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Molecular Simulations of Melittin Induced Membrane Pores Delin Sun, Jan Forsman, and Clifford E. Woodward J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b07126 • Publication Date (Web): 16 Oct 2017 Downloaded from http://pubs.acs.org on October 17, 2017

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The Journal of Physical Chemistry

Molecular Simulations of Melittin Induced Membrane Pores Delin Sun,† Jan Forsman,‡ and Clifford E. Woodward*, † †

School of Physical, Environmental and Mathematical Sciences, University of New South Wales, Canberra ACT 2600, Australia

‡

Theoretical Chemistry, Chemical Centre, Lund University, P.O. Box 124, S-221 00 Lund, Sweden

1 Environment ACS Paragon Plus


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ABSTRACT Membrane-active peptides (MAPs) are able to induce pores in cell membranes via molecular mechanisms, which are still subject to ongoing research. In this work, we present molecular dynamics simulations that suggest that a precursor membrane defect plays an important role in the pore-inducing activity of the prototypical antimicrobial peptide, melittin. The simulations reveal that the hydrophobic N-terminus of melittin is able to recognize and insert into the membrane defect in the lipid bilayer and that this leads to a cascading transfer of adsorbed peptides to the membrane defect, leading to peptide aggregation in the pore. We show that this mechanism also acts in the case of a melittin mutant without the flexible central proline hinge, thus indicating the latter is not crucial to the activity of melittin, which is consistent with experiments.


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1. Introduction Antimicrobial peptides (AMPs) are integral components of the innate mammalian immune system, which defend the host against a wide range of microorganisms, including bacteria, fungi and viruses. Unlike small-molecule inhibitors, which function by shifting the equilibrium between active and inactive structures of target proteins, AMPs appear to kill microorganisms by disrupting the prokaryotic cell membranes. A major class of AMPs is natively unstructured in water but folds into an α-helical form upon adsorbing onto membranes.1 This class of AMPs are the so-called amphipathic α-helical AMPs. The implications of the structural transformation of these peptides are still not well understood. However, numerous studies have established that an amphipathic helix can recognize membrane defects that present hydrophobic lipid tails to the surrounding solution, which then leads to various membrane deformations.2-4 Indeed, one may expect a rich variety of membrane defects due to heterogeneity of lipid composition coupled with thermal fluctuations. For example, the bilayer thickness mismatch at the boundary of segregated domains will expose parts of a lipid’s alkyl chain. This boundary region may act as a target for membrane disrupting peptides, allowing them to gain entry into cells.5 Furthermore, thermal fluctuations of phospholipid head groups and alkyl chains within the bilayer, though energetically unfavorable, can generate short-lived membrane defects, which mediate cell membrane fusion and also insertion of nanoparticles.6-7 It is thus entirely plausible that recognizing and exploiting either stable or transient membrane defects may be a common strategy by which amphipathic helical peptides, including amphipathic AMPs, enter cells. Melittin is an archetypal amphipathic AMP which has already been the subject of extensive experimental and computational investigation.8-15 Previous experimental results have suggested that melittin is able to induce membrane pores with a range of sizes.16 Of note is the work of Lee et al that indicates pore induction by melittin is probabilistic and, furthermore, membrane defect nucleation enhances the probability of pore formation.17 However, it is technically difficult for



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experiments to directly detect the nucleation of membrane defects, due to the small size and the short lifetime of those defects.

Hence, the processes by which the occurrence of membrane defects

promotes the formation of membrane pores remains ambiguous. It is in elucidating these kinds of molecular processes that computational studies, using all-atom models, can provide a valuable adjunct to experimental studies. In this work, we employed all-atom molecular dynamics (MD) simulations to explore the idea that melittin exploits membrane defects, created by thermal fluctuations, in order to induce membrane pores. Our results show that the N-terminus of melittin acts as a type of “defect sensor”. Crucially, it turns out that the rapid diffusion of just one melittin peptide into the defect can prolong its structure for a time sufficient, to allow a cascade of peptide transfer into the defect provided the adsorbed density of peptide is sufficiently high (above a certain threshold concentration). The results presented here shed new light on the underlying mechanism by which amphipathic AMPs disrupts cell membranes.

2. Simulation Methods 2.1 Simulation models The native melittin has the amino acid sequence of NH3+-Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-LeuThr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln-CONH2. Melittin has a central proline residue (proline-14), which is thought to somehow enhance its ability to permeablize the lipid membrane.18 Proline does not hydrogen bond to other residues and thus acts as a molecular “hinge” at the peptide centre. While the added flexibility is often conjectured to help membrane penetration, experiments instead suggest increased lytic activity upon replacement of the proline by a hydrogen-bonding residue.19-20 To investigate the role of proline, we also modelled a mutant melittin wherein proline is replaced with alanine. The two modelled peptides have a net charge of +6. The zwitterionic dipalmitoylphosphatidylcholine (DPPC) lipid bilayer was used as the model membrane. The initial configuration consisted of four identical melittin monomers with an α-helical 4 Environment ACS Paragon Plus

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configuration embedded into the glycerol regions of one leaflet of the bilayer, with their helical axes parallel to the bilayer surface. The simulation systems contained 128 DPPC lipids, 4 melittin peptides, 6186 TIP3P water molecules, 119 chloride ions and 95 sodium ions such that the concentration of salt in the system is 150 mM.

From that initial configuration, we then carried out

400 ns MD simulations in a semi-isotropic ensemble, wherein the system’s volume was allowed to fluctuate independently in the directions parallel and perpendicular to the bilayer.

2.2 Creating a Membrane Defect To test our idea that a precursor membrane defect facilitates the generation of a pore by melittin, we created a membrane defect by slowly pulling (with a rate=0.001 nm/ps) a selected number of lipid head groups towards the lipid bilayer center. If one peptide was found to insert into the defect (see Figure 1), the external force imposed on the lipid head groups was removed. The system was then relaxed using unconstrained MD simulations for two microseconds.

2.3 Potential of Mean Force for Ions Permeating Pores Umbrella sampling simulations21 were used to derive the PMFs for chloride and sodium ions permeating the melittin-stabilized membrane pores. The reaction coordinate was chosen to be the distance between the ion and the center-of-mass of lipid bilayer. 56 sampling windows were used along the reaction coordinate and each window was simulated for 10 ns. In both sets of umbrella sampling simulations, we used the weighted histogram analysis method (WHAM)22 to construct the PMF profiles and to estimate the statistical errors.

2.4 Force Fields and Simulation Parameters All simulations were performed using the GROMACS 4.5.5 package.23 All-atom simulations were run at the temperature of 323 K using a Nosé-Hoover thermostat 4,5 with a coupling time constant of



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0.5 ps. In the unconstrained all-atom MD and umbrella sampling simulations, the system’s volume was allowed to fluctuate according to the semi-isotropic pressure coupling method with both lateral and perpendicular pressures (both 1atm) were independently coupled to the Parrinello-Rahman barostat24 with a coupling time constant of 5 ps and a compressibility of 4.5×10-5 bar-1. Periodic boundary conditions were employed. The simulation time step was 2 fs. CHARMM36 force field25-26 were used to model lipid-peptide interactions and TIP3P model27 was used for water. The electrostatics were evaluated using the particle-mesh Ewald (PME) method.28

3. Results 3.1 Process of Membrane Pore Formation In our MD simulations, four identical peptides were placed randomly onto a model DPPC lipid bilayer (giving P: L = 1/32) and 400 ns of MD simulations were performed. For both cases (native melittin and mutant melittin), the peptides remained adsorbed flat on the bilayer surface with no evidence of membrane pores or precursor defects. To facilitate the precursor defect formation, three adjacent lipids were selected and their head groups were pulled towards the membrane centre with an applied force. The z-direction coordinates for the three displaced phosphorus atoms are -0.37 nm, -0.72 nm, and -1.08 nm, relative to the center of mass of the lipid bilayer. We created such a defect at several places on the leaflet with the adsorbed peptides. In most cases, the defect remained unstable and removal of the force on the head groups after a short time (~ 10 ns) led to rapid reformation of the bilayer on a timescale similar to the bilayer without adsorbed peptide. This is despite the fact that the P: L ratio was quite high. Thus, we concluded, that simply adsorbing melittin peptides to the bilayer surface did not substantially stabilize such membrane defects. On the other hand, if the defect was formed close to the N-terminus of an adsorbed peptide, the peptide immediately “followed” the displaced lipids, inserting the N-terminus into the defect, see Figure 1.


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Furthermore, removing the applied force did not lead to membrane healing. Instead, the defect was sufficiently long-lived to subsequently allow two other nearby peptides to diffuse to and insert their N-terminals into it as well. This occurs on the timescale of hundreds of nanoseconds. This recruitment of other peptides caused the defect to develop into a small pore, which remained stable over the remaining simulation time (~2 µs). It was found that a fourth peptide (shown yellow in Figure 1), which had its cationic C-terminus close to the defect, was unable to fully reorientate and insert into the pore over the simulation period. This is also clearly shown in a movie of the first 650 ns after defect formation for the case of melittin (see MOVIE in Supporting Information). This observation indicates that it is the interactions between the predominately hydrophobic melittin Nterminus and the lipid alkyl chains, exposed in the membrane defect, that provides the driving force by which the peptide reorients and inserts into the membrane defect. This overall qualitative behaviour was similar for both the native melittin and the mutant melittin, indicating that the proline residue is not crucial for the processes we observed. Our results indicate that these peptides are able to recognize and bind to membrane defects, with rapid insertion giving rise to stable pores. We note that the affinity of similar peptides to membrane pores also been observed in recent experiments.29

(A)



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(B) Figure 1. Selected snapshots illustrating the dynamic process of mutant (A) and native (B) melittin insertion into the membrane defect and the development of the defect into a peptide trimer-stabilized membrane pore. For both cases, the pores are formed within 650 ns after the defect is created. Lipid tails are not shown for clarity.

3.2 Pore Structure and Size Our simulations showed that replacement of the proline amino acid affects the membrane pore structure and size. Mutant melittin created a pore with a funnel-like shape, whereas the native melittin (with its central kink) produced a pore with an hourglass-like shape. To estimate the inner diameter of the membrane pore, we assume that the small volume inside the lipid bilayer to be cylindrical and that the density of water molecules in the volume is same to that of bulk water. The height of the volume is taken to be 2 nm for both mutant and native melittin stabilized pores. The time evolution of the number of water molecules in this cylindrical volume is plotted in Figure 2(AB). The time-averaged numbers of water molecules in the mutant and native melittin stabilized pores are 82 and 71, respectively. The inner diameter of the pore (or the diameter of the cylinder) was calculated using the following equation 2∗

18 602


(1)

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where d is the inner diameter of the pore, N is the number of water molecules in the small cylindrical volume, H is the height of the cylindrical volume. The time-averaged inner diameters of the mutant and native melittin stabilized pores were estimated to be ~1.24 nm and ~1.16 nm, which are slightly smaller than the ~1.6 nm obtained in simulations by Leveritt et al.10 Those authors performed simulations of four melittin peptides in a DMPC lipid bilayer pre-inserted into a pore. The outer diameters of the pores were estimated by calculating the center-of-mass distances of the three peptides inserted into the pore in the direction parallel to the membrane plane, and the results are plotted in Figure 2 (C-D). For mutant and native melittin-stabilized pores, the outer diameters are in the range of 1.3-3.5 nm and 1.6-3.4 nm, respectively. The slightly larger inner pore size we see for the mutant peptide is consistent with the observed increased lytic activity of mutated melittin,19-20 but we say this cautiously, as these small pores would not allow the leakage of large molecules (such as fluorescent dyes). However, our results do suggest that pores may indeed grow in size, by diffusive addition of more peptides. This mechanism was recently described by us using coarsegrained simulations that showed surface adsorbed melittin can migrate to a membrane pore, causing its radius to grow to ~4.7 nm.30 Furthermore, solution atomic force microscopy experiments by Pan and Khadka found that melittin-induced membrane pores can gradually expand to a radius of about 4-5 nm over time.11

(A)

(B)



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(C)

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(D)

Figure 2. Time dependent numbers of water, chloride ion and sodium ion in the mutant (A) and native (B) melittin-stabilized membrane pores. The XY plane center-of-mass distances of three mutant melittin peptides (C) and native melittin peptides (D) in the pores.

3.3 Melittin-Induced Membrane Pore is More Permeable to Anions One hypothesis for melittin induced apoptosis is the disruption of ion homeostasis, due to conduction through the formed pores.27 We calculated the potential of mean force (PMF) for translation of sodium and chloride across the peptide-stabilized pores. The full PMF profiles are plotted in Figure 3. The free energy barriers for chloride and sodium ion permeation across the mutant melittin-stabilized pores are 10.5 kJ/mol and 14.7 kJ/mol respectively. These are somewhat lower than the values for the native melittin-stabilized pore, which were 12.8 kJ/mol and 18.0 kJ/mol for chloride and sodium respectively. The free energy results are consistent with the finding that the number of chloride ion in the pores is larger than that of sodium ion, as is shown in Figure 2(A-B). The reason behind the barrier height differences for sodium and chloride ions is likely due to the positive charge of the peptide. However, the size of the free energy barriers for these relatively small pores (~4-5kBT), suggests that ion diffusion is only a relevant mechanism for larger pores, which we predict would eventuate in the case of real systems, via the mechanism described above.


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8 (A)

(B)

Figure 3. Potential of mean force for sodium and chloride ions permeating across the mutant (A) and native (B) melittin-stabilized membrane pores.

4. Discussion The results of our simulations suggest that pore formation induced by melittin (or its mutant) is initiated by a membrane defect formation (i.e., one that exposes the hydrophobic core of the membrane) occurring in the vicinity of the correct part of the adsorbed peptide (the N-terminus). While the presence of adsorbed peptides may, to some extent, increase the frequency of membrane defect formation, adsorption alone does not seem to give rise to long-lived defects, as is often suggested in prevailing theories.2 We propose instead that the crucial step to stable pore formation is to have an optimal spatial coincidence between a membrane defect and an adsorbed melittin (or mutant) that allows rapid response by the peptide via insertion of the N-terminus, thus prolonging the lifetime of the defect. The defect may then possibly accumulate other peptides that diffuse from nearby regions. It is this peptide aggregation, which causes the development of a full pore and not membrane weakening per se. Our suggested mechanism shares some commonalities with the concept of biomolecular recognition often used to describe the binding of ligands to proteins and nucleic acids.


31

The


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conformational selection model proposes that a ligand will bind only to a favoured protein conformation, while the induced fit model posits that binding may occur to a less favourable configuration but causes a shift in the target conformation to the required structure.

While

biomolecular recognition is often invoked to explain the activity of proteins and nucleic acids we suggest that it may also find application in the context of membrane active peptides. That is, fluctuating cell membranes present a substantial conformational ensemble to AMPs, such as melittin. Currently, the dominant hypothesis is that adsorption of AMPs will expand the bilayer and increase the surface tension. This stress is assumed to increase with adsorption density giving rise to spontaneous pore formation above a particular critical peptide:lipid (P:L) ratio.2 This type of collective activity, acting over a large length-scale, does not generally fit with the biomolecular recognition paradigm. However, our results indicate that melittin and its mutant bind strongly to a membrane defect that exposes hydrophobic regions of the membrane. Recently, we also used MD simulations to show that an oligo-arginine peptide could bind to and prolong the lifetime of a thermally induced pore32 in a very similar manner. Finally, there is experimental evidence that certain AMPs may bind strongly to curved sections of membranes, due to the presence of exposed hydrophobic lipid tails.3,

4

Summarizing these observations leads us to suggest that a common

initiating step to pore formation, for many membrane active peptides, may be binding to a thermally induced membrane defect, in a manner consistent with the biomolecular recognition paradigm. In particular, the fact that adsorbed peptides must wait for the “correct” type of fluctuation, i.e., one which is close to the N-terminus and also exposes hydrophobic regions of the membrane, seems to suggest a conformational selection (CS) model. Subsequent changes to the pore upon binding of the peptide could, in principle, be interpreted as more consistent with the induced fit model, but the crucial first step of recognition and binding to an appropriate membrane defect certainly bears the hallmarks of the CS interpretation.


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So how does our proposed mechanism explain why a certain P:L threshold concentration must occur in order for a pore to form? To answer this it is important to note that once a peptide has inserted into the defect, the lifetime of the defect is prolonged. The mechanism by which this occurs is not clear as yet. It is possible that the pore becomes thermodynamically more stable, due to binding of the peptide, but this may only be part of the story. The fact that several lipids are bound to the peptide may also lead to an entropic bottle-neck for pore closure, which slows down the kinetics of the local membrane fluctuations.

In any case, the simulations clearly show a marked

increase in defect lifetime is expected upon addition of peptide. That is, the presence of a peptide (or peptides) in the defect will increase the likelihood that the defect will acquire yet another peptide via diffusion from the nearby regions of the bilayer surface. The time it takes for this peptide to diffuse decreases with increasing P:L ratio. As the defect accumulates peptides it grows to accommodate them to become a pore once it contains a sufficient number of peptides (see MOVIE in Supporting Information). As successive peptide additions to the defect are expected to lead to further increases in its lifetime, a cascade of peptide accumulation will occur, provided the P:L ratio is above a threshold value. We reiterate, that in this model, this threshold concentration is not required to build up sufficient stress on the membrane (as is often proposed), but rather is related to the probability that a defect with a single inserted peptide (with characteristic lifetime of τp) will acquire a second peptide molecule. The lifetime τp can be estimated by the time it takes for a peptide to diffuse out of the defect with characteristic size L. Let D be the diffusion coefficient of the peptide on the bilayer surface, we estimate τp ~ λL2/D. The factor λ (> 1) accounts for the additional attraction between the peptide and the defect compared with the intact bilayer. The characteristic time taken for peptide on the flat bilayer surface to diffuse to a defect can be estimated τp ~ ( Dcp,)-1 where cp is the surface density of peptides, the threshold concentration is thus cp*~ 1/λL2. The defect is expected to involve a few lipid molecules, e.g., we constrained 3 lipids, giving L2~ 3 (in units of lipid cross-section). 13 Environment ACS Paragon Plus


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The factor λ can be estimated from the increased probability of finding a peptide in a defect, compared to the intact bilayer. This is ultimately determined by the hydrophobic interactions between the peptide and the exposed lipid tails, which is difficult to estimate, given that the tails are only partially exposed and the peptide is not able to achieve full contact with them. Experimental estimate of a threshold P:L value of about 1:30 implies λ ~ 5, which is roughly equivalent to one full methyl contact with a hydrophobic surface in water.

5. Conclusions In summary, we have presented an MD study that suggests that an important initiating step for pore formation by melittin (and a mutant) is the binding of the N-terminus to a precursor membrane defect that exposes the hydrophobic interior of the membrane. Above a threshold adsorption concentration, a cascade of peptide insertion into the defect can occur that leads to stable pore formation. We argue that this process can be viewed as a biomolecular recognition, consistent with a local (rather than global) mechanism for peptide activity on the membrane surface. The central proline residue of melittin was shown not to be an essential component of this mechanism and some properties of the native and mutant melittin-stabilized pores were presented, including shape, size and ion permeability. We propose that since melittin is a typical amphipathic AMP, the findings here have relevance to this enigmatic and poorly understood group of amphipathic peptides. Future work will be performed on other amphipathic AMPs in order to derive a general model of action for this class of AMPs.


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ASSOCIATED CONTENT The Supporting Information is available free of charge on the ACS Publications website. Movie showing pore formation within 650 ns after membrane defect formation

AUTHOR INFORMATION Corresponding Author: e-mail: [email protected]. The authors declare no competing financial interest.

ACKNOWLEDGMENT An allocation time from the Lunarc Computing Center at Lund University is gratefully acknowledged. JF acknowledges financial support from the Swedish Research Council.

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