Biophysical Mechanisms of Membrane-Thickness-Dependent MscL

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Biological and Environmental Phenomena at the Interface

Biophysical mechanisms of membrane thickness dependent MscL gating: An all atom molecular dynamics study Hiroki Katsuta, Yasuyuki Sawada, and Masahiro Sokabe Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b02074 • Publication Date (Web): 16 Aug 2018 Downloaded from http://pubs.acs.org on August 20, 2018

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ABSTRACT

The bacterial mechanosensitive channel, MscL is activated by membrane tension, acting as a safety valve to prevent cell lysis against hypotonic challenge. It has been established that its activation threshold decreases with membrane thickness, while the underlying mechanism remains to be solved. We performed all-atom molecular dynamics (MD) simulations for the initial opening process of MscL embedded in four different types of lipid bilayers with different thicknesses: 1,2-Dilauroyl-sn-glycero-3-phosphocholine (DLPC)), 1,2-Dimyristoyl-glycero-3phosphorylcholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2Distearoyl-sn-glycero-3-phosphocholine (DSPC). In response to membrane stretch, channel opening occurred only in the thinner membranes (DLPC, DMPC) in a thickness dependent way. We found that MscL opening was governed by the rate and degree of membrane thinning and that the channel opening was tightly associated with the tilting of transmembrane (TM) helices of MscL towards membrane plane. Upon membrane stretch, the order parameter of acyl chains of thinner membranes (DLPC, DMPC) became smaller, whereas other thicker membranes (DPPC, DSPC) showed interdigitation with little changes in the order parameter. The decreased order parameter contributed much larger to membrane thinning compared with the interdigitation. We conclude that the membrane thickness dependent MscL opening is mainly arisen from structural changes of MscL to match the altered membrane thickness by stretch.

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INTRODUCTION Mechanosensitive channels are a class of ion channels activated by mechanical stresses in the membrane 1. In vertebrates they play major roles in touch 2, hearing3, vestibular sensation4,5 and blood pressure regulation 6,7. Bacterial mechanosensitive channels act as a “safety valve” to keep cell volume against hypoosmotic shock 8, thus preventing them from cell lysis. Two types of bacterial mechanosensitive channels, homopentameric Mechanosensitive channel of Large conductance (MscL) and homoheptameric Mechanosensitive channel of Small conductance (MscS) are most studied and their 3D-crystal structures of the closed state have been solved 9. The 3D-structure of MscL in the closed state was first obtained in Mycobacterium tuberculosis (PDB:1MSL) at 3.5 Å resolution

10

. The structure was refined (PDB: 2OAR) to provide

important information that the N-terminal S1 helices lie along the cytoplasmic surface of the membrane

11

. The subunit of MscL is constituted of two transmembrane helices (TM1 and

TM2,). TM1 helices line the pore and its narrowest part embedded in the inner leaflet of the bilayer forms the gate constituted of hydrophobic amino acids, Lys19 and Val23. In the closed state, MscL does not permeate water and ions by de-wetting due to hydrophobic nature of the gate, which is called vapor lock. Increased membrane tension by stretch breaks down the vapor lock followed by gate expansion, allowing permeation of these molecules12,13. TM2 helices face the lipid bilayer and are in direct contact with lipids. Patch clamp study combined with site directed mutagenesis has shown that some TM2 amino acids near the periplasmic membrane surface are potentially responsible for tension sensing14. Among them phenylalanine 78 (F78) located at the surface is thought to be one of the major tension sensing sites of MscL based on molecular dynamics (MD) study13. Previous fluorescence resonance energy transfer (FRET) and MD simulation studies have shown that during the initial stage of MscL opening, transmembrane


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TM1 and TM2 helices largely tilt against the transmembrane axis upon membrane stretch, followed by an expansion of the gate

12,15

. The size of fully opened pore at the gate is

experimentally estimated at 30-40 Å in diameter 16. MscL is activated by increased membrane tension by stretch, during which membrane thickness around MscL is likely to change. Such changes in the bilayer thickness cause a size mismatch between hydrophobic core of the lipid bilayer and hydrophobic transmembrane (TM) domain of channel proteins, termed “hydrophobic mismatch”. In order to compensate the mismatch to minimize the interaction energy, previous models for “hydrophobic matching” have assumed that the relatively soft lipid bilayer stretches out or compresses so as to match to the hydrophobic core of the protein17–19. Previous studies have investigated this “hydrophobic matching” process of MscL and its effects on MscL gating. For example, MscL gating threshold became smaller in thinner lipid bilayers with shorter acyl chain lengths. Measured changes in the lipid-binding constant of MscL with different acyl chain lengths were much smaller20,21 than that expected from a theoretical study with an assumption that MscL TM helices are rigid bodies 19

. This suggests that MscL itself as well as lipids changes its structure to match altered

membrane thickness, however, the underlying mechanism remains obscure. More specifically, it should be elucidated how membrane tension causes structural changes of the lipids surrounding MscL and how this change induces MscL structural changes leading to gate opening in a membrane thickness dependent way. Experimental methods used in these studies, such as patch clamping, EPR and FRET are difficult to examine the mesoscopic structural changes of MscL and interacting lipids at atomic level. Meanwhile, molecular simulations can overcome this difficulty and actually some such studies have shown at atomic level that MscL structure is altered when it is embedded in thinner membranes22,23.


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This study aimed at clarifying the MscL gating process induced by membrane stretch in the lipid bilayers with different thicknesses using all-atom MD simulations. Since interaction between MscL and membrane is essential to membrane tension sensing, we made fine analyses of structural changes both in MscL and surrounding lipid molecules during the initial phase of channel opening in response to membrane stretch. Based on the obtained results, we discuss biophysical mechanisms of the effect of membrane thickness on the MscL-gating process focusing on the lipid-protein interactions at atomic level.


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EXPERIMENTAL In this study we used MscL from Escherichia coli (Eco-MscL) in the closed state which is based on the crystal structure of Tb-MscL (PDB: 1MSL). Eco-MscL structure was originally modeled by Gullingsrud J. et al.24 based on homology modeling of Sukharev et al.25 This structure was refined later by S. Steinbacher et al. (PDB: 2OAR)11, which showed that Nterminus S1 helices run parallel to the cytoplasmic membrane surface (Figure1). Thus we modified N-terminus S1 helices of Eco-MscL structure in order to match this refinement. The residues in the cytoplasmic region beyond Ala110 of the Eco- MscL, which was suggested not to be essential for MscL gating process26, have been excised to reduce the total size of the system. Figure 1 shows structural model we used in this study. Lipid membranes were modeled by using CHARMM-GUI Membrane Builder

27–31

. In order

to examine the effect of membrane thickness, we used four types of lipids with different acyl chain lengths: 1,2-Dilauroyl-sn-glycero-3-phosphocholine (DLPC, PC12), 1,2-Dimyristoylglycero-3-phosphorylcholine (DMPC, PC14), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC,PC16) and 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC, PC18). The area of each lipid molecule was set at 66 Å2 in all models. The membrane was oriented in the x-y plane with a size of 130 x 130 Å in DLPC and 120 x 120 Å in DMPC, DPPC and DSPC. Water molecules (TIP3P) were added both in outer and inner sides of the membrane as well as in the membrane using VMD Plugin SOLVATE Program32,33. Water molecules were removed from the hydrophobic part of lipids, 10-15 Å apart from the center (0) of the transmembrane (z) axis. The model was ionized with 0.15M KCl by VMD Plugin AUTOIONIZE Program32,33 and an EcoMscL model was embedded by superimposing the channel structure onto the membrane, followed by removal of the lipids and water molecules located within the pore region and


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extensively overlapped with the channel using tcl script. Each system consisted of 175169 (DSPC membrane) to 182446 (DLPC membrane) atoms. A snapshot of a constructed system is shown in Figure 2. All molecular dynamics simulations were carried out by using NAMD Ver. 2.934. Force fields for water and MscL were from CHARMM27, and those for lipids from CHARMM3635–37. Each step of calculation was 2 fs and periodic boundary condition was set for all atoms. Particle Mesh Ewald Method 38 was applied to Coulomb force with a 12 Å cutoff for van der Waals force. Both equilibrium and membrane stretching simulations were carried out in an NPT ensemble at 310 K and 1 atm. Energy minimization was performed for 20000 steps to remove bad contacts and then the energy-minimized system was equilibrated for 30 ns. Membrane tension was increased by decreasing pressure parallel to the membrane plane. The rest of the components of the model, including water and MscL were not subject to the negative pressure. The negative lateral pressure in the lipid bilayer is considered to mimic the stretched membrane patch used in the patch-clamp experiments to activate (open) MscL. We employed relatively large force (tension) in the membrane (75dyn/cm) to save calculation time. Although this value is much stronger than actual patch-clamp experiments (~15 dyn/cm)

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, our previous studies showed that structural

changes of MscL were consistent with other simulation studies, and associated energy profile was also consistent with that of the patch-clamp experiment during the first stage of opening, even with 150 dyn/cm membrane stretch

12,13

. Thus we think that our model is just a speed up

version for the simulation of the initial process of MscL opening. Table 1 shows the number of atoms and lipid molecules, and membrane-stretching simulation time for each model. Pore radius of MscL was calculated by HOLE program using a spherical probe 40. Analysis of interaction energy was conducted by using NAMD Energy Plugin. Coulomb force and van der


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Waals force were considered in this calculation. Each energy profile denotes the sum of the energies from five subunits of MscL protein. In order to obtain the tilt angles of TM2 helices, the orientation of each helix was calculated by measuring the vector passing through the backbone particles in each helix. We defined a vector as the direction from the center of mass for Cα coordinates of the four amino acid residues 93-96 to that of the amino acid residues 83-86 in TM2 helix. When analyzing lipid structural changes, C atom next to carbonyl terminus was defined as C1. Membrane thickness was defined as an average distance between P atoms of lipid molecules in the inner and outer leaflets, respectively. Lipid order parameter, an index of acyl chain flexibility

41 42

,

was calculated by the following equation , where θ denotes the angle

between transmembrane (z) axis and C-H bonding direction of acyl chains. We used MembPlugIn in VMD to calculate the order parameters

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, and evaluated torsional angles of

amino acid residues by Ramachandran plot using VMD plugin. 3 ܿ‫ ݏ݋‬ଶ ߠ − 1 ܵ஼஽ ＝ 2

Images in this paper were made with VMD software which has beens developed with NIH support by the Theoretical and Computational Biophysics group at the Beckman Institute, University of Illinois at Urbana-Champaign.


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RESULTS and DISCUSSION Structural properties of MscL and membrane during 30 ns of equilibration In order to analyze the stability of MscL in the lipid bilayer, we calculated Root Mean Square Deviations (RMSDs) of the Cα atoms of the MscL protein during equilibration process. Figure 3 shows RMSDs with respect to the Cα atoms during 30 ns calculation relative to the initial structure in four different types of membranes, DLPC, DMPC, DPPC and DSPC. It is evident that all the MscL structures are stabilized after 25 ns of equilibration. We also calculated the pore radius of MscL at the gate region, the narrowest part of the pore mainly constituted of L19 and V23. The pore radius after 30 ns equilibration was 1.2 Å in DLPC and 1.5 Å in DSPC (Figure S1), which is consistent with a previous MD simulation study of MscL in different membrane thickness22, where membrane thickness was changed by shortening lipid acyl chain length every 1 ns. In two or three of the five subunits of MscL in DLPC and DMPC membranes, periplasmic side of TM2 helices kink at N81, as shown in the snapshots in Figure 4. We used Ramachandran plot to evaluate this structural change and found that the plot corresponding to the kinked portion was not observed in alpha-helix region (Figure 4). MscL in thicker (DPPC, DSPC) membranes did not show such a kink at N81. In general, helix kink tends to occur at Glycine, Serine, Aspartic acid or Asparagine44, thus this kink at N81 seems to be natural. However, no helices kinked at D84, probably due to strong interaction with K31 in TM1 helices. In our previous study stable interaction between F78 and lipids was suggested to play a critical role in tension sensing12. F78s stably interact with membrane in all models during equilibration process (Figure S2), suggesting that helix kink of TM2 helices at N81 seems to be reasonable and energetically


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favorable for maintaining hydrophobic matching in relatively thin membranes. MscLs of some other bacteria have serine (S) instead of asparagine (N), which can also form a helix kink44,45. It is possible that this region may play an important role for adjusting hydrophobic thickness of MscL to the membrane. Then we analyzed structural changes of lipid molecules in each membrane model. We categorized the membrane forming lipids into two groups: (1) MscL-surrounding lipids within 3 Å from MscL lipid-interacting surface, and (2) bulk lipids more than 10 Å apart from the MscL outer surface. Table 2 shows membrane thickness of both MscL-surrounding and bulk lipids after 30 ns equilibration. No difference in membrane thickness was observed between the two groups. We also calculated lipid order parameter (SCD) of MscL-neighboring lipids in each model after 30 ns equilibration. In DLPC membrane the lipid order parameter with C1-8 of acyl chain had larger value (0.20 to 0.25) than that (0.15 to 0.20 with C1-C16) in DSPC membrane (Figure S3). After 30 ns equilibration, membrane thickness did not show obvious difference between MscLneighboring and bulk parts in all models. However, in a previous study using Tb-MscL in thin membranes, thickness of the membrane that directly border the protein became larger than that of bulk lipids apart from the channel protein22. We do not know the exact reason for this discrepancy. Presumably, it may arise from differences in the protein-lipid interaction caused by the different MscL models employed in our (Eco-MscL) and their (Tb-MscL) studies.

Membrane thickness-dependent pore opening of MscL in response to membrane stretch


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In response to membrane stretch, tilting of transmembrane helices and following expansion of the pore were observed in MscL embedded in DLPC membrane during the first 10 ns of stretch. Snapshots depicting these structural changes of MscL are shown in Figure5. Pore radius of MscL was measured along the transmembrane (z) axis in each model at 10 ns after the onset of stretch using HOLE program that can calculate average distance between the center of the pore and the pore-consisting atoms. The pore radius at the narrowest part of the gate region, which is mainly composed of L19 and V23, was larger in thinner membranes (Figure 6(A); 3.1 Å in DLPC (red line) vs. 1.1 Å in DSPC (green line), clearly showing that MscL opens more easily when embedded in thinner bilayers. To estimate functional opening of the pore, we also analyzed water permeation across MscL during membrane stretch by counting the number of water molecules at the gate region. During 30 ns equilibration process, water could not penetrate into the gate due to dewetting property of the hydrophobic gate region13. When membrane was stretched, MscL allowed continuous water permeation through the pore 9.5 ns after the stretch onset in DLPC membrane and 17.5 ns in DMPC membrane. However, water permeation was not observed in DPPC and DSPC membranes even at 75 ns after the onset of membrane stretch. Water permeation profile of each model is shown in Figure 6 (B). As the pore becomes larger, water permeation increases, thus the result is consistent with the pore size changes described above.

Tilting of TM helices leading to pore opening of MscL We measured tilt angles of TM2 helices because this is the major mesoscopic structural change in MscL that leads to channel opening in response to membrane stretch. Previous patch-clamp46,


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EPR47, FRET15 and MD simulation24 studies have shown that MscL gating (opening) is associated with tilting of transmembrane helices. As TM helices tilt, tensile (dragging) force in the membrane will be applied more efficiently along the long axis of these helices, working as a pulling up force in a radial direction in the membrane plane. This dragging force is thought to be transmitted to TM1 helices via periplasmic loop and/or strong salt bridge between TM1 and TM213. As the inter-helix interaction was stable in all models in this study, force transmission may not be affected by membrane thickness. When transmitting force reaches the TM1 gate region, it will break the hydrogen bond between L19 and Val 23 within a helix to expose a carbonyl oxygen that will form a hydrogen bond with water molecules near the gate, thus atmosphere of the gate changes from hydrophobic to hydrophilic13. In such a way the hydrophobic lock realized by hydrophobic interactions between TM1 helices at the gate that stabilizes the closed state of MscL would be broken39. This will induce a sliding of each hydrophobically interacting portion between TM1 helices towards radial direction, leading to an expansion of the gate. If we can assume that the tilt angles of TM2 and TM1 helices reflect effectiveness of the force transmission to the gate to break the hydrophobic lock, we may be able to explain why thinner membranes give lower threshold for MscL opening. According to our previous study, breakdown of a single interacting portion between TM1 helices and following helix tilting is sufficient to trigger channel opening. Therefore, we calculated the largest tilt angle of a TM2 among the five subunits as an index of tendency for channel opening. Table 3 clearly indicates the negative correlation between the membrane thickness and the maximum TM2 tilt angle under membrane stretch. As TM2 helices tilt, membrane tensile force would be transmitted more efficiently to TM1 helices along the membrane plane. The transmitted force to the gate


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region was large enough for breaking the hydrophobic interactions between amino acids that constitute the gate, which can cause pore expansion toward channel opening.

Structural changes of lipid molecules during membrane stretch We examined lipid dynamics to 75 dyn/cm membrane stretch. Although DLPC and DMPC membranes became unstable and eventually broke up 12 ns in DLPC and 18 ns in DMPC after stretch, as described in Experimental Section, MscL behavior before membrane rupture are consistent with previous simulation and experimental studies12, 13, 22, 39, we considered that lipid behavior before membrane rupture can be usable. Membrane thickness decreased during membrane stretch in all membrane models as in Figure 7. Here we employed averaged thickness over the bilayer as an index of membrane thickness, because the membrane thickness did not show any difference between MscLinteracting and bulk lipids. The rate and amplitude of membrane thinning clearly correlated with acyl chain length during the initial 10 ns membrane stretch (Table 4). Particularly, DLPC and DMPC membranes showed significant continual thinning after 10 ns. By contrast, the rate and amplitude of thinning both in DPPC and DSPC membranes were much smaller and saturated around 20 ns at ca. 30 Å after the onset of membrane stretch (Figure.7). These results are qualitatively consistent with functional channel opening as shown in Figure 6B, where MscL in DLPC or DMPC can permeate water, while that in DPPC or DSPC shows no water permeation. To investigate the effect of lipid configuration on the membrane thickness changes to stretch, we first measured tilt angle of acyl chains of MscL-surrounding lipids in detail. Table 5 shows average tilt angle of lipid acyl chain against z (transmembrane)-axis before and 10 ns after the


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onset of membrane stretch. In DLPC and DMPC membranes the tilt angle became smaller despite membrane stretch, so this structural change did not contribute to membrane thinning. Although tilt angle became slightly larger in DPPC and DSPC membranes, this contributed little to membrane thinning at most 1.0 Å. Generally, we can say that acyl chain tilting had no or little contribution to membrane thinning in our simulation condition. Next, we measured z-coordinates of carbon atoms of acyl chain ends (Table 6) to estimate lipid dislocation along transmembrane axis. In DPPC and DSPC membranes, acyl chain termini of the inner leaflet were above the termini of acyl chains of the outer leaflet, indicating that interdigitation underwent in DPPC and DSPC membranes, where acyl chain termini of inner leaflet wedged into the outer leaflet, and vice versa (Figure 8 (B)). However, no interdigitation was observed in DLPC and DMPC membranes. Then, to check the contribution of acyl chain order to the membrane thinning, we calculated lipid order parameter in each model. Figure 8 (A) shows order parameter of DLPC membrane (0 ns, 5 ns and 10 ns after the onset of membrane stretch) and DSPC membrane (0 ns, 10 ns and 60 ns after the onset of membrane stretch). In DLPC membrane order parameter became smaller as the membrane became thinner. We measured z component of average distance between DLPC phosphate atoms and acyl chain sn-1 or sn-2 end carbon atoms within 3 Å from the outer surface of MscL. The distance was 13.6 ± 1.1 Å (n=47) before membrane stretch and became 11.4 ± 1.0 Å (n=50) at 5 ns and 8.7± 1.4 Å (n=47) at 10 ns after membrane stretch. We can estimate that order parameter change contributed about 10 Å thinning of DLPC membrane during 10 ns stretch, which corresponds to more than 80% of the initial thickness (-12.1 Å). On the other hand, in DSPC membrane order parameter became smaller first, but larger again after 60 ns membrane stretch.


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From these results, changes in the order parameter observed in thin (DLPC, DMPC) membranes caused significant and faster thinning of the lipid bilayer in response to membrane stretch. By contrast, the major structural change in thick (DPPC, DSPC) membranes to stretch was interdigitation, which resulted in slower and limited thinning of the bilayer under our simulation condition. Thus the acyl chain dependent lipid dynamics, particularly alteration of acyl chain order in thinner membranes is the major mechanism for stretch dependent membrane thinning that leads to thickness dependent MscL opening. As lipid interdigitation was observed both in MscL-neighboring and bulk areas of the lipid bilayer, it should arise from the physicochemical nature of the lipids themselves rather than MscL-lipid interaction. In interdigitation phase acyl chain interaction becomes stronger by the increased van der Waals force in comparison with the non-interdigitated phase48, which would increase membrane elastic moduli both in transmembrane and membrane plane axes, requiring significant energy to make membrane thinner. Lipid interdigitation is more likely to occur in thick membranes under increased membrane tension49 and C22:0 lipid can cause interdigitation even under 1 atm, 300K50. Lipid molecules having acyl chains with less than 14 carbon atoms do not have interdigitation phase 59,, which is consistent with our result here. However, it is unclear why thin (C ≤ 14:0) membranes do not undergo interdigitation51, but rather show decreased order parameter in response to membrane stretch. This remarkable different features between thin and thick membranes significantly influences static and dynamic mechanical properties of the membrane, thus would cause distinguished effects on MscL gating. The temperature of each simulation was 310K. At this temperature, DLPC and DMPC membranes are above the solid-liquid transition temperature, while DPPC and DSPC membranes are below their transition temperatures. In order to consider the phase transition effect of each


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lipid, we carried out simulations with DMPC (phase transition temperature is 297K) membrane under 290K. Figure S4 is the snapshots of lipid molecules interacting with MscL after 10 ns stretch. Interdigitation was not observed in DMPC membrane regardless of simulation temperature as shown in the snapshots of lipid molecules interacting with MscL after 10 ns stretch. On the other hand, partial interdigitation was observed in DPPC membrane. From these results, it is unlikely that the lipid interdigitation of DPPC and DSPC membranes arose from phase transition effect.

Dynamics of MscL amino acids coupled with membrane thinning In order to investigate MscL-lipid interaction in detail, we calculated interaction energy between MscL and lipids. As shown in Figure 9 (A), MscL-lipid interaction becomes most unstable in DLPC membrane among the membrane models. We analyzed which amino acid residues were responsible for the unstability by focusing on the interaction energy between lipids and TM2 periplasmic F78, cytoplasmic K97 and K101. This is because F78 is considered as a major tension-sensing amino acid, thus stable interaction between F78 and lipids is crucial for tension sensing 12, and because K97 and K101 are highly conserved among bacterial MscLs and subjected to strong forces during gating process9,23. Figures 9 (B), (C) and (D) show F78-lipid, K97-lipid and K101-lipid interaction energies, respectively. F78 maintained stable interaction with lipids in any types of membranes. By contrast, interaction energy between K97, K101 and lipid molecules decreased by 100 - 200 kcal/mol during the first 5 ns of the simulation in DLPC membrane, suggesting that K97-lipid and K101-lipid interactions became unstable in some subunits.


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We observed that TM2 helices moved toward periplasmic side upon membrane stretch (Figure S5). Both F78 as well as K97 moved from the hydrophobic region of the bilayer toward hydrophobic-hydrophilic boundary matching as membrane became thinner. However, when F78 is exposed to the lipid-water boundary, further moving to periplasmic side is energetically unfavorable, which will cause tilting of TM2 helices towards membrane plane. Based on the present results that F78 maintained stable interaction with hydrophobic region of lipids (Figure 9 (B)), the helix tilting is likely to be caused by unfavorable interaction between F78 and hydrophilic moiety region. Since this structural change occurred about 1 ns before initiating MscL gating, it may trigger MscL gating. In thin membranes F78 interacts with more proximal part of lipids, which results in a little movement to periplasmic side. Meanwhile, in phosphatidylcholine (PC) membranes, positively charged choline head group generates repulsive force against K97 and K101. Thus it is more favorable for these two amino acids to interact with negatively charged phosphate group and/or carbonyl group. Such electrostatic interactions are likely to trigger TM2 helix movement towards periplasmic side. K97 and K101 may play a role as “knot in the rope”45, preventing these hydrophilic amino acid residues from interacting with lipid hydrophobic acyl chains during gating. K97 and K101 seem to play such a role by interacting with negatively charged phosphate group and/or carboxyl group. This hypothesis on the MscL opening is schematically summarized in Figure10. Figure S6 (A) shows snapshots depicting the helix kink around N81 in thick DSPC membrane upon membrane stretch. Result from Ramachandran plot of N81 in DSPC membrane during 50 to 60 ns after membrane stretch indicates that N81 in two of five subunits are not in the α-helix region as well as that in thin DLPC during equilibration process (Figure S6 (B)). The helix kink may occur during the hydrophobic matching process of MscL, because it was observed when


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membrane thickness was (or changed to) about 30 to 32 Å in both DLPC and DSPC membranes. As this kink was observed regardless of lipid species it would not likely to contribute to the opening threshold of MscL. Our results clearly demonstrate that MscL opens more easily in thinner membranes due to their relatively large thinning responses to stretch and following MscL helix tilting caused by the strong and stable interaction with F78 at lipid-water interface. Since lipid molecules constituting plasma membranes in most bacteria have 16 to 20 carbon atoms in acyl chains52, the gating threshold of MscLs in bacteria is relatively high. In E. coli, for example, saturated palmitic acid (C:16;0) is the major fatty acid of lipids at 310K53. Furthermore, lipid head groups in bacterial membrane are mostly composed of phosphatidylethanolamine (PE) and phosphatidate (PA) rather than phosphatidylcholine (PC)54 and gating threshold of MscL is higher in PE than in PC membranes55. Thus, MscLs in bacteria are mostly kept in the closedstate to avoid unfavorable opening by their structural fluctuations or by meaningless small changes in the membrane tension. This is reasonable, considering that MscL is an emergent device to save bacterial life at an extremely dangerous event like subjected to a strong hypotonic challenge. Stable activation of MscL is rather lethal to bacteria.


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CONCLUSIONS The aim of the present study was to elucidate biophysical mechanisms underlying the membrane thickness dependent mechano-gating of the bacterial mechanosensitive channel MscL. To this end we analyzed the relationship between MscL opening and membrane thickness changes to stretch using lipid bilayers with four different thicknesses (DLPC, DMPC, DPPC and DSPC) by performing all-atom molecular dynamics simulations. We found that MscL embedded in thinner lipid bilayers (DLPC, DMPC) were much easier (lower threshold) to open, allowing water permeation through the gate region compared with thicker membranes (OPPC, DSPC). Both the rate and the degree of membrane thinning underwent in an acyl chain length dependent way. Together with the result that time courses of membrane thinning were highly correlated with those of gate expansion, it is suggested that membrane thickness governs the degree of gate expansion through apparent hydrophobic matching of MscL protein to the thickness of the bilayer. Thereupon, we analyzed the structural changes of the bilayers during membrane thinning by stretch and found that completely different mechanisms of membrane thinning between thinner and thicker membranes. In DLPC and DMPC membranes thinning was mainly caused by decreases in the lipid order parameters (SCD), while membrane thinning in DPPC and DSPC membranes was arisen from lipid interdigitation with little change in SCD. We conclude that the membrane thickness dependent MscL gating arises from larger rate and degree of thinning to stretch in thinner membranes due to decreases in the order parameter of acyl chains that cannot occur in relatively thick membranes with long acyl chains. The apparent hydrophobic matching is largely supported by the strong interaction of lipids surrounding MscL with F78 of TM2 helix located at outer surface of the membrane.

The pore opening of MscL is caused by the

combination of the tilting down and dragging of TM helices by thinning lipid bilayers mainly via


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strong and stable interaction between F78 and lipids, and the hydrophobic matching of MscL to the thinning bilayers. Detailed analysis of relative contributions of these two different mechanisms leading to MscL opening remains to be solved.

ACKNOWLEDGMENTS This study was partly supported by Grants-in-Aid for Scientific Research (A) (24247028 to M.S.), and Scientific Research on Innovative Areas (15H0936 to M.S.) from the Ministry of Education, Culture, Sports, Science and Technology. The images in this paper were made with VMD software support. VMD is developed with NIH support by the Theoretical and Computational Biophysics group at the Beckman Institute, University of Illinois at Urbana-Champaign.

COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests.

SUPPORTING INFORMATION 1 Supplemental File, including Figure S1-S6. Pore radius profile of the gating region consisting of L19 and V23 after 30 ns equilibration (Figure S1); time courses of 20 ns F78 of MscL and neighboring lipids interaction energy profile after 10 ns equilibration (Figure S2); order parameter of sn-1 chain of MscL-interacting lipids after 30 ns equilibration (Figure S3); snapshots of DMPC and DPPC MscL-interacting


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lipids upon membrane stretch (Figure S4); trajectories of F78 (A) and K97 (B) along z-axis during 75 dyn/cm membrane stretch (Figure S5); (A) Snapshots of F78 and N81 in DSPC membrane upon membrane stretch, (B) N81 Ramachandran plot of one subunit after 50 to 60 ns membrane stretch in DSPC membrane (Figure S6).


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Moe P, Blount P. Assessment of Potential Stimuli for Mechano-Dependent Gating of MscL: Effects of Pressure, Tension, and Lipid Headgfile:///Users/hiroki/Desktop/20180321-25旅行.docxroups. Biochemistry. 2005;44(36):12239-12244. doi:10.1021/bi0509649


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Side view tension sensing region

Top view gate region

lipid bilayer

Figure 1. Top and side views of 3D closed-state structure of Eco-MscL in our study based on Tb-MscL (PDB: 2OAR). MscL is a homopentamer of a subunit having two transmembrane helices TM1 (green) and TM2 (yellow). The narrowest part of the pore, called “gate region” is indicated by yellow broken rectangular. Main tension sensing region, which is located near the lipid-water interface on the periplasmic side of TM2 helices, is indicated by white dashed ellipse.

X


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Top view

Side view y

z

Figure 2. The side and top views of our simulation model consisting of Eco-MscL (yellow), lipid bilayer (green) and water (blue) molecules. MscL helix structure is shown in a ribbon. The transmembrane axis was defined z-axis here.


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Figure 3. Time-courses of RMSD with respect to the initial structure of Eco-MscL during equilibration process in 4 types of PC bilayers with different acyl chain lengths (DLPC, DMPC, DPPC, DSPC). All the RMSD values reach stable level around 10 ns, suggesting that equilibration time in the present simulation is sufficient. Each line shows time profile of RMSD in DLPC (red), DMPC (blue), DPPC (magenta) and DSPC (green) respectively.


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Figure 4. Ramachandran plot of N81 after 30 ns equilibration in thin (DLPC) and thick (DSPC) membranes. In DLPC (left panel) TM2 helices were kinked at N81 (yellow) in two subunits. In these subunits, backbone dihedral angles were changed and they were not in the area of alphahelix favoring region (red) in the plot. By contrast, helix structure was maintained in all subunits in DSPC. All subunits were near alpha-helix favoring region. Aromatic side chains of F78 (green) in DLPC are in the cytoplasmic side, suggesting that this kink contributes to the stabilization of F78-lipid interaction.


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Figure 5. Snapshots of top and side views of Eco-MscL structural changes embedded in DLPC bilayer at 10 ns after membrane stretch. Left panel shows before stretch and right one at 10 ns after stretch. Gate expansion (shown in white) and tilting of TM2 helices (TM1 in green and TM2 in yellow) are clearly shown in the right panel.


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Figure 6. Pore size and water permeation at the gate region upon membrane stretch. (A) Pore radius profile of the gating region consisting of L19 and V23 amino acids at 10 ns after membrane stretch. Black broken line denotes the pore radius profile in DLPC membrane before membrane stretch. (B) Number of water molecules at the gate region upon membrane stretch. Continuous increase in the number, which indicates water permeation, can be seen after 9 ns in DLPC and 17 ns in DMPC. Water permeation cannot be seen in DPPC and DSPC during our simulations. Each line shows data from DLPC (red), DMPC (blue), DPPC (magenta) and DSPC (green) respectively.


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Figure 7. Time courses of membrane thickness changes upon 75 ns stretch in 4 types of lipid bilayers (DLPC, DMPC, DPPC, DSPC). Membrane thickness was calculated from the distance between phosphorous atoms in inner and outer leaflets, respectively, using MembPlugIn. Membrane thinning undergoes inversely depending on the initial thickness of the membranes. Each line shows time profile of membrane thinning in DLPC (red), DMPC (blue), DPPC (magenta) and DSPC (green) respectively.


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Figure 8. Structural changes of MscL-neighboring lipids during membrane stretch. (A) Changes of lipid order parameter (SCD) at 0, 10 and 60ns after membrane stretch. Vertical axis denotes lipid order parameter (SCD) and horizontal axis carbon number. Order parameter significantly decreases in thin (DLPC) membrane (see also Figure S3), while little change in thick (DSPC) membrane. (B) Snapshots of DLPC and DSPC at 10 ns and 60 ns, respectively after membrane stretch. Interdigitation can be seen in DSPC. Green: outer leaflet, Pink: inner leaflet.


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Figure 9. Time courses of interaction energies between MscL/amino acids and surrounding lipids upon 75 dyn/cm membrane stretch in 4 types of lipid bilayers (DLPC, DMPC, DPPC, DSPC). Vertical axis denotes interaction energy (kcal/mol) and horizontal axis time (ns). (A) Total interaction energy between MscL and lipids, (B) F78 (periplasmic side) and lipids, (C) K97 (cytoplasmic side) and lipids, (D) K101 (cytoplasmic side) and lipids. The energy here consists of electrostatic and van der Waals interactions. Each line shows data from DLPC (red), DMPC (blue), DPPC (magenta) and DSPC (green) respectively


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Figure 10. Hypothetical cartoon of structural changes of MscL at different membrane thicknesses. (1) First, TM helices moves relatively toward periplasmic side in response to membrane stretch (thinning). (2) The movement of hydrophobic F78 toward hydrophilic moiety makes it energetically unstable. (3) To compensate this unfavorable situation, TM helices tilt toward membrane plane, reestablishing stable interaction with lipids. This tilting accelerates efficient transmission of dragging force from lipids to the helices, eventually leading to gate expansion.


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Table 1. Numbers of atoms and lipid molecules, and simulation time of each model under 75 dyn/cm membrane tension.

type of membrane

Number of atoms

Number of lipid molecules

simulation time under 75 dyn/cm stretch (ns)

DLPC

182446

424

12.2

DMPC

177185

365

18.8

DPPC

177191

365

75.0

DSPC

175169

359

75.0

Table 2. Membrane thicknesses of MscL-surrounding and bulk lipids after 30 ns equilibration. MscL-surrounding lipids are the lipid molecules within 3 Å from MscL outer surface. Bulk lipids are out of 10 Å from MscL outer surface. Both types of lipids showed no difference in their thicknesses among all models

Lipid Molecules

MscL-Surrounding (Å)

Bulk (Å)

DLPC

31.7 ± 0.7

31.4 ± 0.3

DMPC

32.3 ± 0.8

32.2 ± 0.4

DPPC

32.9 ± 0.9

35.5 ± 0.4

DSPC

37.0 ± 0.8

37.8 ± 0.4


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Table 3. Largest tilt angles among five TM2 helices after 10 ns membrane stretch. The tilt angles were well correlated with changes of membrane thickness, suggesting that TM2 helix tilting was governed by membrane thinning.

Type of membrane

Largest tilt angle of TM2 among five subunits

DLPC

51.4°

DMPC

41.8°

DPPC

39.6°

DSPC

30.4°

Table 4. Changes of membrane thickness during early phase of membrane stretch. Membrane thickness was calculated in the same way as in Figure 7. The rate and degree of membrane thinning inversely depend on acyl chain length.

membrane

before membrane stretch (Å)

after 5 ns membrane stretch (Å)

after 10 ns membrane stretch (Å)

DLPC

32.0

23.4 (-8.6)

19.9 (-12.1)

DMPC

32.6

26.3 (-6.3)

24.0 (-8.6)

DPPC

35.6

29.7 (-5.9)

27.6 (-8.0)

DSPC

38.2

34.7 (-3.5)

32.7 (-5.5)


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Langmuir

Table 5. Tilt angles (θ) of sn-1 acyl chain of MscL-surrounding lipids before (A) and after 10 ns stretch (B) in 4 types of model membranes.

z

membrane

before membrane stretch (A)

after 10 ns membrane stretch (B)

(B) - (A)

DLPC

44.2 ± 2.8°

37.8 ± 6.3°

-6.4°

DMPC

41.7 ± 3.3°

39.8 ± 2.8°

-1.9°

DPPC

41.3 ± 2.9°

44.7 ± 2.9°

3.4°

DSPC

39.9 ± 2.8°

41.6 ± 2.5°

1.7°

ી

Table 6. Z-coordinates of acyl chain terminal carbon atoms of MscL-surrounding lipids before and after stretch. In DPPC and DSPC membranes, the carbon atoms in the inner leaflet are above those in the upper leaflet, indicating that interdigitation occurred in these two membranes.

before membrane stretch

after 10 ns membrane stretch

after 60 ns membrane stretch

outer

-1.8 ± 0.4

0.2 ± 0.5

NA

inner

-5.7 ± 0.6

-1.9 ± 0.5

NA

outer

-2.1 ± 0.5

-2.1 ± 0.6

NA

inner

-5.0 ± 0.7

-1.5 ± 0.6

NA

outer

-1.4 ± 0.8

-1.8 ± 0.7

3.3 ± 0.6

inner

-3.8 ± 0.7

-0.9 ± 0.7

3.8 ± 0.8

outer

-2.7 ± 0.7

-1.9 ± 0.7

0.0 ± 0.5

inner

-5.0 ± 0.7

-2.6 ± 0.7

3.9 ± 0.9

membrane DLPC

DMPC

DPPC

DSPC


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Table of Contents Graphics


43

Biophysical Mechanisms of Membrane-Thickness-Dependent MscL

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