Article pubs.acs.org/Langmuir
Interplay between Curvature and Lateral Organization of Lipids and Peptides/Proteins in Model Membranes Qing-Yan Wu and Qing Liang* Center for Statistical and Theoretical Condensed Matter Physics and Department of Physics, Zhejiang Normal University, Jinhua 321004, PR China ABSTRACT: Membrane curvature plays a crucial role in the realization of many cellular membrane functions such as signaling and trafficking. Here, using coarse-grained molecular dynamics (MD) simulation, we present an effective method of producing curved model membranes and systematically investigated the interplay between the curvature and lateral sorting of lipids and transmembrane (TM) peptides/proteins in the model membranes. We first confirmed the experimental results of the lateral organization of lipid domains in curved ternary membranes. Then, we focused on exploring the lateral sorting of TM peptides/proteins with symmetric shape in the curved membranes. The results showed that the lateral inhomogeneous packing of lipids induced by the curvature and/or the component heterogeneity drives the peptides/proteins to accumulate in the curved regions in both the unary and ternary membranes. However, whether the peptides/proteins can stably and compactly reside in the curved regions is determined by their final packing configuration, which may be influenced by the membrane curvature in the curved regions. Additionally, the insertion of peptides/proteins may enhance the membrane curvature. This work provided some theoretical insights into understanding the mechanism of the interplay of membrane curvature and lateral organization (especially the lateral sorting of the peptides/proteins with symmetric shape) in the biomembrane in some biological processes.
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INTRODUCTION Membrane curvature is associated with many biological processes such as membrane fusion,1−3 membrane fission,4−6 membrane budding,7,8 and endocytosis.9−11 One of the most significant roles of membrane curvature in these biological processes is to control the lateral organization of the components in the membranes and the activity of the cell.8,12 For example, in endosomes, poorly degradable phospholipid lysobisphosphatidic acid is enriched in the membranes of the highly curved multivesicular elements.13−15 In the ternary supported model membranes and giant unilamellar vesicles (GUV), it was also found that the liquid-disordered (ld) domains enriched in unsaturated lipids with low bending rigidity spontaneously reside in the curved regions whereas the liquid-ordered (lo) domains enriched in saturated lipids and cholesterols with high bending rigidity spontaneously localize in the flat regions.16−19 Additionally, in the lipid bilayer containing two kinds of lipids with different effective shapes, the lipids with asymmetric shape preferentially localize in the curved regions.8,20−22 Besides the lipids, the membrane curvature may also affect the lateral sorting of proteins in the membranes. When the shape of the TM protein was conical, the protein was found to favor distributing in the curved regions of the membrane as a result of its shape asymmetry.8,23 Moreover, the membrane curvature can promote the aggregation of the TM proteins.24−26 © 2014 American Chemical Society
Conversely, the lateral sorting of lipids, the binding of peripheral proteins, and the insertion of asymmetric TM proteins may induce or enhance the curvature of the cellular membranes.8,12−14,23−27 Many biological functions of the cellular membranes are determined by the interplay of the membrane curvature and the lateral sorting of lipids and proteins in the membranes.28 However, because of the complexity of the structure and the dynamic property of the natural cellular membranes, the mechanism of the interplay between the membrane curvature and the lateral organization of the membranes remains elusive in many biological processes. In particular, in natural cellular membranes, many TM proteins, for instance, the 5-lipoxygenase-activating protein (FLAP),29 the bacteriorhodopsin (bR),30 the bacterial oxalate transporter (OXlT),31 and the sucrose-specific porin (ScrY),32 are cylindrical. How the lateral organization of these symmetric cylindrical TM proteins interplays with the membrane curvature was rarely examined in previous work. In this work, using coarse-grained molecular dynamics simulation, we investigated the interplay of the membrane curvature and the lateral sorting of TM peptides/proteins with symmetric shape and lipids in model membranes. First, we examined the lateral organization of a curved ternary lipid Received: October 9, 2013 Revised: December 11, 2013 Published: January 13, 2014 1116
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solved by 55 745 coarse-grained water beads whereas the ternary membrane is composed of 990 DPPC, 690 DLiPC, and 720 cholesterol molecules and solved by 52 703 coarse-grained water beads. To explore the interplay between membrane curvature and the lateral sorting of TM peptides (proteins) in the model membrane, a number of model WALP23 (GWW(LA)8LWWA) peptides and bR proteins were uniformly inserted into the above planar membranes, respectively. The effective shapes of WALP23 and bR are both cylindrical in the natural state, and each bR is composed of 7 TM αhelixes.39,42 In the initial state of the simulation, all of the TM peptides or proteins were perpendicular to the membrane plane. Because of the insertion of WALP23s or bR’s, some of the lipids and/or cholesterols would be overlapped by the peptides or bR’s, and these overlapped lipids and/or cholesterols must be removed before simulation. For each system, after a short energy minimization, the membrane was successively equilibrated by an NVT simulation and an NpT simulation at a high temperature between 323−330 K to form randomly laterally organizing and curved membranes as an initial state in the following simulation (e.g., Figure 1A). Once the disordered curved membrane was formed, the system was cooled to 295 K to perform the real simulations. For the pure DPPC membrane, the real gel−liquid transition temperature of DPPC is 314.4 K.43,44 However, in the MARTINI model, the gel−liquid transition temperature of DPPC has been estimated to be 295 K if both the system size and the simulation time are comparable to the experimental conditions.45,46 In the relatively small system examined in the present work, the gel− liquid transition temperature of DPPC was estimated to be 280−290 K.45−47 Thus, the pure DPPC membrane in the present system is in the fluid phase at 295 K. For the DPPC/DLiPC/cholesterol ternary membranes, the temperature of 295 K was proposed as an optimal temperature for the observation of lo/ld domain formation in the MARTINI model in ref 38. All systems simulated in this work are summarized in Table 1. Data Analysis. To examine the lateral sorting process of lipids, we calculated the variation in the fraction of various components in the curved region with the simulation time. To explore the effect of the insertion of WALP23 peptides on the curvature of the membranes, we compared the curvature of the curved regions of the membranes with and without WALP23. To measure the curvature of the curved regions of the membrane, we fitted the xz-plane projection points of the position of the beads at the ends of lipid tails to curved lines and then calculated the curvature of the curved part of the lines. In the present system, we focused on examining the sorting of lipids and peptides/ proteins in the curved membrane regions whose curvature is larger than 0.13 nm−1. Additionally, the packing density of the lipids in different regions was also calculated to reveal the influence of the curvature on the lateral organization of lipids.
bilayer consisted of saturated lipids, unsaturated lipids, and cholesterols and found as expected that the lo domains reside in the flat regions whereas the ld domains reside in the curved regions. This result is consistent with the relevant experimental results.16−18 Second, we examined the interplay between the curvature and lateral sorting of TM WALP23 peptides and bacteriorhodopsin (bR) proteins in the lipid bilayers. The results indicated that the lateral sorting of the peptides/proteins is cooperatively determined by the lateral inhomogeneous packing of lipids and the final effective packing configuration of peptides/proteins, and the insertion of the peptides/proteins can obviously influence the membrane curvature.
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MODEL AND METHODS
Molecular Dynamics Simulations with the MARTINI Force Field. All of the simulations were performed using the Gromacs package (version 4.6).33 The lipid, peptide, protein, cholesterol, and water molecules in the systems were described with the coarse-grained MARTINI force field, which uses a 4-to-1 mapping scheme to coarse grain the molecules; that is, on average 4 heavy atoms together with associated hydrogen atoms are represented by 1 interaction bead.34−38 In addition, the molecules diffuse about 4 times faster in the MARTINI model than in the real systems.34 Thus, the time in the experimental systems is approximately equal to the simulation time multiplied by a factor of 4. In this work, however, except for being pointed out explicitly, all of the times mentioned are the simulation times. In the MD simulation, the time step of integration was set as 20 fs. For the nonbonded interactions, the standard MARTINI interaction parameter scheme was adopted: the Coulomb interaction was shifted to zero between 0 and 1.2 nm, the Lennard-Jones interaction was shifted to zero between 0.9 and 1.2 nm, and the relative dielectric constant was set at 15. The systems were simulated with the constant particle number, pressure, and temperature (NpT) ensemble. The lipid, cholesterol, peptide (protein), and water molecules were separately coupled to a heat bath with a relaxation time of τT = 1 ps to maintain the system temperature at 295 K. The bilayer (xy) plane and the normal (z) direction were separately coupled to a 1 bar pressure bath with a relaxation time of τp = 2 ps to ensure that the lipid bilayer is tensionless.39 The compressibility was set to zero in the xy plane and to 3 × 10−5 bar−1 in the z direction. Thus, the box size can be varied only in the z direction, and the area and shape of the xy plane were fixed during the simulation.40,41 System Setup. In this work, the initial planar membranes were constructed using the methods proposed in ref 38. We mainly examined two kinds of membrane systems: the unary membrane composed of saturated dipalmitoyl-phosphatidylcholine (DPPC) and the ternary membrane composed of DPPC, unsaturated dilinoleoyl-PC (DLiPC), and cholesterol. To obtain a curved membrane, we first built up a planar lipid bilayer in the xy plane with the lipid packing density being 1.5−1.8 times larger than the lipid packing density in ref 38. Thus, the lateral pressure of the lipid bilayer is very high in the initial stage of the simulation. With the increase in simulation time, because the size of the simulation box in the xy plane is fixed, the membrane could not extend in the lateral (xy) plane and it would be bent in the normal (z) direction to release the tension in the lateral plane of the membrane and a curved membrane would be formed. The final morphology of the membrane is determined by the lipid packing density in the initial planar membrane. If the initial lipid packing density is not very high, then the membrane may include only one curved region. With the increase in the lipid packing density, two curved regions may appear in the membranes. In the present system, with the above-mentioned packing density, all membranes contain two curved regions. Additionally, the periodic boundary conditions were used in all three directions. To provide sufficient space for the extension of the membrane in the z direction and the formation of an ideal curved membrane, the simulation box in the z direction was set to 26 nm and the final size of the simulation box is about 33 × 10 × 26 nm3. The unary membrane contains 1680 DPPC molecules and is
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RESULTS AND DISCUSSION
Lateral Organization of Lipids in the Curved Ternary Lipid Bilayer without Peptides. First, we examined the lateral organization of lipid domains in the curved DPPC/ DLiPC/cholesterol ternary membrane, and the results are shown in Figure 1. As expected, the randomly laterally organizing membrane (Figure 1A) begins to undergo a phase separation at ∼1 μs (Figure 1B), and the ld domains enriched in DLiPC and the lo domains enriched in DPPC and cholesterol appear. For subsequent simulation times, the ld domains with a smaller bending rigidity gradually aggregate into the curved regions whereas the lo domains with a larger bending rigidity accumulate into the planar regions. At ∼9 μs of simulation, most of the ld domains resided in the curved regions whereas most of the lo domains were excluded to the planar regions, and this laterally organizing structure stably exists in the rest time of the simulation (Figure 1C). The demixing process of the lipids in the curved regions can also be 1117
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examined from the variation of the fraction of components over simulation time as shown in Figure 1D. The fraction of DLiPC in the curved regions increases from ∼0.3 in the initial stage to ∼0.8 in the later stage of the simulation, which indicates the self-organizing process of the ld domains in the curved regions. These results agree well with the relevant experimental results,16−18 and this agreement confirms the validity of the methods and model used in the present work. Lateral Sorting of TM Peptides/Proteins in Model Membranes. Next, we explored the effect of membrane curvature on the lateral sorting of TM peptides/proteins in the curved membranes. We first considered a DPPC lipid bilayer inserted with 30 WALP23 peptides. The WALP23s were randomly inserted into the membrane at the beginning of the simulation as shown in Figure 2A. With the increase in
Figure 1. Front view (upper) and top view (lower) of the snapshots for the DPPC/DLiPC/cholesterol ternary lipid bilayer at simulation times of (A) 0, (B) 1, and (C) 9 μs and (D) the time evolution of the fractions of the components in the curved regions. Here, DPPC, DLiPC, and cholesterol are shown in green, red, and gray, respectively. Note that this color scheme is used throughout the article. Additionally, for structural completeness, each snapshot contains two simulation box periods. In the following figures, all of the snapshots are shown with two simulation box periods.
Table 1. Overview of the Systems Simulated in This Work system (molecule number (ratio))
simulation time (μs)
DPPC/DLiPC/cholesterol (990:690:720) DPPC/DLiPC/cholesterol/WALP23 (750:510:540:60) DPPC (1680) DPPC/WALP23 (1470:30) DPPC/DLiPC/cholesterol/bR (771:508:587:6)
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Figure 2. Front and top views of the snapshots for the DPPC bilayer inserted with 30 WALP23s at simulation times of (A) 0, (B) 3, and (C) 8 μs and (D) the time evolution of the number of WALP23s in the curved regions. Here, WALP23 is shown in yellow.
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simulation time, some of the WALP23s aggregate together to form peptide clusters (Figure 2B). Most interestingly, after ∼7.5 μs, all of the WALP23 peptides partition into the curved regions of the membrane as shown in Figure 2C. The aggregation process of WALP23s in the curved regions is clearly presented in Figure 2D with the variation of the WALP23 number in the curved regions over simulation time. Here, we find that more and more WALP23s accumulate in the curved regions in the initial 7.5 μs. In the remaining simulation
8 8 20
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membrane and the asymmetric distribution of the lipids in the two membrane leaflets in the curved regions. The packing of lipids in the outer leaflet in the curved regions is loosened by the curvature. In the ternary DPPC/DLiPC/cholesterol membrane in the presence of TM peptides,42 it was found that if the peptides are inserted into the well-ordered lo domains, although the insertion of the TM peptides will disorder the lipids in the lo domains and increase the entropy of the lipids, the enthalpic penalty induced by the insertion of the peptides outweighs the increase in entropy. On the contrary, if the peptides are inserted into ld domains, then the enthalpic penalty is much smaller than that of inserting peptides into the ordered lo domains and the entropy of the lipid does not significantly increase. Therefore, WALP23 prefers to be distributed in the ld domains where the unsaturated DLiPCs are loosely packed compared to being distributed in the lo domains where the saturated DPPCs are packed in a compact, orderly manner. In the present system, although there are no ld domains in the membrane, the structural property of curved regions (especially the outer leaflet) where DPPCs are relatively loosely packed and disordered is similar to that of the ld domains in the ternary membrane. Therefore, WALP23s spontaneously aggregate in the curved region because of the effect of enthalpy. Additionally, we examined the packing configuration of the peptides in the curved regions. We found that most of the peptides are perpendicularly aligned to the membrane plane and no obvious tilting of the peptides with respect of the membrane normal was observed. Interestingly, because of the asymmetric packing of the lipids in the two membrane leaflets in the curved region, the configuration of WALP23 is drastically changed: in the outer leaflet where the packing of DPPC is loose, two tryptophanes at the end of WALP23 are stretched away from the backbone of the peptide whereas the two tryptophanes at the other end of WALP23 in the inner leaflet where the packing of DPPC is compact are folded toward the backbone of the peptides as shown in Figure 4A. As a result, the
time, the WALP23 number remains unchanged, which implies that the membrane structure with the peptides residing in the curved regions as shown in Figure 2C is stable and WALP23 favors being distributed in the curved regions of the unary DPPC membrane. In previous relevant work,8,23 it was proposed that the TM peptides/proteins with a conical or wedge shape may prefer to aggregate into curved regions of the membranes because of the asymmetry of their shape. However, WALP23 is a cylindrical TM peptide, and the residence preference of WALP23 in the curved regions could not be attributed to the effect of shape asymmetry. For the TM protein/peptide with a symmetric shape such as WALP23, possible reasons for the preference of the distribution of the peptides/proteins in the curved regions include the asymmetric distribution of peptides/proteins in the two leaflets of the membrane (i.e., the peptides/proteins are inserted into only one of the leaflets of the membranes) and the positive mismatch between the hydrophobic length of the peptides/ proteins and the hydrophobic thickness of the lipid bilayer (i.e., the hydrophobic length of the peptides/proteins is greater than the hydrophobic thickness of the lipid bilayer).26 To verify whether the preference of the residence of WALP23 in the curved regions is caused by these mechanisms, we examined the position of WALP23 in the membrane in the later stage of the simulation. We projected the positions of the beads at the configuration center of WALP23s and the beads at the end of tails of DPPC and DLiPC in the curved region onto the xz plane as shown in Figure 3 and found that the positions of the
Figure 3. xz-plane projection of the positions of the beads at the configuration center of WALP23s and the beads at the tail ends of the lipids in the upper curved region of the snapshot shown in Figure 2C.
WALP23 centers and the tail ends of the lipids coincide well with each other. This implies that the WALP23s symmetrically distribute in the two leaflets of the membranes during the simulation. Additionally, the DPPC bilayer thickness (∼3.27 nm) in the present system is larger than the hydrophobic length of WALP23 (∼2.55 nm).48 Therefore, the preference of the distribution of WALP23 in the curved regions in the present system also could not be attributed to the asymmetric distribution of the peptides and the hydrophobic mismatch effect. To explain the preference of the residence of cylindrical WALP23 in the curved regions of the membrane, we calculated the number density of DPPC in the planar and curved regions in the pure DPPC membrane presented in Table 1 and found that the number density of DPPC is ∼1.75 nm−2 in the planar regions, ∼1.45 nm−2 in the outer leaflet of the curved region, and ∼2.00 nm−2 in the inner leaflet of the curved regions. This means that the curvature of the membrane leads to both the lateral inhomogeneity of the lipid packing in the whole
Figure 4. Packing structures of (A) single WALP23, (B) a WALP23 dimer, and (C) a WALP23 trimer in the curved regions of the membrane. For clarity, expect for the tryptophanes, the other amino acids are not shown. The backbone of WALP23 is shown in pink, and the tryptophanes are shown in yellow.
effective shape of the WALP23 transfers from the symmetric cylinder to an asymmetric wedge. This asymmetric shape of WALP23 geometrically matches the asymmetric packing of DPPC in the two membrane leaflets in the curved regions and ensures that the peptides can be stably distributed in the curved regions. Besides the single WALP23 monomer, the configuration deformation of the WALP23 also leads to the WALP23 dimer (Figure 4B) and trimer (Figure 4C), and clusters containing more WALP23s (not shown) also display asymmetric packing configurations in the curved regions. 1119
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leaflet (∼1.56 nm2); therefore, the configuration of the peptides is also deformed into an asymmetric wedge shape as shown in Figure 4. Additionally, we also compared the curvature of the curved regions of the ternary membrane with and without WALP23 and found that the insertion of WALP23s also obviously influences the curvature of the membranes (Figure 7).
Consequently, we can conclude that the membrane curvature can not only mediate the lateral sorting of the TM peptides but also influence the packing configurations of the TM peptides. Furthermore, in Figure 2C, the curvatures of the upper and lower curved regions in the membrane are not completely identical, and the curvature of the upper curved regions is larger than that of the lower region. The curvature of the membrane may also provide a driving force for the aggregation of the WALP23 clusters. The larger the membrane curvature, the more compact the WALP23 aggregation. Therefore, the WALP23s aggregate more compactly in the upper curved region with larger curvature than in the lower curved region with smaller curvature as shown in Figure 2C. To explore the influence of TM peptide insertion on the membrane curvature, we compared the curvature of the pure DPPC bilayer with that of the DPPC bilayer in the presence of 30 WALP23s, and the results are shown in Figure 5. We found
Figure 7. Variation of the curvature of the upper curved regions in the DPPC/DLiPC/cholesterol ternary membrane and the DPPC/DLiPC/ cholesterol membrane inserted with 60 WALP23s over the simulation time.
Finally, we examined the lateral sorting of bR proteins in the curved DPPC/DLiPC/cholesterol ternary membrane (Figure 8). In previous work,39,49 it was found that bR’s prefer to reside Figure 5. Variation of the curvature of the upper curved regions in the pure DPPC membrane and the DPPC membrane inserted with 30 WALP23s over the simulation time.
that the curvature of the DPPC bilayer inserted with WALP23s is obviously larger than that of the pure DPPC bilayer in the later stage of the simulation. Therefore, the insertion of the TM peptides with asymmetric packing configurations in the curved regions enhances the asymmetry of the two membrane leaflets and bends the membrane more strongly. For completeness and comparison with the results of the DPPC/DLiPC/cholesterol ternary curved membrane, we also examined the lateral organization of the curved DPPC/DLiPC/ cholesterol membrane inserted with 60 WALP23 peptides. Because the lipids are packed more loosely in the ld domain than in the lo domains and the bending rigidity of ld domains is small,16−18 the ld domains together with the majority of WALP23s expectedly reside in the curved regions whereas the lo domains are distributed in the planar regions of the membranes as shown in Figure 6. The membrane curvature also induces the density of the DLiPC in the outer leaflet of the curved regions (∼1.24 nm2) to be smaller than that in the inner
Figure 8. Front and top views of the snapshots for the DPPC/DLiPC/ cholesterol membrane inserted with six bR’s at simulation times of (A) 0, (B) 6, and (C) 20 μs. Here, bR is shown in yellow.
in the ld domains because of the enthalpic effect. Because of this enthalpic affinity between bR’s and ld domains, the randomly distributed bR’s as shown in Figure 8A also tend to accumulate into the curved regions enriched in ld domains (Figure 8B). However, different from the cases of curved unary and ternary membranes inserted with WALP23s as shown in Figures 2 and 6, even after a 20 μs simulation, some bR’s do not yet completely partition into the upper curved regions with large curvature but reside on the boundary of the lo and ld
Figure 6. Front and top views of the final snapshot of the 12 μs simulation on the DPPC/DLiPC/cholesterol lipid bilayer inserted with 60 WALP23s. 1120
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Langmuir domains (Figure 8C). A careful examination of the packing configuration of bR’s in the curved regions with large curvature reveals that the packing configuration of bR’s is nearly uninfluenced by the membrane curvature and remains symmetric (Figure 8C). Thus, the bR clusters do not match well and even will disturb the asymmetric packing of the lipids in the curved regions, and some bR’s are excluded from the curved regions as shown in Figure 8C.
ACKNOWLEDGMENTS
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REFERENCES
This work was supported by the National Natural Science Foundation of China under grant no. 11004173 and the Starting Research Fund for Doctors from Zhejiang Normal University. We thank Dr. Wen-De Tian for helpful discussions. The computational resources provided by the Department of Computer Science and Technology at Zhejiang Normal University is greatly appreciated.
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CONCLUSIONS In this work, we presented an effective method to build up curved membranes and investigated the interplay between the curvature and the lateral organization of lipids and TM peptides/proteins in the curved model membranes using coarse-grained molecular dynamics simulations. In the DPPC/DLiPC/cholesterol ternary membrane, the ld domains with a smaller bending rigidity prefer to distribute in the curved regions whereas the lo domains with a larger bending rigidity favor residence in the planar regions of the membrane. This result agrees very well with the experimental results. In the curved membrane inserted with cylindrical model peptide WALP23s, on the one hand, in both the unary and ternary membranes, the lipids are packed inhomogeneously in the lateral plane of the membrane, and this inhomogeneity in the lateral distribution of lipids provides a driving force for the aggregation of WALP23s in the curved regions. On the other hand, the membrane curvature also leads to an asymmetric packing of the lipids in the outer and inner membrane leaflets in the curved regions, and the lipids are packed more loosely in the outer leaflet than in the inner leaflet. To match this lipid packing asymmetry, the packing configuration of the peptides or peptide clusters transfers from a symmetric cylinder to an asymmetric wedge. This configuration deformation stabilizes the residence of the peptides in the curved regions. In the membrane inserted with larger bR proteins, the configuration of the bR’s is hardly influenced by the membrane curvature, and the bR’s cannot stably accumulate in the curved regions but distribute in both the curved regions and on the boundary of the lo and ld domains. Comparing the lateral sorting of WALP23s and bR’s in the curved membrane, we can conclude that the configuration deformation is crucial to the stable residence of TM peptides or proteins in the curved regions of the membranes. Furthermore, the membrane curvature also provides a driving force for the aggregation of peptides/ proteins in the curved regions. The larger the membrane curvature, the more compactly the peptides/proteins aggregate. Besides the influence of the curvature on the lateral organization of the lipids and peptides or proteins, the insertion of the peptides or proteins can conversely enhance the curvature of the membranes. This work provides a possible mechanism for the interplay between the curvature and the lateral organization of lipids and peptides/proteins (especially the lateral sorting of TM peptides/proteins) in the curved membranes and a theoretical reference for relevant experimental studies.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Notes
The authors declare no competing financial interest. 1121
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Separation on Curvature-Driven Lipid Sorting. J. Biophys. Chem. 2011, 2, 268−284. (20) Callan-Jones, A.; Sorre, B.; Bassereau, P. Curvature-Driven Lipid Sorting in Biomembranes. Cold Spring Harbor Perspect. Biol. 2011, 3, a004648. (21) Liang, Q.; Ma, Y.-q. Curvature-Induced Lateral Organization in Mixed Lipid Bilayers Supported on a Corrugated Substrate. J. Phys. Chem. B 2009, 113, 8049−8055. (22) Cooke, I. R.; Deserno, M. Coupling between Lipid Shape and Membrane Curvature. Biophys. J. 2006, 91, 487−495. (23) Phillips, R.; Ursell, T.; Wiggins, P.; Sens, P. Emerging Roles for Lipids in Shaping Membrane-Protein Function. Nature 2009, 459, 379−385. (24) Reynwar, B. J.; Illya, G.; Harmandaris, V. A.; Muller, M. M.; Kremer, K.; Deserno, M. Aggregation and Vesiculation of Membrane Proteins by Curvature-Mediated Interactions. Nature 2007, 447, 461− 464. (25) Parton, D. L.; Klingelhoefer, J. W.; Sansom, M. S. Aggregation of Model Membrane Proteins, Modulated by Hydrophobic Mismatch, Membrane Curvature, and Protein Class. Biophys. J. 2011, 101, 691− 699. (26) Yue, T.; Li, S.; Zhang, X.; Wang, W. The Relationship Between Membrane Curvature Generation and Clustering of Anchored Proteins: A Computer Simulation Study. Soft Matter 2010, 6, 6109− 6118. (27) Antonny, B. Membrane Deformation by Protein Coats. Curr. Opin. Cell Biol. 2006, 18, 386−394. (28) Cho, W.; Stahelin, R. V. Membrane-Protein Interaction in Cell Signaling and Membrane Trafficking. Annu. Rev. Biophys. Biomol. Struct. 2005, 34, 119−151. (29) Ferguson, A. D.; McKeever, B. M.; Xu, S.; Wisniewski, D.; Miller, D. K.; Yamin, T.-T.; Spencer, R. H.; Chu, L.; Ujjainwalla, F.; Cunningham, B. R.; Evans, J. F.; Becker, J. W. Crystal Structure of Inhibitor-Bound Human 5-Lipoxygenase-Activating Protein. Science 2007, 317, 510−512. (30) Lewis, B. A.; Engelman, D. M. Bacteriorhodopsin Remains Dispersed in Fluid Phospholipid Bilayers over a Wide Range of Bilayer Thicknesses. J. Mol. Biol. 1983, 166, 203−210. (31) Hirai, T.; Heymann, J. A.; Shi, D.; Sarker, R.; Maloney, P. C.; Subramaniam, S. Three-Dimensional Structure of a Bacterial Oxalate Transporter. Nat. Struct. Biol. 2002, 9, 597−600. (32) Forst, D.; Welte, W.; Wacker, T.; Diederichs, K. Structure of the Sucrose-Specific Porin ScrY from Salmonella Typhimurium and Its Complex with Sucrose. Nat. Struct. Mol. Biol. 1998, 5, 37−46. (33) Hess, B.; Kutzner, C.; Van Der Spoel, D.; Lindahl, E. GROMACS 4: Algorithms for Highly Efficient, Load-Balanced, and Scalable Molecular Simulation. J. Chem. Theory Comput. 2008, 4, 435− 447. (34) Marrink, S. J.; Risselada, H. J.; Yefimov, S.; Tieleman, D. P.; de Vries, A. H. The MARTINI Force Field: Coarse Grained Model for Biomolecular Simulations. J. Phys. Chem. B 2007, 111, 7812−7824. (35) Monticelli, L.; Kandasamy, S. K.; Periole, X.; Larson, R. G.; Tieleman, D. P.; Marrink, S. J. The MARTINI Coarse-Grained Force Field: Extension to Proteins. J. Chem. Theory Comput. 2008, 4, 819− 834. (36) Periole, X.; Marrink, S.-J. In Biomolecular Simulations: Methods and Protocols; Monticelli, L., Salonen, E., Eds.; Methods in Molecular Biology; Humana Press: New York, 2013; Vol. 924, pp 533−565. (37) Marrink, S. J.; Tieleman, D. P. Perspective on the Martini Model. Chem. Soc. Rev. 2013, 42, 6801−6822. (38) Risselada, H. J.; Marrink, S. J. The Molecular Face of Lipid Rafts in Model Membranes. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 17367− 17372. (39) Domański, J.; Marrink, S. J.; Schäfer, L. V. Transmembrane Helices Can Induce Domain Formation in Crowded Model Membranes. Biochim. Biophys. Acta, Biomembr. 2012, 1818, 984−994. (40) Wang, H.; De Joannis, J.; Jiang, Y.; Gaulding, J. C.; Albrecht, B.; Yin, F.; Khanna, K.; Kindt, J. T. Bilayer Edge and Curvature Effects on
Partitioning of Lipids by Tail Length: Atomistic Simulations. Biophys. J. 2008, 95, 2647−2657. (41) Jiang, Y.; Kindt, J. T. Simulations of Edge Behavior in a MixedLipid Bilayer: Fluctuation Analysis. J. Chem. Phys. 2007, 126, 045105− 045105. (42) Schäfer, L. V.; de Jong, D. H.; Holt, A.; Rzepiela, A. J.; de Vries, A. H.; Poolman, B.; Killian, J. A.; Marrink, S. J. Lipid Packing Drives the Segregation of Transmembrane Helices into Disordered Lipid Domains in Model Membranes. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 1343−1348. (43) Lewis, R. N. A. H.; Mak, N.; McElhaney, R. N. A Differential Scanning Calorimetric Study of the Thermotropic Phase Behavior of Model Membranes Composed of Phosphatidylcholines Containing Linear Saturated Fatty Acyl Chains. Biochemistry 1987, 26, 6118− 6126. (44) De Young, L. R.; Dill, K. A. Solute Partitioning into Lipid Bilayer Membranes. Biochemistry 1988, 27, 5281−5289. (45) Marrink, S. J.; Risselada, J.; Mark, A. E. Simulation of Gel Phase Formation and Melting in Lipid Bilayers Using a Coarse Grained Model. Chem. Phys. Lipids 2005, 135, 223−244. (46) Marrink, S. J.; de Vries, A. H.; Mark, A. E. Coarse Grained Model for Semiquantitative Lipid Simulations. J. Phys. Chem. B 2004, 108, 750−760. (47) Prates Ramalho, J. P.; Gkeka, P.; Sarkisov, L. Structure and Phase Transformations of DPPC Lipid Bilayers in the Presence of Nanoparticles: Insights from Coarse-Grained Molecular Dynamics Simulations. Langmuir 2011, 27, 3723−3730. (48) Kim, T.; Im, W. Revisiting Hydrophobic Mismatch with Free Energy Simulation Studies of Transmembrane Helix Tilt and Rotation. Biophys. J. 2010, 99, 175−183. (49) Kahya, N.; Brown, D. A.; Schwille, P. Raft Partitioning and Dynamic Behavior of Human Placental Alkaline Phosphatase in Giant Unilamellar Vesicles. Biochemistry 2005, 44, 7479−7489.
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Interplay between Curvature and Lateral Organization of Lipids and Peptides/Proteins in Model Membranes Qing-Yan Wu and Qing Liang* Center for Statistical and Theoretical Condensed Matter Physics and Department of Physics, Zhejiang Normal University, Jinhua 321004, PR China ABSTRACT: Membrane curvature plays a crucial role in the realization of many cellular membrane functions such as signaling and trafficking. Here, using coarse-grained molecular dynamics (MD) simulation, we present an effective method of producing curved model membranes and systematically investigated the interplay between the curvature and lateral sorting of lipids and transmembrane (TM) peptides/proteins in the model membranes. We first confirmed the experimental results of the lateral organization of lipid domains in curved ternary membranes. Then, we focused on exploring the lateral sorting of TM peptides/proteins with symmetric shape in the curved membranes. The results showed that the lateral inhomogeneous packing of lipids induced by the curvature and/or the component heterogeneity drives the peptides/proteins to accumulate in the curved regions in both the unary and ternary membranes. However, whether the peptides/proteins can stably and compactly reside in the curved regions is determined by their final packing configuration, which may be influenced by the membrane curvature in the curved regions. Additionally, the insertion of peptides/proteins may enhance the membrane curvature. This work provided some theoretical insights into understanding the mechanism of the interplay of membrane curvature and lateral organization (especially the lateral sorting of the peptides/proteins with symmetric shape) in the biomembrane in some biological processes.
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INTRODUCTION Membrane curvature is associated with many biological processes such as membrane fusion,1−3 membrane fission,4−6 membrane budding,7,8 and endocytosis.9−11 One of the most significant roles of membrane curvature in these biological processes is to control the lateral organization of the components in the membranes and the activity of the cell.8,12 For example, in endosomes, poorly degradable phospholipid lysobisphosphatidic acid is enriched in the membranes of the highly curved multivesicular elements.13−15 In the ternary supported model membranes and giant unilamellar vesicles (GUV), it was also found that the liquid-disordered (ld) domains enriched in unsaturated lipids with low bending rigidity spontaneously reside in the curved regions whereas the liquid-ordered (lo) domains enriched in saturated lipids and cholesterols with high bending rigidity spontaneously localize in the flat regions.16−19 Additionally, in the lipid bilayer containing two kinds of lipids with different effective shapes, the lipids with asymmetric shape preferentially localize in the curved regions.8,20−22 Besides the lipids, the membrane curvature may also affect the lateral sorting of proteins in the membranes. When the shape of the TM protein was conical, the protein was found to favor distributing in the curved regions of the membrane as a result of its shape asymmetry.8,23 Moreover, the membrane curvature can promote the aggregation of the TM proteins.24−26 © 2014 American Chemical Society
Conversely, the lateral sorting of lipids, the binding of peripheral proteins, and the insertion of asymmetric TM proteins may induce or enhance the curvature of the cellular membranes.8,12−14,23−27 Many biological functions of the cellular membranes are determined by the interplay of the membrane curvature and the lateral sorting of lipids and proteins in the membranes.28 However, because of the complexity of the structure and the dynamic property of the natural cellular membranes, the mechanism of the interplay between the membrane curvature and the lateral organization of the membranes remains elusive in many biological processes. In particular, in natural cellular membranes, many TM proteins, for instance, the 5-lipoxygenase-activating protein (FLAP),29 the bacteriorhodopsin (bR),30 the bacterial oxalate transporter (OXlT),31 and the sucrose-specific porin (ScrY),32 are cylindrical. How the lateral organization of these symmetric cylindrical TM proteins interplays with the membrane curvature was rarely examined in previous work. In this work, using coarse-grained molecular dynamics simulation, we investigated the interplay of the membrane curvature and the lateral sorting of TM peptides/proteins with symmetric shape and lipids in model membranes. First, we examined the lateral organization of a curved ternary lipid Received: October 9, 2013 Revised: December 11, 2013 Published: January 13, 2014 1116
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solved by 55 745 coarse-grained water beads whereas the ternary membrane is composed of 990 DPPC, 690 DLiPC, and 720 cholesterol molecules and solved by 52 703 coarse-grained water beads. To explore the interplay between membrane curvature and the lateral sorting of TM peptides (proteins) in the model membrane, a number of model WALP23 (GWW(LA)8LWWA) peptides and bR proteins were uniformly inserted into the above planar membranes, respectively. The effective shapes of WALP23 and bR are both cylindrical in the natural state, and each bR is composed of 7 TM αhelixes.39,42 In the initial state of the simulation, all of the TM peptides or proteins were perpendicular to the membrane plane. Because of the insertion of WALP23s or bR’s, some of the lipids and/or cholesterols would be overlapped by the peptides or bR’s, and these overlapped lipids and/or cholesterols must be removed before simulation. For each system, after a short energy minimization, the membrane was successively equilibrated by an NVT simulation and an NpT simulation at a high temperature between 323−330 K to form randomly laterally organizing and curved membranes as an initial state in the following simulation (e.g., Figure 1A). Once the disordered curved membrane was formed, the system was cooled to 295 K to perform the real simulations. For the pure DPPC membrane, the real gel−liquid transition temperature of DPPC is 314.4 K.43,44 However, in the MARTINI model, the gel−liquid transition temperature of DPPC has been estimated to be 295 K if both the system size and the simulation time are comparable to the experimental conditions.45,46 In the relatively small system examined in the present work, the gel− liquid transition temperature of DPPC was estimated to be 280−290 K.45−47 Thus, the pure DPPC membrane in the present system is in the fluid phase at 295 K. For the DPPC/DLiPC/cholesterol ternary membranes, the temperature of 295 K was proposed as an optimal temperature for the observation of lo/ld domain formation in the MARTINI model in ref 38. All systems simulated in this work are summarized in Table 1. Data Analysis. To examine the lateral sorting process of lipids, we calculated the variation in the fraction of various components in the curved region with the simulation time. To explore the effect of the insertion of WALP23 peptides on the curvature of the membranes, we compared the curvature of the curved regions of the membranes with and without WALP23. To measure the curvature of the curved regions of the membrane, we fitted the xz-plane projection points of the position of the beads at the ends of lipid tails to curved lines and then calculated the curvature of the curved part of the lines. In the present system, we focused on examining the sorting of lipids and peptides/ proteins in the curved membrane regions whose curvature is larger than 0.13 nm−1. Additionally, the packing density of the lipids in different regions was also calculated to reveal the influence of the curvature on the lateral organization of lipids.
bilayer consisted of saturated lipids, unsaturated lipids, and cholesterols and found as expected that the lo domains reside in the flat regions whereas the ld domains reside in the curved regions. This result is consistent with the relevant experimental results.16−18 Second, we examined the interplay between the curvature and lateral sorting of TM WALP23 peptides and bacteriorhodopsin (bR) proteins in the lipid bilayers. The results indicated that the lateral sorting of the peptides/proteins is cooperatively determined by the lateral inhomogeneous packing of lipids and the final effective packing configuration of peptides/proteins, and the insertion of the peptides/proteins can obviously influence the membrane curvature.
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MODEL AND METHODS
Molecular Dynamics Simulations with the MARTINI Force Field. All of the simulations were performed using the Gromacs package (version 4.6).33 The lipid, peptide, protein, cholesterol, and water molecules in the systems were described with the coarse-grained MARTINI force field, which uses a 4-to-1 mapping scheme to coarse grain the molecules; that is, on average 4 heavy atoms together with associated hydrogen atoms are represented by 1 interaction bead.34−38 In addition, the molecules diffuse about 4 times faster in the MARTINI model than in the real systems.34 Thus, the time in the experimental systems is approximately equal to the simulation time multiplied by a factor of 4. In this work, however, except for being pointed out explicitly, all of the times mentioned are the simulation times. In the MD simulation, the time step of integration was set as 20 fs. For the nonbonded interactions, the standard MARTINI interaction parameter scheme was adopted: the Coulomb interaction was shifted to zero between 0 and 1.2 nm, the Lennard-Jones interaction was shifted to zero between 0.9 and 1.2 nm, and the relative dielectric constant was set at 15. The systems were simulated with the constant particle number, pressure, and temperature (NpT) ensemble. The lipid, cholesterol, peptide (protein), and water molecules were separately coupled to a heat bath with a relaxation time of τT = 1 ps to maintain the system temperature at 295 K. The bilayer (xy) plane and the normal (z) direction were separately coupled to a 1 bar pressure bath with a relaxation time of τp = 2 ps to ensure that the lipid bilayer is tensionless.39 The compressibility was set to zero in the xy plane and to 3 × 10−5 bar−1 in the z direction. Thus, the box size can be varied only in the z direction, and the area and shape of the xy plane were fixed during the simulation.40,41 System Setup. In this work, the initial planar membranes were constructed using the methods proposed in ref 38. We mainly examined two kinds of membrane systems: the unary membrane composed of saturated dipalmitoyl-phosphatidylcholine (DPPC) and the ternary membrane composed of DPPC, unsaturated dilinoleoyl-PC (DLiPC), and cholesterol. To obtain a curved membrane, we first built up a planar lipid bilayer in the xy plane with the lipid packing density being 1.5−1.8 times larger than the lipid packing density in ref 38. Thus, the lateral pressure of the lipid bilayer is very high in the initial stage of the simulation. With the increase in simulation time, because the size of the simulation box in the xy plane is fixed, the membrane could not extend in the lateral (xy) plane and it would be bent in the normal (z) direction to release the tension in the lateral plane of the membrane and a curved membrane would be formed. The final morphology of the membrane is determined by the lipid packing density in the initial planar membrane. If the initial lipid packing density is not very high, then the membrane may include only one curved region. With the increase in the lipid packing density, two curved regions may appear in the membranes. In the present system, with the above-mentioned packing density, all membranes contain two curved regions. Additionally, the periodic boundary conditions were used in all three directions. To provide sufficient space for the extension of the membrane in the z direction and the formation of an ideal curved membrane, the simulation box in the z direction was set to 26 nm and the final size of the simulation box is about 33 × 10 × 26 nm3. The unary membrane contains 1680 DPPC molecules and is
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RESULTS AND DISCUSSION
Lateral Organization of Lipids in the Curved Ternary Lipid Bilayer without Peptides. First, we examined the lateral organization of lipid domains in the curved DPPC/ DLiPC/cholesterol ternary membrane, and the results are shown in Figure 1. As expected, the randomly laterally organizing membrane (Figure 1A) begins to undergo a phase separation at ∼1 μs (Figure 1B), and the ld domains enriched in DLiPC and the lo domains enriched in DPPC and cholesterol appear. For subsequent simulation times, the ld domains with a smaller bending rigidity gradually aggregate into the curved regions whereas the lo domains with a larger bending rigidity accumulate into the planar regions. At ∼9 μs of simulation, most of the ld domains resided in the curved regions whereas most of the lo domains were excluded to the planar regions, and this laterally organizing structure stably exists in the rest time of the simulation (Figure 1C). The demixing process of the lipids in the curved regions can also be 1117
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examined from the variation of the fraction of components over simulation time as shown in Figure 1D. The fraction of DLiPC in the curved regions increases from ∼0.3 in the initial stage to ∼0.8 in the later stage of the simulation, which indicates the self-organizing process of the ld domains in the curved regions. These results agree well with the relevant experimental results,16−18 and this agreement confirms the validity of the methods and model used in the present work. Lateral Sorting of TM Peptides/Proteins in Model Membranes. Next, we explored the effect of membrane curvature on the lateral sorting of TM peptides/proteins in the curved membranes. We first considered a DPPC lipid bilayer inserted with 30 WALP23 peptides. The WALP23s were randomly inserted into the membrane at the beginning of the simulation as shown in Figure 2A. With the increase in
Figure 1. Front view (upper) and top view (lower) of the snapshots for the DPPC/DLiPC/cholesterol ternary lipid bilayer at simulation times of (A) 0, (B) 1, and (C) 9 μs and (D) the time evolution of the fractions of the components in the curved regions. Here, DPPC, DLiPC, and cholesterol are shown in green, red, and gray, respectively. Note that this color scheme is used throughout the article. Additionally, for structural completeness, each snapshot contains two simulation box periods. In the following figures, all of the snapshots are shown with two simulation box periods.
Table 1. Overview of the Systems Simulated in This Work system (molecule number (ratio))
simulation time (μs)
DPPC/DLiPC/cholesterol (990:690:720) DPPC/DLiPC/cholesterol/WALP23 (750:510:540:60) DPPC (1680) DPPC/WALP23 (1470:30) DPPC/DLiPC/cholesterol/bR (771:508:587:6)
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Figure 2. Front and top views of the snapshots for the DPPC bilayer inserted with 30 WALP23s at simulation times of (A) 0, (B) 3, and (C) 8 μs and (D) the time evolution of the number of WALP23s in the curved regions. Here, WALP23 is shown in yellow.
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simulation time, some of the WALP23s aggregate together to form peptide clusters (Figure 2B). Most interestingly, after ∼7.5 μs, all of the WALP23 peptides partition into the curved regions of the membrane as shown in Figure 2C. The aggregation process of WALP23s in the curved regions is clearly presented in Figure 2D with the variation of the WALP23 number in the curved regions over simulation time. Here, we find that more and more WALP23s accumulate in the curved regions in the initial 7.5 μs. In the remaining simulation
8 8 20
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membrane and the asymmetric distribution of the lipids in the two membrane leaflets in the curved regions. The packing of lipids in the outer leaflet in the curved regions is loosened by the curvature. In the ternary DPPC/DLiPC/cholesterol membrane in the presence of TM peptides,42 it was found that if the peptides are inserted into the well-ordered lo domains, although the insertion of the TM peptides will disorder the lipids in the lo domains and increase the entropy of the lipids, the enthalpic penalty induced by the insertion of the peptides outweighs the increase in entropy. On the contrary, if the peptides are inserted into ld domains, then the enthalpic penalty is much smaller than that of inserting peptides into the ordered lo domains and the entropy of the lipid does not significantly increase. Therefore, WALP23 prefers to be distributed in the ld domains where the unsaturated DLiPCs are loosely packed compared to being distributed in the lo domains where the saturated DPPCs are packed in a compact, orderly manner. In the present system, although there are no ld domains in the membrane, the structural property of curved regions (especially the outer leaflet) where DPPCs are relatively loosely packed and disordered is similar to that of the ld domains in the ternary membrane. Therefore, WALP23s spontaneously aggregate in the curved region because of the effect of enthalpy. Additionally, we examined the packing configuration of the peptides in the curved regions. We found that most of the peptides are perpendicularly aligned to the membrane plane and no obvious tilting of the peptides with respect of the membrane normal was observed. Interestingly, because of the asymmetric packing of the lipids in the two membrane leaflets in the curved region, the configuration of WALP23 is drastically changed: in the outer leaflet where the packing of DPPC is loose, two tryptophanes at the end of WALP23 are stretched away from the backbone of the peptide whereas the two tryptophanes at the other end of WALP23 in the inner leaflet where the packing of DPPC is compact are folded toward the backbone of the peptides as shown in Figure 4A. As a result, the
time, the WALP23 number remains unchanged, which implies that the membrane structure with the peptides residing in the curved regions as shown in Figure 2C is stable and WALP23 favors being distributed in the curved regions of the unary DPPC membrane. In previous relevant work,8,23 it was proposed that the TM peptides/proteins with a conical or wedge shape may prefer to aggregate into curved regions of the membranes because of the asymmetry of their shape. However, WALP23 is a cylindrical TM peptide, and the residence preference of WALP23 in the curved regions could not be attributed to the effect of shape asymmetry. For the TM protein/peptide with a symmetric shape such as WALP23, possible reasons for the preference of the distribution of the peptides/proteins in the curved regions include the asymmetric distribution of peptides/proteins in the two leaflets of the membrane (i.e., the peptides/proteins are inserted into only one of the leaflets of the membranes) and the positive mismatch between the hydrophobic length of the peptides/ proteins and the hydrophobic thickness of the lipid bilayer (i.e., the hydrophobic length of the peptides/proteins is greater than the hydrophobic thickness of the lipid bilayer).26 To verify whether the preference of the residence of WALP23 in the curved regions is caused by these mechanisms, we examined the position of WALP23 in the membrane in the later stage of the simulation. We projected the positions of the beads at the configuration center of WALP23s and the beads at the end of tails of DPPC and DLiPC in the curved region onto the xz plane as shown in Figure 3 and found that the positions of the
Figure 3. xz-plane projection of the positions of the beads at the configuration center of WALP23s and the beads at the tail ends of the lipids in the upper curved region of the snapshot shown in Figure 2C.
WALP23 centers and the tail ends of the lipids coincide well with each other. This implies that the WALP23s symmetrically distribute in the two leaflets of the membranes during the simulation. Additionally, the DPPC bilayer thickness (∼3.27 nm) in the present system is larger than the hydrophobic length of WALP23 (∼2.55 nm).48 Therefore, the preference of the distribution of WALP23 in the curved regions in the present system also could not be attributed to the asymmetric distribution of the peptides and the hydrophobic mismatch effect. To explain the preference of the residence of cylindrical WALP23 in the curved regions of the membrane, we calculated the number density of DPPC in the planar and curved regions in the pure DPPC membrane presented in Table 1 and found that the number density of DPPC is ∼1.75 nm−2 in the planar regions, ∼1.45 nm−2 in the outer leaflet of the curved region, and ∼2.00 nm−2 in the inner leaflet of the curved regions. This means that the curvature of the membrane leads to both the lateral inhomogeneity of the lipid packing in the whole
Figure 4. Packing structures of (A) single WALP23, (B) a WALP23 dimer, and (C) a WALP23 trimer in the curved regions of the membrane. For clarity, expect for the tryptophanes, the other amino acids are not shown. The backbone of WALP23 is shown in pink, and the tryptophanes are shown in yellow.
effective shape of the WALP23 transfers from the symmetric cylinder to an asymmetric wedge. This asymmetric shape of WALP23 geometrically matches the asymmetric packing of DPPC in the two membrane leaflets in the curved regions and ensures that the peptides can be stably distributed in the curved regions. Besides the single WALP23 monomer, the configuration deformation of the WALP23 also leads to the WALP23 dimer (Figure 4B) and trimer (Figure 4C), and clusters containing more WALP23s (not shown) also display asymmetric packing configurations in the curved regions. 1119
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leaflet (∼1.56 nm2); therefore, the configuration of the peptides is also deformed into an asymmetric wedge shape as shown in Figure 4. Additionally, we also compared the curvature of the curved regions of the ternary membrane with and without WALP23 and found that the insertion of WALP23s also obviously influences the curvature of the membranes (Figure 7).
Consequently, we can conclude that the membrane curvature can not only mediate the lateral sorting of the TM peptides but also influence the packing configurations of the TM peptides. Furthermore, in Figure 2C, the curvatures of the upper and lower curved regions in the membrane are not completely identical, and the curvature of the upper curved regions is larger than that of the lower region. The curvature of the membrane may also provide a driving force for the aggregation of the WALP23 clusters. The larger the membrane curvature, the more compact the WALP23 aggregation. Therefore, the WALP23s aggregate more compactly in the upper curved region with larger curvature than in the lower curved region with smaller curvature as shown in Figure 2C. To explore the influence of TM peptide insertion on the membrane curvature, we compared the curvature of the pure DPPC bilayer with that of the DPPC bilayer in the presence of 30 WALP23s, and the results are shown in Figure 5. We found
Figure 7. Variation of the curvature of the upper curved regions in the DPPC/DLiPC/cholesterol ternary membrane and the DPPC/DLiPC/ cholesterol membrane inserted with 60 WALP23s over the simulation time.
Finally, we examined the lateral sorting of bR proteins in the curved DPPC/DLiPC/cholesterol ternary membrane (Figure 8). In previous work,39,49 it was found that bR’s prefer to reside Figure 5. Variation of the curvature of the upper curved regions in the pure DPPC membrane and the DPPC membrane inserted with 30 WALP23s over the simulation time.
that the curvature of the DPPC bilayer inserted with WALP23s is obviously larger than that of the pure DPPC bilayer in the later stage of the simulation. Therefore, the insertion of the TM peptides with asymmetric packing configurations in the curved regions enhances the asymmetry of the two membrane leaflets and bends the membrane more strongly. For completeness and comparison with the results of the DPPC/DLiPC/cholesterol ternary curved membrane, we also examined the lateral organization of the curved DPPC/DLiPC/ cholesterol membrane inserted with 60 WALP23 peptides. Because the lipids are packed more loosely in the ld domain than in the lo domains and the bending rigidity of ld domains is small,16−18 the ld domains together with the majority of WALP23s expectedly reside in the curved regions whereas the lo domains are distributed in the planar regions of the membranes as shown in Figure 6. The membrane curvature also induces the density of the DLiPC in the outer leaflet of the curved regions (∼1.24 nm2) to be smaller than that in the inner
Figure 8. Front and top views of the snapshots for the DPPC/DLiPC/ cholesterol membrane inserted with six bR’s at simulation times of (A) 0, (B) 6, and (C) 20 μs. Here, bR is shown in yellow.
in the ld domains because of the enthalpic effect. Because of this enthalpic affinity between bR’s and ld domains, the randomly distributed bR’s as shown in Figure 8A also tend to accumulate into the curved regions enriched in ld domains (Figure 8B). However, different from the cases of curved unary and ternary membranes inserted with WALP23s as shown in Figures 2 and 6, even after a 20 μs simulation, some bR’s do not yet completely partition into the upper curved regions with large curvature but reside on the boundary of the lo and ld
Figure 6. Front and top views of the final snapshot of the 12 μs simulation on the DPPC/DLiPC/cholesterol lipid bilayer inserted with 60 WALP23s. 1120
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Langmuir domains (Figure 8C). A careful examination of the packing configuration of bR’s in the curved regions with large curvature reveals that the packing configuration of bR’s is nearly uninfluenced by the membrane curvature and remains symmetric (Figure 8C). Thus, the bR clusters do not match well and even will disturb the asymmetric packing of the lipids in the curved regions, and some bR’s are excluded from the curved regions as shown in Figure 8C.
ACKNOWLEDGMENTS
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REFERENCES
This work was supported by the National Natural Science Foundation of China under grant no. 11004173 and the Starting Research Fund for Doctors from Zhejiang Normal University. We thank Dr. Wen-De Tian for helpful discussions. The computational resources provided by the Department of Computer Science and Technology at Zhejiang Normal University is greatly appreciated.
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CONCLUSIONS In this work, we presented an effective method to build up curved membranes and investigated the interplay between the curvature and the lateral organization of lipids and TM peptides/proteins in the curved model membranes using coarse-grained molecular dynamics simulations. In the DPPC/DLiPC/cholesterol ternary membrane, the ld domains with a smaller bending rigidity prefer to distribute in the curved regions whereas the lo domains with a larger bending rigidity favor residence in the planar regions of the membrane. This result agrees very well with the experimental results. In the curved membrane inserted with cylindrical model peptide WALP23s, on the one hand, in both the unary and ternary membranes, the lipids are packed inhomogeneously in the lateral plane of the membrane, and this inhomogeneity in the lateral distribution of lipids provides a driving force for the aggregation of WALP23s in the curved regions. On the other hand, the membrane curvature also leads to an asymmetric packing of the lipids in the outer and inner membrane leaflets in the curved regions, and the lipids are packed more loosely in the outer leaflet than in the inner leaflet. To match this lipid packing asymmetry, the packing configuration of the peptides or peptide clusters transfers from a symmetric cylinder to an asymmetric wedge. This configuration deformation stabilizes the residence of the peptides in the curved regions. In the membrane inserted with larger bR proteins, the configuration of the bR’s is hardly influenced by the membrane curvature, and the bR’s cannot stably accumulate in the curved regions but distribute in both the curved regions and on the boundary of the lo and ld domains. Comparing the lateral sorting of WALP23s and bR’s in the curved membrane, we can conclude that the configuration deformation is crucial to the stable residence of TM peptides or proteins in the curved regions of the membranes. Furthermore, the membrane curvature also provides a driving force for the aggregation of peptides/ proteins in the curved regions. The larger the membrane curvature, the more compactly the peptides/proteins aggregate. Besides the influence of the curvature on the lateral organization of the lipids and peptides or proteins, the insertion of the peptides or proteins can conversely enhance the curvature of the membranes. This work provides a possible mechanism for the interplay between the curvature and the lateral organization of lipids and peptides/proteins (especially the lateral sorting of TM peptides/proteins) in the curved membranes and a theoretical reference for relevant experimental studies.
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