. The presence of aqueous cavity causes localized but strong bilayer perturbation. The tilting of lipid nearer to the prepore allows local bilayer thinning. The flipped lipid head is now able to line the surface of the penetrating aqueous phase. From the conformation of lipids in the vicinity of the prepore, it can be classified as toroidal-shaped. Moreover, we are also able to capture the movement of chloride ions inside the aqueous cavity, although no such evidence is observed for sodium ions. The chloride ions entered into the prepore due to strong electrostatic attraction by charged residues located at its wall. We also investigate the diffusive nature of chloride ions in bulk as well as in the prepore interior. The slower diffusion coefficient obtained in the later case points toward the confined motion of Cl- ions near the center of the prepore. The selectivity of the cationic prepore toward anion is compatible with an experimental approach. Thus, we are able to produce the molecular mechanism related to the initiation of the ion-permeable water pore by AMP melittin. Acknowledgment. We are thankful to the Department of Chemistry, University of Calcutta, and the UPE project for the computational facilities. M.M. is thankful to CSIR, India, for the fellowship through CSIR-NET. This work is also supported by the “Council for Scientific and Industrial Research” [No. 01(2035)/06/EMR-II], Government of India. Supporting Information Available: More data regarding the equilibration of the pure POPC bilayer in NPT (area per lipid and order parameter profile) and NγPT (area per lipid and atom density distribution) ensembles, conformational studies of melittin (secondary structure analysis, interhelical bend angle, and backbone rmsd), snapshots of lipid-peptide complexes, lipid conformations away and near the center of the prepore, bilayer structure of the melittin/lipid complex system (headgroup P-N vector and atom density distribution), representation of the prepore structure in the X-Y plane, membrane permeability (water and ion penetration, RDF of CL-), and results of the additional simulations. Two movies showing the initiation of cell lysis by melittin and the chloride ion penetration into membrane core. This material is available free of charge via the Internet at http://pubs.acs.org.

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