Article pubs.acs.org/Langmuir
Insights into Binding of Cholera Toxin to GM1 Containing Membrane Ipsita Basu and Chaitali Mukhopadhyay* Department of Chemistry, University of Calcutta, 92, A. P. C. Road, Kolkata − 700009, India S Supporting Information *
ABSTRACT: Interactions of cholera toxin (CT) with membrane are associated with the massive secretory diarrhea seen in Asiatic cholera. Ganglioside GM1 has been shown to be responsible for the binding of the B subunit of cholera toxin (CT-B), which then helps CT to pass through the membrane, but the exact mechanism remains to be explored. In this work, we have carried out atomistic scale molecular dynamics simulation to investigate the structural changes of CT upon membrane binding and alteration in membrane structure and dynamics. Starting from the initial structure where the five units of B subunit bind with five GM1, only three of five units remain bound and the whole CT is tilted such that the three binding units are deeper in the membrane. The lipids that are in contact with those units of the CT-B behave differently from the rest of the lipids. Altogether, our results demonstrate the atomistic interaction of CT with GM1 containing lipid membrane and provide a probable mechanism of the early stage alteration of lipid structure and dynamics, which can make a passage for penetration of CT on membrane surface.
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INTRODUCTION Cholera toxin produced by Vibrio cholerae is the important factor responsible for diarrhea seen in Asiatic cholera due to huge salt and water secretion without epithelial damage.1 The secretory diarrhea is induced by the direct action of cholera toxin (CT) on intestinal epithelial cells and is accompanied by an increase in intracellular cAMP concentration induced by adenylate cyclize.2,3 The toxin consists of a B domain (CT-B) containing five identical binding subunits (CTB5) and a single A-domain (CT-A), which after proteolytic cleavage produces the enzymatically active A1-chain, that activates adenylyl cyclase by catalyzing the ADP-ribosylation of the α subunit of the heterotrimeric GTP-binding protein Gs.4 This process thereby increases the intracellular level of cAMP, which is responsible for the distorted transport rate of NA+ and Cl−.5 The B-subunit of the toxin binds specifically to the glycosphingolipid ganglioside-monosialylated (GM1) present in the outer leaflet of the plasma membrane. Movement of CT into cell by endocytosis is solely dependent upon the binding with GM1 and interaction with the target cell.6 To induce toxicity, binding of CT to GM1 of the target cell is mandatory.7 Therefore, the atomic-level detail of the interaction of the B subunit of CT with GM1 containing membrane is crucial. CT belongs to the subfamily of AB5 of the family AB toxins in an assembly of stoichiometry ratio 1:5 to form the holotoxin where CT-A is enzymatically active and causes toxicity, and CT-B is responsible for binding with the cell through GM1, which then mediates toxin entry into the cell.8 Five identical B subunits, each containing 103 residues, form a highly stable ring-like assembly with each having a single binding site for the plasma membrane receptor, GM1.9 Residues from two adjacent B subunits are involved in each of the binding sites. The A subunit, extending well above the pentameric plane, is © 2014 American Chemical Society
composed of two domains: compact N terminal A1 domain and extended C terminal A2 domain, which are linked by a disulfide bridge and extensive noncovalent interaction.10 The A2 polypeptide occupies the central channel and goes through the B-subunit, tethering together A1 and B domains. A2 domain possesses a C-terminal KDEL (Lys/Asp/Glu/Leu) motif, which is believed to play a role in retrograde trafficking from Golgi to the endoplasmic reticulum.11 A1 is a catalytic polypeptide possessing mono-ADP ribosyltransferase activity, thus responsible for the toxicity of CT.12 Prior to 1990, it was believed that the A domain is reduced to A1 and A2 domains and only the A1 domain entered the cell interior by crossing the membrane barrier.13,14 Yet the current view that toxin action depends on entry into host cells by membrane traffic is established on the evidence obtained from the recent studies.15,16 Toxin action and endocytosis were first correlated in the study on rat hepatocytes intoxicated with CT in vivo, which showed that reduction of the A subunit and activation of adenylyl cyclase associated with the appearance of the A1-peptide is an intracellular but not a plasma membrane function.17,18 To cause disease, CT must enter into the cytosol of the host cell from the plasma membrane through the transGolgi and endoplasmic reticulum (ER). In the ER, reduction of the disulfide bond makes A1 sub domain free from the entire toxin, which then enters to the cytosol, that induces toxicity.10 It is well established that CT first has to bind to GM1 at the cell surface to start its action.19 This binding then tethers the toxin to the membrane and results in the association of CT with membrane microdomains rich in cholesterol and Received: September 12, 2014 Revised: November 25, 2014 Published: November 26, 2014 15244
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glycosphingolipids,20,21 which is necessary for its function.7,22,23 Several studies regarding the trafficking pathway of CT from PM to ER have been reported.22,23 The most informative transport mechanism from PM to ER has been done using Stx B (Shiga toxin) subunit that contains an attached KDEL motif.24,25 It has been proposed that it is possible that CT follows the same route from PM to ER as shiga toxin.26 Despite rigorous experimental focus on CT upon the transport mechanism, the molecular mechanism behind how CT enters to the Golgi, crossing the cell membrane is still unexplored. One of the great challenges of molecular modeling is to provide an atomistic-level insight into such a biologically significant complex phenomenon, which is tricky to attain by the usual experimental techniques.27 Molecular dynamics simulation is the widely used technique to study complex biological systems.28 Although a large number of experiments have been performed on the mechanism of action of CT, only a very limited computational approach is reported on CT.29 Moreover, E. coli type I heat labile toxin (LTI) and CT are closely related toxins in terms of structure and function.30 Like CT, LT1 binds to membrane GM1, enters the cell by endocytosis, and follows the same retrograde pathway into Golgi and then ER.31 Yet how LT1 crosses the membrane barrier is not undertaken in the past studies. So, focusing on the mechanisms of the entry into the cell of both toxins may be a good approach to study comparatively the local lipid alterations for toxin penetration. In the present work, we have performed Molecular Dynamics Simulation of CT, GM1−POPC monolayer system to study the effect of binding with GM1 on lipid membrane morphology and the pretransitional structural conversion of CT. As GM1 resides in the outer layer of membranes, a monolayer modeling the outer layer of the membrane is constituted. In the past decade, monolayer has been used to illustrate the interfacial interactions of proteins with lipids, and they constitute an excellent experimental setup of protein−monolayer interactions where the effects of protein insertion on the lipids can be accurately quantified. Moreover, it is useful to simulate CT in a monolayer system as it allows a direct judgment with experiment of CT−monolayer interactions, which suggests that the mechanism of membrane translocation by the toxin may be assisted by lipid packing arrangement.32 We have also performed another simulation taking a mutated LT1, GM1− POPC system to find if the binding causes different effects from that of CT or not. The enzymatic action of CT is initiated after the proteolytic cleavage of disulfide bridge in the A domain, which is an intracellular function, and the structure of CT remains intact in the plasma membrane.17,18 The initial attachment of the CT to GM1 containing membrane will then act as a step where lipid rearrangements occur around the protein.32 In our study, we monitor the early modification of the lipid environment. The main focus of the work is on how the lipids in the cell membrane adjacent to the protein adjust themselves to induce the entry of the toxin and how the toxin starts to penetrate. We emphasize our study on CT; besides this, we monitor the mutated LT1 system to compare the similarities and dissimilarities between the two systems, and thus we can make a summary of the mechanism of the toxin entry into the cell. We observed that three copies of the pentameric CT-B subunit penetrate deeper on the surface, which makes the entire CT tilted with respect to the membrane surface as if some force is acting upon the CT to the direction of the lipid layer. In case
of the mutated LT1, no such observation is found. The structure and dynamics of the lipids near the subunits have been changed as compared to the rest of the lipids indicating the passage for the CT, whether this change is less significant in case of mutated LT1 system.
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METHODS
System Setup. Monolayer Model. A monolayer was constructed using POPC molecules. To stabilize the monolayer system, an additional monolayer was built up and rotated such that the lipid head groups are facing each other.33 Water then was added between the gap of the two monolayers to represent the system as lipid tail−lipid head−water−lipid head−lipid tail with simulation box size 120 Å × 120 Å × 190 Å. TIP3P water model was used.34 The system then was equilibrated for 50 ns. Protein Structure. Initially, cholera toxin B pentamer structure complexed with GM1 pentasaccaride (PDB ID: 3CHB) was downloaded from the Protein Data Bank and was placed above one of the monolayers. Now 5 GM1 was added to that monolayer such that the pentasaccharide groups of the 5 GM1 merged on the pentasaccharide group of the protein containing structure. To do this, 5 POPC from that layer were removed to avoid steric clashes. The pentasaccharide group of the complexed structure then was removed. Cholera toxin AB5 structure (PDB ID: 1XTC) was downloaded, and the missing residues near the terminal A2 domain were added using PDB swiss modeler35 and placed such that the B subunits are superimposed, and then the B subunit from the previous structure was removed. For Enterotoxin, we have taken the double mutated structure of the wild-type LT1 (PDB ID: 1LT3), where addition of the extra disulfide bond increases the thermo stability of the structure. The reason to start with this structure is that as we do not observe any significant structural changes in CT system, so here our focus was just to observe the lipid alteration and the overall penetration mechanism. In this structure, one GAL (BETA-D-GALACTOSE) connected with BGC (BETA-D-GLUCOSE) was attached to each of the copies in the B subunits, which make an extra advantage to start with this structure. We placed the protein on the GM1−POPC monolayer system such that the GAL and BGC molecules can merge with the Glc-GAL group of the GM1. The BGC then attached with GAL are removed from the system. After the toxins were placed, overlapping water molecules were removed. Five NA+ ions were added to both systems to maintain electro neutrality. The protein−monolayer system then was equilibrated for 50 ns, keeping the backbone of the protein fixed. System preparation was done using CHARMM, and system equilibration was done using NAMD_2.736 package. The standard CHARMM27 force field37 including dihedral cross term corrections (CMAP)38 was used for the lipids and the proteins, which was found to be suitable in many lipid-protein molecular dynamics studies.39 As in the CT system, we found more prominent changes in the structure and dynamics of the lipids that are closer to the bound CT, and we have carried out an additional simulation where the whole CT is remained fixed, referred as CT-fixed. To do this, we have taken the structure after 300 ns simulation, which was used as the initial structure of the simulation. As the CT is fixed there, the molecular dynamics simulation will only affect the structural and dynamic properties of the lipids by which we can understand how the lipids will influence the protein to penetrate into the membrane. This simulation was then carried out for 200 ns. Simulation Protocol. All MD simulations were carried out under the isobaric−isothermal (NPT) ensemble with imposed 3D periodic boundary conditions. A time step of 2 fs was used to integrate the equation of motion. The temperatures were maintained for the simulations using Langevin dynamics, while the pressure was kept constant at 1 bar using a Nose−Hoover−Langevin piston.40 The smooth particle mesh Ewald method was used to calculate long-range electrostatic calculations.41 Short-range interactions were cut off at 10 Å. All bond lengths involving hydrogen atoms were held fixed using the RATTLE442 and SETTLE43 algorithm. The trajectory analysis was 15245
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performed with CHARMM (Chemistry at Harvard Macromolecular Mechanics),44 and the snapshots were generated by VMD.45
Atom density distribution and distance between the subunits and the lipid headgroup calculation of CT (Figures 2 and 3) also support that B1, B2, and B5 are closer to GM1 as well as to the membrane headgroup region than the other two.
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RESULTS AND DISCUSSION As we are interested in the local lipid adjustments and the structural changes of B subunit, upon binding with GM1, we have defined the five monomers in the CT-B of both toxins as B1, B2, B3, B4, and B5 to make it simple. In reported studies of translocation of molecules in lipid bilayer, it was observed that the simulations suffered from long correlation time and insufficient sampling.46,47 The initial attachment of the CT to GM1 containing membrane will act as a step where lipid rearrangements occur around the protein32 and protein translocation starts after that. In our study, we monitor the early modification of the lipid environment, initiated within the early stage of the simulation, for which we do not have to suffer from the problem of insufficient sampling. Toxins in Lipid Membrane Environment. Figure 1a represents the time variable snapshots of the CT bound membrane system.
Figure 2. Atom density distribution of individual parts of lipid monolayer and individual subunits of CT-B along membrane normal (Z axis) for the CT-monolayer system.
Figure 1. Snapshots showing the (a) CT-monolayer and (b) mutatedLT1-monolayer systems with respect to time. POPC lipids and GM1 molecules are shown as silver lines and cyan bonds, respectively. Only B subunit of both toxins is shown for clarity. Individual subunits such as B1, B2, B3, B4, and B5 are presented separately as red, blue, green, mauve, and yellow vdw spheres, respectively. The image rendering is done with VMD.
Figure 3. Distances between the center of mass (COM) of the individual subunits of CT-B with the center of mass (COM) of the headgroup of the POPC contact monolayer as a function of time. The calculation was done from the last 500 ns simulation trajectory after the equilibration.
The figure shows that the CT-B resides at the pentasaccharide headgroup of GM1. Specially, three of five subunits of B (B1, B2, and B5) approached the membrane headgroup region, while the rest remained partly exposed to the aqueous environment. Because of this, the protein becomes tilted with respect to the bilayer normal. Tilting is increased with time (Figure 1a and Figure S1 of the Supporting Information). Such orientation of CT may be attributed to CT-induced membrane perturbation for CT penetration. It is well-known that binding to GM1 is crucial for CT activity,7 although addition of GM1 to POPC lipid monolayer enhances the rigidity of the layer, which may reduce the chance of crossing the lipid layer. In case of mutated LT1, each copy interacted almost equally with GM1, and no tilting of B subunit is observed (Figure 1b).
From the snapshots and from the above analyses, it appeared that three of the five subunits of CT-B approach nearer to the lipid surface, which makes it tilted. From the atom density distribution and distance calculation (Figures S2 and S3 of the Supporting Information), no such preference of the individual units of B subunit is observed, which is in accordance with the snapshots viewed in mutated LT1. Lipid Protein Interaction. Because we know lipid−protein interaction is crucial for many cellular processes,48 we have calculated the interaction energies of individual units of B subunit with surrounding lipids of the contact monolayer and with GM1 and partition of the total interaction energy into electrostatic and van der waal terms (Figure S4a and Figure 15246
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S4b, respectively, of the Supporting Information). We have observed that unit B5 interacts most strongly with the lipids followed by units B1 and B2. As from the previous findings in our work, it was examined that the above three units of the five copies are in close proximity with the contact monolayer, which is again reflected from the energy calculation. To elucidate the source of columbic interaction, we have calculated the H-bond occupancy (given in Table S1 of the Supporting Information). As B2 has the highest occupancy of forming H-bonds with GM1, it contributes most to the electrostatic energy among the five. Similarly, highest electrostatic energy is contributed by the interaction between unit B5 and the lipids as they have the highest H-bonding occupancy. The higher vdw interaction energy contributed for both GM1 and lipids from B1, B2, and B5 is the result of the nonbonded interaction between the hydrophobic segments of the protein and the hydrophobic part of the lipids. Altogether, the result shows that the maximum contributions are from B1, B2, and B5, which is similar to the previous findings. We have also calculated the interaction energies between the five copies of B subunit with contact lipids and with GM1 (Supporting Information Figure S5a and b, respectively) for mutated LT1. The energy calculations illustrate that except for B4 all of the subunits interact strongly with both lipids and GM1. As we have found that the main contribution in the interaction with the lipids is from B1, B2, and B5 subunits for the CT system, however, in the mutated LT1, we found that all five subunits contributed more or less equally, which is in agreement with the previous findings in our work. For a more focused examination of lipid environment response to cholera toxin, we have further calculated the number of contacts between the B subunit with 5 GM1 and lipid molecules (Supporting Information Figure S6). It shows that the maximum number of contacts with the lipids is contributed from B5, and with GM1, it is from B1. The above results indicate that the interaction of the three units (B1, B2, and B5) of CT-B with the lipid layer is initially liable in the preliminary penetration of cholera toxin. Protein Structure. Protein conformational study is necessary to determine the influence of binding of the protein to the lipid matrix on the protein structure and dynamics. CT is a member of AB family of toxins where the A subunit is enzymatically active and induces toxicity, and the B subunit is responsible for binding to cell and mediates toxin entry. As we have focused to determine the mechanism occurring for toxin entry in the cell membrane and find out the role of the B subunit in the early insertion process, the knowledge about the conformation of the binding B subunit is needed. The detail of the conformational drift was obtained by computing the rootmean-square deviation (RMSD) for the Cα atoms of the individual units of the B subunit of both CT and muatated LT1 relative to their starting structure49,50 (Figure 4a and b, respectively). The rmsd values of CT-B (in Figure 4a) ranges from 1 to 3 Å, indicating almost stable structure after binding to the membrane in accordance with previous studies that the five copies of the B subunit form a highly stable ring assembly.51 Calculated RMSD for the individual units of the B subunit of the mutated LT1 indicates their stability in the system after binding except for B5 (Figure 4b). Effects of Binding of CT on Membrane Structure and Dynamics. In the previous sections, we have discussed the effect of CT binding on the conformational changes of the
Figure 4. Root mean square deviation of individual units of B domain of CT (a) and mutated LT1 (b) as a function of time. The calculation was done from the last 500 ns simulation trajectory after the equilibration.
binding subunit of CT, and from the lipid protein interactions we have identified the roles of lipids inducing the protein to penetrate. Earlier studies revealed that penetration of hydrophobic subunits of CT into the lipid bilayer matrix requires structural rearrangements of lipid molecules.52 A detailed understanding of CT-induced changes of the lipids of the contact monolayer is required to gain insight into their capability of disrupting the packing arrangements of the lipid molecules. We have highlighted the effect on the bilayer structural properties (conformational ordering, headgroup orientation, lipid in-plane distribution) as well as on the dynamic changes of the local lipids (if the phosphorus atom of the phosphate group of the lipid is within 10 Å of any heavy atom of the B subunit of the toxin, then it is referred to as local lipid and the rest are referred to as bulk lipids). Lipid Tail Order. To simplify the analysis, we have divided the POPC lipids into two categories: local (those with a lipid non-hydrogen atom within 10 Å from any non-hydrogen atom of B-subunit) and bulk (the rest of the lipids).53 Figure 5a represents the order parameter profile for both palmitoyl and oleyol tails of CT system. It depicts that the value of the parameter is almost similar for the local and bulk lipids, but the trend for the local lipids is little disturbed. Thus, our results suggest that binding of CT influences the structure of the POPC lipids around the contact region with CT. The lipids that are not in contact with the CT behave like pure POPC 15247
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Figure 5. (a) Order parameter for the palmitoyl (red line) and oleoyl (black) chains of different categories of POPC lipids in the contact monolayer of CT system. (b) The surface distribution of lipid headgroup orientation. (c) The snapshot showing lipid curvature change after 500 ns of simulation trajectory in the contact monolayer. Only the tilted B1, B2, and B5 are shown for simplicity as red, blue, and yellow cartoons, respectively. The phosphorus atoms of POPC lipids are presented as silver spheres, and GM1 molecules are shown as cyan bonds. (d) The in-plane distribution of the change in local lipid thickness, which is defined as the Z position of phosphorus atom of individual POPC lipids as a function of their average (over the last 100 ns of simulation) in-plane position. The blue dots stand for the five copies of CT-B, and the green dots are for five GM1.
other cell binding or cell penetrating peptides.57,58 The effect is much localized, which strongly indicates that a preliminary stage of the alteration of lipid structure that may enhance the possibility of CT insertion is started. For the mutated LT1, the lipid headgroup orientation and lipid in-plane distribution were calculated to monitor the effects of binding on lipid structure and dynamics (Figure 6a and b, respectively). It was found that the lipids around all five copies of B subunit are oriented almost parallel with that of the monolayer surface and local lipid thinning is observed near the B subunit. From Figure 5b, we observe that the angle of the vector connecting P and N atoms of lipid headgroup with the membrane normal [plotted along the vertical axis Z] is ≅90° in the immediate vicinity of the B1, B2, and B5 subunits. However, the angle varies from 120°−150° for other lipids including those near B3, B4, making the head-groups nearly parallel to the membrane normal. From the lipid in-plane distribution shown in Figure 5d, it is demonstrated that the P atoms of the lipids near the B1, B2, and B5 subunits reside below −4, −3, and −2.5 Å, respectively, from the average position of the plane formed by the P atoms of all of the lipids, which may signify the change in lipid curvature. Finally, we have represented both of the results schematically in Figure 5c, where the difference in the orientation of P−N vector of
lipids as the value of the order parameter of POPC lipids agrees well with previous findings.54 Thus, local lipid perturbation induced by CT is reflected in the asymmetry in the order parameter profile. Lipid Head Group Orientation. To characterize the impact of bound B subunit on lipid, we have calculated the angle between the outward membrane normal (along Z axis) and the vector joining P and N atoms of POPC headgroup.55 The values of the average angle are then plotted as the function of the in-plane position of the phosphorus atoms of the lipids (Figure 5b). The adjacent lipids of the B subunits, especially the B1, B2, and B5 (which are more interacting with the lipids), are oriented almost parallel with the lipid surface. It is quite clear that, although the presence of GM1 on that region (denoted by green circle) gives additional rigidity on that layer, the CTinduced perturbation dominates over it. Lipid In-Plane Distribution. The local perturbation of the lipids of the contact monolayer induced by CT together with the lipid head tilting can cause a local lipid thinning. The membrane landscape is defined by the equation ΔZi = Zi − ⟨Z⟩, where Zi is the Z-coordinate of the phosphorus atom of the ith lipid in the contact monolayer and ⟨Z⟩ is the average Z position of the phosphorus atoms of the surface.56 Subunits B1, B2, and B5 depress the lipids by 1−3.5 Å in its immediate vicinity (Figure 5c and d). A similar effect was reported previously for 15248
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Figure 7. Difference in the cross-sectional area of CT-B on the lipid layer and the same for the headgroup of lipids, which reside under the pentameric hole of CT-B.
of the above-mentioned two is ≅5500 Å2, which is enough for the entry of CT. We have also noticed the change of the number of lipids near and far from the toxin with time in mutated LT1 (Figure S8b of the Supporting Information). This change is very low in case of mutated LT1. So this again proves that lipids near the CT are crucially involved to alter their structure and dynamics as if the toxin may enter into the host cell. These alterations of lipids are not observed in mutated LT1 except for those that are usual after protein binding. Rotational Autocorrelation Function. The amount of rigidity can be understood from the rotational autocorrelation function of P−N vector of headgroup of lipids, plotted as Figure 8a and b for CT and mutated LT1, respectively, and is calculated from the given equation:60
Figure 6. (a) The surface distribution of lipid headgroup orientation of mutated LT1 system. (b) The in-plane distribution of the change in local lipid thickness of the same system. The blue dots are for the five copies of B pentamer. Analyses were done using the last 100 ns of simulation trajectory.
headgroup of local and bulk lipids and the position of the P atoms are clearly observed from which the occurrence of lipid curvature change can be ascertained. Accumulative results obtained from Figure 5b−d suggest the change of lipid curvature near the vicinity of those subunits, which approach more to the headgroup of lipids. It has been found from previous reports59 that a change in lipid curvature has an effect on protein interaction and lipid curvature change is the key step of endocytosis. So it can be supposed that the binding of CT to the lipid layer induces curvature change, which was not found in the mutated LT1 system (Figure S7 of the Supporting Information), and the orientation of CT changes naturally. We have calculated the cross-sectional area of CT-B on the lipid layer and the same for the headgroup of lipids, which reside under the pentameric hole of CT-B, and the difference between the above two is plotted in Figure 7. The plot shows that the difference between the two was almost the same initially but it increases with time. In addition, we have calculated the number of lipids near and far from the toxins (Figure S8a of the Supporting Information for CT). It is evident from the plot that the number of lipids near the CT reduces with time while the number of bulk lipids increases. This means the local lipids move from the side of CT-B and thus make free space for the passage for entry of the toxin. The average cross-sectional area (averaged over last 20 ns) occupied by CT-B on the lipid layer is 5373 Å2, and the difference in area
Figure 8. Decay of rotational relaxation of P−N vector of POPC lipid molecule for bulk (black line) and local lipids (gray line) of CT (a) and mutated LT1 (b) systems. 15249
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̂ ·μ(̂ t )) Γl(t ) = Pl(μ(0)
Diffusion Coefficient. Again, the lateral mobility of the lipid molecules can be obtained from their translational diffusion coefficient.62 The diffusion coefficient, which is estimated from the slope of the time series of the mean square displacement of the lipids in the membrane plane, is given in Table 3. We find the lateral diffusion coefficient is lower for the local lipids in case of the CT system, which means more restricted mobility than the bulk, that is in agreement with the rotational reorientation dynamics of the lipid headgroup. In case of mutated LT1, the difference in the diffusion coefficient value is not as significant. Results Obtained from Simulation CT-Fixed. This simulation was performed only to asses the lipid structural and dynamic changes made after binding CT, which helps to penetrate CT through the membrane. To observe this, only the lipid properties are calculated. We have emphasized the lipid structural properties such as lipid in-plane distribution, lipid headgroup orientation near the protein, as well as the lipid dynamic properties, which are given in Figures S9 and S10, respectively, in the Supporting Information. We observed that lipid thinning is more prominent near the B1, B2 subunits than in the case of CT system, and lipids are completely parallel with the membrane surface where lipids have negative ΔZ. Although reorientational dynamics of lipid headgroup (Figure S11 and Table S2 of the Supporting Information) does not show a significant difference between the local and bulk lipids, the diffusive motion (Table S3 of the Supporting Information) observed from the lateral diffusion coefficient value of the local lipids is more restricted than the bulk than in the case of CT system.
(1)
where Pl are the Legendre polynomials of the order l and μ̂ (t) is the unit vector along the P−N molecular axis at time t; the brackets “⟨⟩” denote time average. We have investigated the second-order Legendre polynomial, that is, P2. The calculation is averaged over the last 100 ns of simulation. The figure shows that the decay becomes slower for local lipids than the bulk, more prominently for the case of mutated LT1. To asses the restriction in rotation, we have calculated the time of decay by fitting a biexponential decay with a fixed part as follows: Γl(t ) = A 0 + A1t / τ1 + A 2t / τ2
(2)
where A1 and A2 are fractions of the fast and slow components of motions to the decay, and A0 is a constant indicating the stable component of decay and indicative of restriction of rotation.61 The rotational autocorrelation correlation time τ1 and τ2 along with their relative contributions A1, A2, and A0 are shown in Tables 1 and 2. Table 1. Fitting Parameters for the Rotational Autocorrelation Function of Lipid P−N Vector CT
A0
A1
τ1 (ns)
A2
τ2 (ns)
local bulk
0.042 0.38
0.131 0.148
0.054 0.072
0.810 0.452
3.351 1.571
Table 2. Fitting Parameters for the Rotational Autocorrelation Function of Lipid P−N Vector LT1 (mutated)
A0
A1
τ1 (ns)
A2
τ2 (ns)
local bulk
0.60 0.51
0.091 0.14
0.041 0.075
0.301 0.32
1.281 1.15
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CONCLUSION In this work, we have carried out atomistic scale molecular dynamics simulation to investigate the structural changes of CT upon membrane binding and alteration in membrane structure and dynamics as well as for mutated LT1. The enzymatic activity of CT is generated after the proteolytic cleavage of disulfide bridge in A domain, which is an intracellular function. The initial attachment of the CT to GM1 containing membrane will then act as a step where lipid rearrangements occur around the protein. In our study, we focus on monitoring the early alteration of the lipid structure and dynamics. Starting from the initial structure where the five units of B subunit of CT bind with five GM1, only three of the five units remain bound, and the whole CT is tilted such that the three binding units reach deeper into the membrane. No such preference of the copies of the B subunit is shown in case of mutated LT1, and hence no tilting of the toxin is observed. These two units are more stable than the other three due to interaction with lipids. The lipids that are in contact with those units of the B subunit behave differently from the bulk lipids in case of CT. From the CT-fixed system, we have observed the changes of the local lipids become more considerable than in the case of the CT system. So, taking together these observations, we can
It is observed from Table 1 that A0 is almost 10 times higher for bulk lipids, which shows the restriction of rotation is higher for those. The shorter component is much faster (τ1 = 0.054 ns with A1 = 0.131) for the local lipids than the bulk lipids (τ1 = 0.072 ns with A1 = 0.148), whereas the longer component is slower (τ2 = 3.351 ns with A2 = 0.810) for local lipids than the bulk (τ2 = 1.571 ns with A2 = 0.452). The longer decay time indicates that the head groups of the local lipids are more restricted than the bulk in case of CT bound lipid system. In case of mutated LT1, the A0 is more or less equal for local and bulk lipids. The shorter component for both types of lipids is almost the same. The longer component is a little slower for local lipids. So the rotational autocorrelation of headgroup indicates that in case of mutated LT1, there is no such difference in the rotational behavior of the headgroup of the local lipids than the bulk. Yet the local lipids in the CT-system show different rotational behavior, which may indicate the lipids attached to the CT play a crucial role in the penetration process of the CT, which is not directly observed in the mutated LT1 system. Table 3. Diffusion Coefficient of Lipid
system CT lateral diffusion coefficients (10−7 cm2/s)
LT1 (mutated)
local
bulk
local
bulk
7.15(±0.032)
8.955(±0.034)
6.35(±0.037)
5.9(±0.038)
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conclude that if it will be possible for us to do more prolonged simulation, we may observe the complete alteration of structure and dynamics of lipids near the toxin, which make the complete penetration of toxin. Moreover, we did not find a significant difference in the local and bulk lipids in mutated LT1 from that of CT, which forces us to conclude that LT1 does not follow the same mechanism as CT. The most important observation is the indication of change in lipid curvature from the middle stage of the simulation in case of the CT system, which is known as crucial for protein endocytosis. We have also monitored the difference in the cross-sectional area occupied by the CT-B on the lipid layer and the headgroup of lipids lying under the pentameric CT-B hole. We have noticed that the difference is large enough for the CT penetration. Altogether our results demonstrate from the atomistic interaction of CT with GM1 containing lipid membrane that, after binding of CT to the GM1 containing lipid layer, lipid curvature and protein orientation change. The lipids that are lying just under the CT-B pentameric hole then move to the corner of the lipid monolayer and thus make a void so that CT can pass through it. Thus, our results suggest a mechanism of the early stage alteration of lipid structure and dynamics, which can make a passage for penetration of CT on the membrane surface. As we were interested in monitoring the nature of changes in the membrane that is induced by the CT, our simulation length is believed to be adequate to capture the nature of changes in the lipids surrounding the toxin, which can further throw light on the possible mechanism of action of the toxin.
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ASSOCIATED CONTENT
S Supporting Information *
Data regarding the hydrogen-bond occupancy between lipid and GM1 headgroup with CT-B, the tilt angle of CT-B with lipid monolayer normal, atom density and distance calculation in mutated LT1 system, energy calculations of both CT and mutated LT1 systems, the effect on lipid structure and dynamics upon binding of CT on the fixed-CT system, and the snapshot of cholera toxin with structural details. This material is available free of charge via the Internet at http:// pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected],
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work is partially supported by the Department of Science and Technology, Government of India [project no. SR/S1/PC60/2009], and a fellowship to I.B. through UGC-NET. We are also thankful to the NANO Project (CONV/002/NANORAC/ 2008) of the Department of Chemistry, University of Calcutta, Kolkata, India.
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REFERENCES
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