emphasize that the free-energy of the stalk intermediate, i.e. the stalk barrier, mainly depends on the distance between the opposing leaflets and is rather independent of the leaflet’s curvature [20,39,69]. Thus, the important role of the dimple is to bring the leaflets into close proximity. To this aim, a dimple lowers the energetic cost of leaflet approach, i.e. the inter- membrane repulsion, both by reducing the effective contact area of the fusion site and by increasing its surface hydrophobicity (i.e., curvature stress) [39]. Its direct observation, however, suggests that the peptide-coated dimple might not suffice in bringing the membranes sufficiently close. In these experiments [6,7], fusion was only observed after formation of a funnel-like intermediate in the target membrane. Notably, a bundle allows an additional proximity by tilting the membrane and such a ‘volcano’ structure substantially eases stalk formation (see Fig. S3). To this end, our simulations offer a new interpretation for a number of known features of the fusion reaction mediated by the prototype fusion protein, influenza hemagglutinin, and might bring new insights into mechanisms of other viral fusion reactions. We hypothesize that such a line-tension controlled fusion mechanism, which closely resembles the mechanism of electrofusion [43], might be the direct evolutionary consequence of the substantial free-energy barriers [4,5,27] that the viral fusion machinery faces in its attempt to fuse with membranes that are, in turn, evolutionary designed to be largely resistant to (viral) fusion. It is a great pleasure to thank Michael Schick, Reinhard Jahn, Leonid Chernomordik and Volker Knecht for stimulating discussions and constructive comments. Financial support has been provided by the DFG under grant SFB 803/B2-B3. Methods Pore- and Stalk Formation The hydrophilic pore of radius R = 2.0 nm, located in one of the bilayers, was stabilized using an repulsive potential U (ri) with U (ri)~1=2k(ri{R)2, if rivR and U (ri)~0 if ri§R, where ri denotes the distance of the center of mass of the lipid from the pore center and k a force constant (k = 50 kJ nm{2 mol{1) [41,70]. Likewise, we induced the initial stalk in the bilayer fusion setup by applying an external field. Here, we applied the same harmonic potential to induce a R = 1.0 nm ‘void’ in the solvent layer between the bilayers. The hydrophobic nature of the void attracts the lipid tails in the adjacent leaflets and results in the formation of a stalk. After the stalk formation, the external field has been removed to allow for an additional 4 ns equilibration of the stalk structure. Peptide Model All peptides used consisted of 20 aminoacids: GLFGAIAG- FIENGWEGMIDG (wild-type). The Martini coarse-grained model [28,29] captures the specific nature of each individual amino acid but does not predict secondary structure. The secondary structure and protonation state of the wild-type peptide (pdb:1IBN) and mutants (pdb:16OP, pdb:2DCI, pdb:16OO, pdb:2L4G) were derived from the NMR-resolved structures [15,71]. The secondary structure was modeled by both restraining proper dihedrals between four neighboring backbone beads with an harmonic potential and by altering the non- bonded interactions according to the imposed secondary structure (free in solution, or in a coil or bend the backbone has a more polar character than in a helix or b-strand). Further details concerning this methodology can be found in the original publication [29]. To reproduce the experimentally observed Influenza Fusion Mechanism ‘fixed’ angle between the two helices (the boomerang shape) [15,22,71] additional dihedral angle potentials were explicitly introduced for the backbone beads of residue 8 to 16. Further details are given in Ref. [23]. Recent atomistic simulations suggested that the ‘kink’ is in fact less preserved in the G1S mutant than in the wild-type [22]. We corrected for the latter effect by allowing a slightly larger flexibility between the two helices (i.e., without the additional dihedral restraints). For mutant G13A, an additional elastic network (Force constant of 500 kJ nm{2 Mol{1) between the nearest backbone beads (cutoff between 0.5–0.9 nm) was applied to conserve its shallow 150 degrees tilt angle. Atomistic simulations of the wild-type in an implicit membrane environment suggested that the NMR- derived kink (secondary structure) is altered when the peptides closely pack into an trans-membrane arranged bundle/oligomer [72]. Although such prediction is beyond the capability of our model, we emphasize that both a linear and kinked wild-type peptide stabilizes the bundle in our simulations (see the section about mutant G1V), whereas a linear or kinked G1V mutant does not. Peptide Bundle Stable peptides pores were formed by simulating 8 peptides, adhered to the membrane surface of a 10|10 nm POPC bilayer (128 lipids), in the presence of externally stabilized hydrophilic pores with a diameter of 2–3 nm (see above). The peptides (both wild types and mutants) showed a remarkable attraction towards the pore’s rim. The external pore-stabilizing potential was removed after all peptides were assembled into the pore. We found that at least 4 peptides were required to form self-stabilized bundles. We additionally tested such scenario in pure DOPC, pure DOPE and pure POPE bilayers and found that the formed bundles were stable in all cases. Furthermore, these wild-type bundles were found to be stable at both 310 and 350 K in the course of the 10 ms simulation (in total about 20 simulations were preformed). In addition, we also tested the stability of the wild- type bundle with all the peptides oriented in the same direction, and the stability of the bundle in the polar Martini forcefield [73] in the presence of long-range electrostatic effects (PME). In all cases the bundle was found to be stable. To investigate the stability of the mutant bundles, the G1S, G1V, and W14A mutations were performed on the stable and equilibrated wild-type bundle (at 350 K). Statistics was collected by performing multiple simulations (5 for each mutant) with different starting velocities. Bundle-facilitated Stalk Elongation The bundle-facilitated stalk elongation was performed within the same system as the hydrophilic pore facilitated stalk elongation (DOPC, 1152 lipids per membrane, 16 water molecules per lipid within the inter-membrane space). Here, a pre-formed and equilibrated bundle was carefully embedded in a constructed pore and was additionally equilibrated for 40 ns. Then the stalk was induced according to the procedure described above. To be able to capture the bundle-facilitated stalk elongation (which is a nucleated event) within the limited time scales of the simulation, the simulations were performed at a slightly elevated temperature of 350 K (instead of 310 K). We emphasize that the absence of the bundle or pore also destabilizes the stalk at 350 K. The G1S mutation was performed on the readily equilibrated ‘stalk-bundle’ setup of the wild-type. Statistics was collected by performing multiple simulations (5 with for each) with different starting velocities. PLoS ONE | www.plosone.org 12 June 2012 | Volume 7 | Issue 6 | e38302