To gain insight into the atomistic details of membrane fusion induced by fusogenic peptides, molecular dynamic simulations of synthetic peptides, derived from viral fusion proteins, contained in lipid bilayers were performed. A 20 amino acid peptide from the N-terminus of the influenza HA fusion peptide (WT20) assumed the oblique orientation at the interface between water and the membrane made up of dipalmitoylphosphatidylcholine (DPPC)/palmitic acid (PA), as reported previously for different membranes. Simulations of WT20 embedded in bilayer membranes made up of dioleoylphos-phatidylethanolamine (DOPE) and DPPC/PA showed a positive curvature-inducing effect, whereas WT20 showed a negative curvature-inducing effect on a DPPC bilayer. In phase re-constitution analyses starting from a random mixture of DPPC, PA and water molecules, WT20 weakly stabilized an inverted hexagonal phase. In the latter analyses WT20 preferentially assumed a transmembrane orientation as opposed to the interfacial orientation, regardless of the phase to which the system settled (lamellar vs. inverted hexagonal). In another set of analyses using systems containing a water layer between the apposed DPPC/PA (and DOPE) monolayers, the behavior of WT20 during the formation of an intermembrane connection (or stalk) was examined. Comparison among the mutants supports a view that the oblique orientation of WT20 facilitates the perturbation of the lipid-water interface and the stalk formation. Taken together, these results imply that the influenza HA fusion peptide can have substantial effects on the membrane curvature and can assume a wide range of orientation/position in membranes depending on the local environment of the lipid/water system. Its movability and oblique orientation appear to be associated with its ability to perturb membrane/water interfaces.
Fusion of viral and plasma or endosomal membranes is mediated by envelope glycoproteins, known as fusion proteins. For many viruses, fusion requires major structural modification of the fusion protein. Such modification has been established, for example, for influenza virus hemagglutinin (HA) and HIV gp41 proteins [1-6]. The ectodomain of HA has been shown to consist of six polypeptides forming a trimer of HA1-HA2 complexes. The N-terminus of HA2, critical for HA fusion activity, has a highly conserved, hydrophobic sequence, referred to as the fusion domain or fusion peptide (FP) [7,8]. In response to low pH in the endosome, HA changes conformation and projects its fusion peptide towards the target cell membrane [2,9].
Sequence and mutational analyses have identified and characterized FPs of fusion proteins for a number of viruses. Many findings support the direct involvement of FP, which is typically located at the N-terminus, in fusion between viral and cellular membranes (e.g. [7,10-15]). Studies of the full-length fusion protein and in vitro assay of fusion peptide activities provided important clues on protein-induced membrane fusion events. In addition, synthetic FPs have been shown to exhibit membrane fusion and lytic activities [16-26].
The FP of the Newcastle Disease Virus was shown to adopt an oblique angle in a DPPC monolayer [
Recent computational molecular simulations have been extended to studies of phase transitions of lipids, providing opportunities to study how the formation of nonlamellar phases is affected by lipid composition and peptides [
Given the progress in molecular dynamics simulations, one may ask whether membrane fusion induced by FPs can be studied with MD simulations. Several simulation studies of FPs with explicit treatment of the water/lipid membrane have been carried out [45-48]. They showed that the oblique and kinked structure of FPs is stable for at least 5-18 ns in DMPC (dimyristoyl-phosphatidylcholine) bilayer [
First, the equilibrium position/orientation in the bilayer membranes of HAFP was analyzed (in the “equilibrium analysis” section). Second, using relatively large patches of membranes, the effect of HAFP on the membrane curvature was examined (“curvature analysis”). Third, using the phase reconstitution system made up of DPPC/PA/water, the effect of HAFP on the phase behavior was analyzed (“phase analyses”). Finally, in the Discussion section, we show some non-equilibrium MD simulation results to examine the behavior of HAFP and MPER during the formation of an intermembrane connection (“stalk propensity analyses”). Overall results are consistent with recent experimental and theoretical findings, suggesting the potential usefulness of the atomistic simulation approach for studying peptide-induced fusion. However, given the previous simulation studies of HAFP with the DMPC and POPC membranes [46-48], we here attempted to examine the peptide dynamics in lipid structures involving non-lamellar ones. Therefore, the simulation conditions are not physiological and corresponding experiments involving the peptides have not been done. Since this approach cannot stand alone, experimental and structural studies that solidify the hypothesis are necessary.
The GROMACS 3.3.1 program was used for MD simulations [
WT20:GLFGAIAGFIENGWEGMIDG
W14A:GLFGAIAGFIENGAEGMIDG
G1V:VLFGAIAGFIENGWEGMIDG
WT13:GLFGAIAGFIENG
WT8:GLFGAIAG
MPER: ELDKWASLWNWFNITNWLWYIK
Note that W14A and G1V are identical with WT20 except for the altered residue shown with an underline. For the peptides and their mutants, the GROMOS-96 parameter set was used. For water, the simple-point charge (SPC) model [
As stated in the Introduction, this study consists of the equilibrium analysis, the curvature analysis, the phase analysis and the stalk propensity analysis. For all DPPC/PA membranes, the initial coordinates were created by randomly placing the DPPC and PA in a manner shown in [
For the equilibrium analysis (
For the curvature analysis (
water/WT20 1:2:40:1 system with the dihedral restraints 100 kJ/(deg)2 at 283 K (a), 323 K (b) and 353 K (c). (d, e) Results of the same system as in (a-c) but with using 20 kJ/(deg)2 restraints at 323 K (d) and 353 K (e). (f, g) Results of the same system as (a-c) but without dihedral restraints at 323 K (f) and 353 K (g). The average positions of the phosphorus atoms in lipid molecules are shown by arrowheads. (h) G1V data at 323 K with 20 kJ/(deg)2 restraints. (i) G1V data at 323 K without restraints. (j) G1V data at 323 K restrained to the 1IBN (pH 5 NMR-structure of WT20) with 20 kJ/(deg)2 dihedral restraints. (k) W14A at 323 K with 20 kJ/(deg)2 restraints. (l) W14A at 323K without restraints. (m) DPPC/PA/water/MPER 1:2:40:1 system with the 20 kJ/(deg)2 restraints, at 323 K. (n) Same as (m), but without the restraints. In each figure, an open triangle represents the average position of the COM of phosphorus atoms of DPPC, whereas a closed triangle represents that of carbonyl oxygen atoms of DPPC.
bilayer midplane and the COM of the group of the lipid molecules whose x-coordinate of the COM is located within the range covered by the three peptides as shown by the cartoon in the
For the phase analysis (
For the stalk propensity analysis (
One feature of this study is that many simulations utilized restraints on the f and y dihedral angles of the peptide backbone, using the initial structures (i.e., the models from 1IBN and 2PV6) as the reference structures. For the restraints, a coefficient of 100 kJ/(deg)2 (strong restraint) and 20 kJ/(deg)2 (weak restraint) were tried. With the weak restraints at the high temperature (353 K), the helicity of WT20 was preserved, but the kink-like structure was less prominent (
In this study, the z-axis is perpendicular to bilayer. For the secondary structure analysis, the DSSP program was used based on [
First, the equilibrium position and structure of the peptides were analyzed. The positions of the COM of the amino acid sidechains in DPPC/PA/water/WT20 1:2:40:1 simulations were analyzed (Figures 1(a)-1(g)). The composition was chosen because the phase behavior (i.e., lamellar vs. inverted hexagonal) has been studied by both experiments [
As previous simulation studies have described [46-48], the overall structure was V-like in shape, but the angle of V was larger than the pH 5 NMR-structure (PDB code 1IBN) by Han et al. [
When the restraints were weakened to 20 kJ/(deg)2, the general orientations were similar to those found with 100 kJ/(deg)2 restraints (Figures 1(d) and 1(e)). The α-helix content was also similar to that with 100 kJ/(deg)2 restraints (Figures 2(a) and 2(b)). At 353 K, α-helical content was 63.5 ± 10.3% for 100 kJ/(deg)2 and 68.1 ± 8.1% for 20 kJ/(deg)2 for the last 5 ns (Figures 2(a) and 2(b)), similar to ~66.8% for the pH 5 structure (1IBN).
However, when no dihedral restraints were used at 353 K, the orientation was flatter and the helical content decreased to ~45% (
Taken together, without the restraints, WT20 exhibits substantial deformation at 353 K from the pH 5 NMRstructure. Removal of the dihedral restraints resulted in a substantial change in the overall structure and an increase in α-helical content also in the case with G1V and W14A mutants (Figures 1(h), 1(i), 1(k), 1(l) and Figures 2(d), 2(e).). Of note, for G1V and W14A, the restraints based on the reported structures (PDB code 1XOP and 2DCI, respectively) were used. Considering the potential importance of structure, simulations were performed mainly with dihedral restraints of 20 kJ/(deg)2 in the following, whereas some simulations were carried out without the restraints.
The second line of the analyses examined the effect of WT20 on the curvature of bilayer membranes. As
The effect of WT20 on ‘phase behavior’ of the DPPC/ PA/ water system was analyzed in the manner described in [
The effect of WT20 was more pronounced in the simulations performed at 313 K (set-R3 and -R4) than in simulations performed at 333 K; the presence of WT20 increased the propensity of hexagonal phase formation from 35% (set-R3) to 65% (set-R4). These simulations were performed with the 20 kJ/(deg)2 dihedral restraints on WT20 to the 1IBN structure. When the dihedral restraints were removed and 313 K simulations performed, the results were largely similar (set-R5) with 60% were judged to be an inverted hexagonal phase. Overall, although the effect appeared to be subtle, the presence of WT20 caused a small shift toward the hexagonal phase. One notable feature of the results was that the WT20 tended to reside in the hydrophobic part of the bilayer or hexagonal structures, protruding into the lipid acyl chains, with the Nand C-termini interacting with distinct water columns (or layers) (Figures 4(d) and 4(e)). In fact, such position/orientation was observed for all the simulations that assumed an inverted hexagonal phase and for 13 out of the 14 simulations that assumed a lamellar phase. Of course, many more simulations have to be carried out to draw any conclusion regarding the equilibrium position because the amount of water seems to be a critical factor governing the equilibrium position of the peptide. Nonetheless, these features are interesting in that such versatility of WT20 positioning may be relevant to its function in later stages of membrane fusion, such as hemifusion formation and rupture of hemifusion diaphragm.
In the above, the results of the equilibrium analysis, the curvature analysis and the phase analysis of WT20 were shown. In the equilibrium analysis, the oblique orientation of WT20 was largely similar to that reported
previously, despite of our use of the DPPC/PA membrane. At the high temperature, the orientation was less oblique than at the low temperature, yet the introduction of the dihedral restraints restored the oblique angle. This finding may have technical implications for future simulation studies of peptide-induced membrane fusion.
Does WT20 alter the membrane curvature? Experiments using dipalmitoleoylphosphatidylethanolamine (DiDOPE) by Epand and Epand [
The transmembrane orientation of WT20 observed in the phase analysis may be relevant to the later steps of liposome fusion (e.g., the formation of hemifusion and the hemifusion diaphragm rupture), which deserve future analyses. The previous studies using EPR and NMR have unambiguously shown that WT20 resides in the outer leaflet of the bilayer membranes [
HAFP and many other peptides derived from virus fusion proteins exhibit lipid destabilizing and fusion activity against liposomes. So, one could be tempted to ask whether the fusogenicity of such peptides in vitro experiments can be studied in silico. For the DPPC/PA system, the stalk formation is the critical (rate-limiting) step in the transition from a lamellar to an inverted hexagonal phase [
For all five simulations in set 1, a stalk was formed promptly at t = ~1-5 ns (
with the headgroups of lipid molecules of the apposed membrane. Our analyses showed that, once formed, a
stalk consisting of 3-4 lipid molecules tends to grow thicker consisting of > 8 molecules. Stalk formation was typically accompanied by a subtle upward movement of WT20. When the z-position of Asn12 Cα in WT20 was restrained relative to the z-position of the COM of lipid phosphorus atoms of the same (i.e., cis) monolayer, the stalk propensity was weak (set 7). Similar analyses were carried out for the MPER. Figures 1(m) and 1(n) show the equilibrium position of the MPER at 353 K. For both cases with and without the restraints, the helical structure was stable and the orientation was largely parallel to the membrane surface. The stalk propensity of MPER was small (set 13).
The system containing one peptide in each of the apposed monolayers was also examined (i.e., two-peptide analysis). Despite the relatively high hydration level was used (DPPC/PA/water/WT20 = 1:2:30:2, the same as set 4), the stalk propensity was > 0.8 regardless of the configuration. Typically, the stalk formation was preceded by self-association of WT20 (Figures 5(b) and 5(c)). When the z-position of the Asn12 Cα of both WT20 molecules was restrained, no stalk formation occurred. Compared with the one-WT20 simulations, stalk formation was much quicker in the two-WT20 simulations. The association between the two WT20 peptides was typically mediated by ~2-3 hydrogen bonds formed by sidechains of residues Glu11, Asn12 and Glu15 of both peptides (data not shown). When simulations were performed on WT8 (i.e., a segment of eight residues at the N-terminus of WT20), no stalk propensity was found (set 8 and data not shown for the two-peptide system), consistent with the experimental finding [
The following findings implicate the structure and orientation of the peptide for its ability to perturb the membrane-water interface. For most of the stalk propensity analysis, we introduced 20 kJ/(deg)2 restraints on the peptide dihedral angles based on the 1IBN structure. When the restraints were removed, structural variation was large (Figures 1(f) and 1(g) and data not shown) and WT20 showed weak stalk propensity (set 10. The stalk propensity was 0.3 for the 1:2:30:2 system). Further insights may be obtained from mutants G1V and W14A. Gly1, the residue at the N-terminus, is critical for functions of the HA protein [7,33,67]. In the equilibrium analysis, G1V adopted a linear or slightly U-shaped structure (e.g., Figures 1(h) and 1(i)). With the 20 kJ/(deg)2 dihedral restraints at its reported (linear) structure (PDB code: 1XOP), the stalk propensity of G1V was small (set 11. The stalk propensity was 0.3 for the 1:2:30:2 system). When the backbone dihedrals of WT20 were restrained to those of the NMR-determined G1V structure (i.e., linear), WT20 adopted a linear structure and the stalk-inducing activity became very weak, the propensity being 0.1 for the 1:2:30:2 system. Strikingly, when the dihedrals of G1V were restrained to those of the 1IBN (i.e., v-shape), the stalk propensity was high (0.9 for 1:2:30:2). When W14A was examined in the one-peptide system, the propensity was weak (set 12). When two W14A mole cules in anti-parallel and parallel configurations were examined in the 1:2:30:2 system (dihedral restraints set to the reported W14A structure, 2DCI), there was no significant stalk-inducing effect, the propensity being 0.1.
Overall, these results are consistent with the experimental results [
While our stalk propensity results were consistent with experiments, it should be borne in mind that the membranes rich in palmitic acid or DOPE are unphysiological. It should also be stressed that, while we started simulations from lamellar structures, the membrane we used have ‘per se’ a propensity to form non-bilayer structures at the temperature based on the analysis by Knecht et al. [
The amount of water was critical in this study, emphasizing the importance of the dehydration for the membrane fusion. Future directions may involve oligomerization of peptides (not limited to the N-terminal fusion peptides) because the oligomerization may help the dehydration. β-sheet aggregation of HAFP may recruit HA trimers into a fusion site [
To summarize, the dynamics of the HAFP within lipid membranes was studied. When the HAFP was located at the DPPC/PA membrane-water interface, it assumed an oblique orientation as previously reported. The HAFP exhibited a significant effect on the membrane curvature. The effect was dependent on the lipid composition of the membranes: i.e., a positive curvature for the DPPC/PA membrane and the DOPE membrane whereas a negative curvature for the DPPC membrane. In the phase reconstitution analysis starting from the random DPPC/PA system, the HAFP exhibited a weak stabilization of an inverted hexagonal phase, which is suggestive of enhancement of the negative curvature. The distinct effects (i.e., positive vs. negative curvature) observed in the two DPPC/PA systems is likely to be related to the result that in the latter system the HAFP assumed a transmembrane orientation more frequently than an interfacial orientation. It can be envisaged that HAFP induces different types of curvature, depending on lipid composition and on the location and orientation within membrane. HAFP also increased the rate of stalk-formation in our nonequilibrium MD simulations in which the spontaneous stalk formation is slow without peptides. However, there are clearly many difficulties if one tries to interpret rigorously the non-equilibrium simulation results. Many more efforts to bring the system closer to a physiological system and to deal with longer time scales are necessary.
The authors thank the anonymous reviewers for their helpful suggestions. This work was supported by Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.