Simulations Suggest Possible Novel Membrane Pore Structure

Article pubs.acs.org/Langmuir

Simulations Suggest Possible Novel Membrane Pore Structure Robert Vácha*,† and Daan Frenkel‡ †

National Centre for Biomolecular Research, Faculty of Science and CEITEC - Central European Institute of Technology, Masaryk University, Kamenice 5, 625 00 Brno-Bohunice, Czech Republic ‡ Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom S Supporting Information *

ABSTRACT: Amphiphilic proteins and peptides can induce the formation of stable and metastable pores in membranes. Using coarse-grained simulations, we have studied the factors that affect structure of peptide-stabilized pores. Our simulations are able to reproduce the formation of the well-known barrel-stave or toroidal pores, but in addition, we find evidence for a novel “double-belt” pore structure: in this structure the peptides that coat the membrane pore are oriented parallel to the membrane plane. To check the predictions of our coarsegrained model, we have performed more detailed simulations, using the MARTINI force field. These simulations show that the double-belt structure is stable up to at least the microsecond time scale.

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INTRODUCTION The inside of a cell is isolated from the outside by a phospholipid membrane. Pores in these membranes allow the cell to exchange molecules between the cytosol and the environment. The formation and control of pores can be an essential part of the functioning of a healthy cell. However, external factors that induce pore formation may cause cell death, as is the case for some antimicrobial agents. A wide variety of proteins and peptides have been shown to induce the formation of stable or metastable membrane pores.1,2 This group includes antimicrobial peptides that are part of the natural defense system of many organisms against foreign micro-organisms.3 However, sometimes organisms produce peptides that are harmful to eukaryotic cells as well. This is the case for amyloid forming peptides associated with neurodegenerative and related diseases. Such peptides have been found to induce pore formation in membranes, and these pores have been implicated in the pathogenic nature of amyloidogenic peptides.4−8 The best known transmembrane pore structure is a cylindrical barrel, where the peptides are oriented perpendicular to the plane of the membrane. Because barrel-shaped pores tend to be fairly rigid structures, they are comparatively easy to image. In fact, most of the experimental evidence for peptideinduced pore formation relates to such barrel pores. The most direct evidence for this structure originates from atomic force microscopy(AFM) experiments that allow direct visualization of the ringlike structure of barrel pores.7,9−15 Computer simulations show that tetramers and hexamers of peptides are locally stable conformations in agreement with AFM measurements.16 Additional evidence comes from electron microscopy measurements that support the AFM results.11,12,14 Conductivity measurements show that peptide-based barrel pores © XXXX American Chemical Society

can act as switchable ion channels that can be in either their “on” or “off” state.4−8 In addition to the barrel pore, another more transient and more variable pore structure has been identified: the so-called toroidal pore. Whereas in the case of barrel pores the structure of the surrounding lipid bilayer is barely perturbed, the lipid structure around a toroidal pore is strongly affected as some of the lipids participate directly in the pore with their head groups being present along the pore rim, in contact with the peptides.17,18 In this toroidal structure, the peptides can be either perpendicular or tilted with respect to the plane of the membrane. As toroidal pores are transient and tend to fluctuate in shape, they cannot easily be imaged in experiments, and much of the evidence for such structures comes from computer simulations and NMR experiments.19−26 A very disordered version of the toroidal pore, where peptides only partly participate in the pore structure, has also been proposed.27−30 While all the above mechanisms (summarized in Figure 1) were proposed for specific peptides, the general understanding of peptides properties necessary for each mechanism is missing. To our knowledge, there is as yet no experimental evidence for membrane pore structures stabilized by peptides that are aligned parallel to the plane of the membrane. Yet, a priori, such structures do not seem to be excluded. In fact, the complementary structure is known to exist: a membrane disk stabilized on its edge by a “belt” of amphiphilic proteins that are aligned parallel to the membrane plane.31,32 The aim of the present study is to explore under what conditions a peptide belt can form inside a pore. As we are Received: June 7, 2013 Revised: September 9, 2013

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Figure 1. Schematic drawing of the mechanism of interaction between amphiphilic peptides and phospholipid membranes. realistic values for the compressibility and bending modulus of phospholipid membranes. The hydrophobic stripe on the model peptides has an attractive interaction with the hydrophobic tail beads. The interactions with all hydrophilic groups are purely repulsive. All repulsive interactions are described using a Weeks−Chandler−Andersen “repulsive LennardJones” potential. The attractive part has a cosine squared profile, as described in ref 33 with a switching range (distance from the potential minimum to the cutoff) of 1.6 nm for lipids and 1.0 nm for peptides. The depth of the attractive potential is 1 kT per unit length of 1 nm and 1 kT for lipid tail beads. The angle dependence of the interaction is described following the approach described in ref 33. All simulations with patchy spherocylinders were carried out using the Metropolis Monte Carlo method with rotations and displacements of single particles and/or lipid chains. In our simulations, we considered a system of 500 lipid molecules placed in a prismatic cell with periodic boundary conditions. At the beginning of the simulation, the lipid bilayer was assembled in xyplane, and the system was equilibrated at zero lateral tension, yielding a box size of 17 × 17 × 50 nm3. To study the preferred orientations of peptides on the pore surface, we prepared extended membranes with sharp edges. These were created by taking a system with the same number of phospholipids and stretching the simulation box in the x-direction to a length of 50 nm and kept fixed. This procedure resulted in the formation of a single membrane stripe per simulation box with a width of 17 nm in the xdirection and periodically repeated in the y-direction. We used this artificial membrane edge as a substrate onto which model peptides could bind in various orientations. Initially, only a single peptide was added to this membrane edge. The center of mass of this peptide was kept at the membrane edge. The orientational distribution of the peptide was computed using free energy calculations with the Wang− Landau method35 while the bilayer was left free to move. To verify our findings, we then placed four peptides on both membrane edges and let the system fully equilibrate. There are several ways to represent the orientational distribution of the peptide. In one case, the orientational distribution was plotted as a function of cos θ, where θ is the angle between the axis of the peptide and the normal of the membrane. This choice of coordinates is natural if the peptide is free to rotate in the azimuthal direction. In fact, if all peptide orientations would be equally likely, the fee energy as a function of cos θ would be flat. In contrast, if the peptide can only rotate in a plane (e.g., parallel to the membrane edge), then the logical coordinate to compute the fee energy would be θ, as the free energy of a particle that can freely rotate in a plane is flat when potted against θ. In practice, we expect the orientational distribution of the peptide to be in between the 2D and the 3D limits. We have plotted the peptide free energy as a function of cos θ, but it is easy to apply the Jacobian correction to the free energy that is needed to plot the free energy as a function of θ: apart from a constant shift, the correction to the free energy is kBT ln|sin θ|. In the Supporting Information we show the free energy profiles as a function of θ.

interest in the generic features of peptides that might form intrapore belts, we use a coarse-grained model of amphiphilic prolate peptides33 to explore the region in peptide parameter space where belt-stabilized pores could exist. The advantage of the coarse-grained model is that it is sufficiently cheap to allow a systematic exploration of parameter space. The disadvantage is, of course, that it cannot be mapped directly onto a real peptide. It is for this reason that, in the final part of this paper, we consider a higher-resolution model for a real peptide that is expected to have the necessary features to form a double-belt stabilized pore. For more details on the coarse-grained model, see the Methods section.

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METHODS

We consider a system of prolate, amphiphilic peptides interacting with a membrane. The coarse-grained peptide model that we use is described in ref 33. This model is designed to mimic prolate peptides with amphiphilic character. The peptide is represented as a patchy spherocylinder (PSC) with a diameter of about 1.2 nm, which is the characteristic diameter of an α-helix. The length of the cylindrical part varies between 3 and 7 nm. The amphiphilic character of the peptides was accounted for by an attractive stripe parallel to the axis of the spherocylinder. We considered two variants of the patchy spherocylinder model: both had an attractive stripe along the cylindrical part of the particle, but while one version of the model (AE-PSC) had attractive end-caps, the other version of the model (NE-PSC) had noattractive end-caps (see Figure 2). The opening angle of the attractive stripe was 180° (i.e., the surface of the model peptide was 50% hydrophobic and 50% hydrophilic in AE model).

Figure 2. Snapshots of the employed coarse-grained model of amphiphilic peptides. Spherocylinders, shown in orange, have attractive stripe on its side (colored gray). On the left side is the model has attractive ends (AE), while on the right side is the model with no-attractive ends (NE).

The model phospholipids that form the membrane consisted of three-bead chains.34 The bead representing the hydrophilic headgroup is purely repulsive. The other two beads represent the hydrophobic tail and have a relatively long-ranged attraction. The properties of the model particles can be mapped onto those of real phospholipids by assuming that the bead diameter (1σ) is approximately 1 nm. The membrane thickness is then ∼5 nm. This implicit solvent model yields B

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To study the properties of several peptide in a circular pore, we subsequently prepared a system with a finite pore by stretching the lateral dimensions of the original simulation box from 17 × 17 nm to 19 × 19 nm. In addition to these highly coarse-grained simulations, we also performed a study of a more realistic model, namely a membrane in contact with apolipoprotein Apo-A1. We chose this specific protein because it is known to form a “belt” around lipid nanodisks. The apolipoprotein Apo-A1 was parametrized using the MARTINI force field.36,37 The initial structure of Apo-A1 was taken from a simulation of a lipid nanodisk, with two copies of the mainly α-helical protein wrapped around it38 and converted to coarse-grained model with martinize script. We simulated 189 resides known to be involved in membrane binding.39 The sequence is the following: STFSKLREQL GPVTQEFWDN LEKETEGLRQ EMSKDLEEVK AKVQPYLDDF QKKWQEEMEL YRQKVEPLRA ELQEGARQKL HELQEKLSPL GEEMRDRARA HVDALRTHLA PYSDELRQRL AARLEALKEN GGARLAEYHA KATEHLSTLS EKAKPALEDL RQGLLPVLES FKVSFLSALE EYTKKLNTQ. In the MARTINI force field, the secondary structure of the Apo-A1 protein is kept fixed, with the exception of unstructured loops. As the loops are unconstrained, the rigid alpha-helices were free to rotate. Helical structure was recognized in residues 6−39, 51−60, 72−83, 95−105, and 117−184. These are further referred as helices of Apo-A1. The first and the last helix are considered as long, while the other three helices with length about 10 residues are referred as short helices. The proteins were inserted into a circular hole cut out of an equilibrated sheet of a DPPC membrane bilayer resulting in system of 876 lipid molecules, 2 APO-A1 proteins, 19 680 water molecules, 45 sodium ions, and 35 chloride anions. The system was kept at 325 K using velocity-rescaling thermostat40 and at zero tension using the Berendsen barostat.41 A simulation of 2 μs was carried out in the NPγT ensemble. The time step was 20 fs. Note that in MARTINI model the diffusion of lipid molecules is about 4 times faster than reality.36,37 Therefore, a simulation of 2 μs effectively corresponds roughly to 8 μs. This molecular dynamic simulation was carried out using the Gromacs 4.5.5. program package.42 We also constructed a spherocylinder model of Apo-A1. As the protein is formed of several connected helices the PSC model is chain of six AE-PSC of length 5 nm. The individual PSC are connected with harmonic bonds of length 1 nm between the ends of PSC. No angular restraints were applied between PSCs.

Figure 4. Free energy profiles of peptide orientation with both attractive ends AE-PSC (A) and no-attractive ends NE-PSC (B) models and with the peptide length from 3 to 7 nm. Two distinct peptide orientations are depicted for clarity. cos(θ) = −1 corresponds to the perpendicular orientation of peptide to the membrane plane, while cos(θ) = 0 stands for the parallel orientation.

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RESULTS AND DISCUSSION Representative snapshots of the model amphiphilic peptide at the membrane edge (in the pore) are shown in Figure 3, where

for the perpendicular orientation. In contrast, medium and long peptides, with length of 5 and 7 nm, respectively, have the free energy minima when aligned parallel to the membrane plane. The depth of the free energy minimum was about 4 kT for L = 5 nm and about 12 kT for L = 7 nm. For the NE-PSC model, short and medium peptides (L = 3 and 5 nm) have the lowest free energy minimum (≈5 kT) for a perpendicular orientation. The long peptide have a free energy minimum for tilted orientations with cos(θ) = 0.4. The perpendicular orientation is unfavorable (5 kT above the minimum), while parallel orientation lies is only slightly less favorable then the optimal orientation (free energy cost: 1 kT). We can see that the preferred orientation (position of free energy minimum) changes with the increasing peptide length and with the choice of the model employed. In general, short peptides tend to be oriented perpendicular to the membrane plane, while the long peptides are more likely to be oriented tilted or parallel to the plane. Note that the peptides with parallel orientation tend to accumulate in the upper or lower part of the membrane edge rather than in the middle. Interestingly, for peptides with intermediate lengths (L = 5 nm), which is similar to membrane thickness, the preferred orientation depends the most on the attractiveness of the endcaps. Peptides with attractive ends prefer to be parallel to the

Figure 3. Representative snapshots of a single amphiphilic peptide at the membrane pore of an infinite size. Three different orientations of PSC toward the membrane plane are depicted (from left to right): perpendicular, tilted, and parallel. At parallel orientation of PSC are typically located in upper or lower half of the membrane. Color coding: orange = spherocylinder; gray = attractive patch; blue = lipid headgroup; red = lipid tails.

three different orientations are displayed. The free energy difference with the change of the peptide orientation with respect to the normal vector of the membrane plane was calculated for both models of patchy spherocylinders with attractive ends (AE-PSC) and with nonattractive ends (NEPSC). The obtained free energy profiles at the rim of a membrane pore with zero curvature are depicted in Figure 4. Short peptides (L = 3 nm) described by the AE-PSC model have an almost flat free energy profile, with a 1−3 kT minimum C

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core. Similarly, peptides with a hydrophobic part longer than the membrane thickness tend to tilt to fit within the hydrophobic part of membrane. Here we extend the simple hydrophobic mismatch picture to membrane pores, which are represented as cylindrical holes in hydrophobic layer. In this picture, peptides coating the pore adopt an orientation that minimizes hydrophobic mismatch. This simple geometrical argument is helpful to rationalize our observations but is clearly oversimplistic as the membrane can deform locally and change its properties, e.g., thickness and orientation lipids. Our coarse-grained model reproduces the results of earlier studies that reported the observation of perpendicular and tilted orientations of peptides in membrane pores.1,2,16,26−29 Illya et al. used a similar level of coarse-grained model to show that, depending on amount of hydrophobicity, rodlike helices form barrel-stave or toroidal pores.29 The studied helices had hydrophilic ends and thus corresponding to our PSC-NE model, which indeed prefers perpendicular or tilted orientation. However, we are not aware of evidence for a situation where peptides inside a pore align parallel to the membrane plane as do our model peptides with attractive ends. Yet, this possibility should be considered as there are natural proteins that are oriented along the membrane edge. For example, in nature, high-density lipoprotein particles, which is a membrane disk with two amphiphilic proteins, the proteins enclose the membrane disk, with an orientation parallel to the membrane plane. Of course, a disk is not the same as a pore, yet the observation suggests that there are situations where peptides in pores could be expected to be oriented parallel to the membrane plane. In what follows, we refer to such a structure as a “double-belt” pore following the terminology from the double-belt membrane disks. To explore whether a realistic mode could reproduce the formation of double-belt pores, we performed an higherresolution (MARTINI forcefield) simulation of protein Apo-A1 in a pore. This protein is known to stabilize double-belt membrane disks, and in our simulations we tested if it can also stabilize a membrane pore. The final snapshot of a 2 μs simulation is displayed in Figure 6. Even though distorted from the perfect double-belt pore this structure remained without substantial changes for 1 μs, which suggests at least local stability of the pore. The overall shape of the pore is determined by long helices, which remained parallel to the membrane plane. Short helices adopted various orientations in agreement with our more coarse-gained model (see Figure 6B). On the time scale of our “MARTINI” simulations, we could not observe the spontaneous breakup or the spontaneous formation of double-belt pores. To see that, we needed to use the (cheaper) coarse-grained model. Interestingly, we find that when we model protein of similar character as Apo-A1, i.e., protein made of few connected amphiphatic helices (PSC-AE), a double-belt pore form spontaneously. Initially, we placed one helical chain above each side of membrane, which resulted in fast symmetric membrane adsorption, and consequently a pore structure was spontaneously formed (for trajectory see Supporting Information or ref 47). This observation suggests that, at least for the coarse-grained model, the double-pelt pore is thermodynamically stable (see Figure 7). Moreover, as the more detailed simulations support the idea that double-belt pore can be longlived, it is clearly of interest to search experimentally for such structures in systems of proteins or peptides that have the same overall features as our coarse-grained model.

membrane plane, while peptides with nonattractive ends prefer to be oriented perpendicular to the membrane plane. In order to explore the implications of the above findings for membranes containing a higher peptide concentration, additional simulations were performed. A small number of peptides were placed on the membrane edge, where they were free to move and rotate. The resulting conformations are depicted in Figure 5A, and the observed orientations are in accord with the

Figure 5. Representative snapshots of peptides in the membrane pore of (A) infinite size and (B) radius of 4 nm. A cut through the middle of the finite pore is shown for clarity. Color coding is the same as in previous figures.

calculated free energy profiles in Figure 4. We verified that these findings are robust with respect to the detailed representation of the coarse-grained model using a bead model of amphiphilic peptides (see Supporting Information). To investigate the effect of the pore size, additional simulations were performed for a membrane with a pore that had radius about 4 nm. The final snapshots (Figure 5B) display that the pore curvature influences the orientation with enhanced preference of orientations perpendicular to the membrane plane. This can be easily understood since long peptides do not fit into small pores if they are orientated parallel to the membrane plane. Yet, also for finite-size pores the peptides with attractive ends (AE) are more likely to be parallel to the membrane plane than those described by the NE model. We stress that, if anything, our coarse-grained model is likely to underestimate the tendency of peptides in pores to align parallel to the membrane plane because our model peptides are rigid and cannot adjust to the curvature of the pores. In contrast, real proteins are flexible and even a single αhelix can bend. We can study the effect of flexibility by considering model peptides compose of several subunits. The behavior of such peptides in pores depends crucially on the bending rigidity of the connection between the subunits. We tested this by performing a simulations of a system of long peptides (6 nm) that were composed of two short (3 nm) peptides connected by a harmonic springs without any orientation restraints. As expected, we find that the orientational distribution of the subunits of these peptides in a pore differs from that of a rigid 6 nm peptide but is very similar to that of isolated 3 nm peptide. Part of our findings can be rationalized in terms of hydrophobic mismatch, which is well established for transmembrane proteins.43−46 In the hydrophobic mismatch picture, the protein tilt is determined by the fit of the hydrophobic part of the protein into the hydrophobic layer of the membrane D

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Figure 7. Top view of the final structure of a spontaneously formed double-belt pore obtained with our PSC coarse-grained model. Protein helices are colored in orange, and lipid head groups are blue. Color coding: orange = spherocylinder; gray = attractive patch; blue = lipid headgroup; red = lipid tails.

To complete our study, we constructed similar protein but made of PSC-NE model, which means that the ends of helices were not attractive (hydrophilic). Again, we placed one helical chain above each side of membrane and observed spontaneous formation of a pore. In this case, individual helices were oriented perpendicular or tilted to the membrane plane (for trajectory see Supporting Information or ref 48).

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CONCLUSIONS In summary, using coarse-grained simulations, we have studied the organization of amphiphilic peptides in pores in phospholipid membranes. We find that peptides can be oriented perpendicular, parallel, or tilted with respect to the membrane plane, depending on the length of the peptide and its hydrophobicity distribution. The tilt from a perpendicular orientation is larger for peptides with a longer hydrophobic part. This observation can be rationalized in terms of the hydrophobic mismatch. Surprisingly, we find that isolated peptides with hydrophobic ends and a length of 5 nm or more tend to align parallel to the membrane plane, yet inside the membrane pore. This orientation preference is also observed when there are multiple peptides in the pore. Our simulations suggest the existence of membrane pores coated with peptides or proteins oriented parallel to the membrane plane. We performed both coarse-grained and high-resolution simulations that support this hypothesis. In coarse-grained simulations, we even observe the spontaneous formation of such a “doublebelt” pore.

Figure 6. Final snapshot of the Apo-A1 protein in the pore of DPPC membrane after 2 μs simulation: (A, C) top view and (B) side view of a cut through the pore (a cut plane is displayed in part A as a black line and arrow shows the view direction). Long helices remained in parallel orientation with membrane in double-belt pore structure. DPPC lipids are displayed as tubes (licore) with color coding: cyan = hydrocarbons, tan = phosphate, and blue = choline. Helical part of protein backbone is depicted with orange. Residues are depicted with color representing their character (yellow = hydrophobic, red = positively charged, blue = negatively charged, green = polar). Helices are represented as orange spherocylinders in part C to clarify the similarity to our PSC model.

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ASSOCIATED CONTENT

S Supporting Information *

Figures S1−S5 and two movies. This material is available free of charge via the Internet at http://pubs.acs.org. E

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected], [email protected] (R.V.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work of RV was supported by European Regional Development Fund (CZ.1.05/1.1.00/02.0068 - project CEITEC) and by the EU Seventh Framework Programme (Contract No. 286154 - SYLICA project). D.F. acknowledges ERC support (Advanced Grant Agreement 227758), EPSRC Programme Grant EP/I001352/1, and support from a grant of the Royal Society of London (Wolfson Merit Award). We acknowledge computing facilities provided by MetaCentrum the program “Projects of Large Infrastructure for Research, Development, and Innovations” LM2010005.

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REFERENCES

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dx.doi.org/10.1021/la402727a | Langmuir XXXX, XXX, XXX−XXX