Molecular View of the Role of Fusion Peptides in Promoting Positive Membrane Curvature

Fusion peptides are moderately hydrophobic segments of viral and nonviral membrane fusion proteins that enable these proteins to fuse two closely apposed biological membranes. In vitro assays furthermore show that even isolated fusion peptides alone can support membrane fusion in model systems. In addition, the fusion peptides have a distinct effect on the phase diagram of lipid mixtures. Here, we present molecular dynamics simulations investigating the effect of a particular fusion peptide, the influenza hemagglutinin fusion peptide and some of its mutants, on the lipid phase diagram. We detect a systematic shift toward phases with more positive mean curvature in the presence of the peptides, as well as an occurrence of bicontinuous cubic phases, which indicates a stabilization of Gaussian curvature. The wild-type fusion peptide has a stronger effect on the phase behavior as compared to the mutants, which we relate to its boomerang shape. Our results point to a different role of fusion peptides than hitherto assumed, the stabilization of pores rather than stalks along the fusion pathway

(A) Overlap between the coarse-grained model (backbone red and side-chains yellow) of the wild-type influenza fusion peptide and the NMR structure [<b>71</b>].

<p>The two helices are joined by a linker region at a slightly bent angle (boomerang-shape). (B) The wild-type influenza fusion peptides (side-chains not shown) aggregate into a stable hexameric bundle. The bundle interior is depleted in solvent (colored blue) and lipid head groups. For sake of clarity, the first backbone residue (Gly1) is colored yellow. (C) Top view of the bundle. The bundle’s interior is mainly composed of the hydrophilic residues Glu11 (colored blue) and Asn12 (colored green) that are located in the kinked region of the peptide and which point toward the central axis of the bundle.</p

Interaction between multiple peptide bundles.

<p>(upper panel) Four wild-type bundles (top-view). The bundles strongly repel each other and maximize their separation distance in the course of the simulation. Eventually one of the bundles vanishes. (middle panel) Four G13A mutant bundles. The bundles are attractive and their coalescence results in a ‘super’ bundle consisting of 10 trans-membrane arranged peptides. (lower panel) Aggregation number of the largest bundle in the course of the simulation (Only the trans-membrane arranged peptide are counted). The brown line shows a separate simulation where the G13A mutation is reversed after 20 s (G13A -> wild-type). The wild-type ‘super’ bundle readopts its usual size in the course of the simulation.</p

A -shaped hemifusion diaphragm (-HD) which is generated by a stalk that has encircled a membrane pore.

<p>A -shaped hemifusion diaphragm (-HD) which is generated by a stalk that has encircled a membrane pore.</p

Standard stalk-hemifusion pathway (cross-section, side-view): The initial stalk (I) radially expands (II) forming an H-shaped hemifusion diaphragm (H-HD) after the trans-leaflets (colored yellow) meet (III).

<p>When the H-HD ruptures a fusion pore is formed (IV).</p

The G1S mutation reverses the stalk elongation process facilitated by the wild-type.

<p>Notice the removal of solvent (colored blue) and lipid head-groups (colored tan) from the membrane interior when the peptide bundle ‘reseals’ itself – the stalk and peptide are competitive lineactants.</p

Evolution of a stalk in the absence and presence of a pore.

<p>For sake of clarity the size of the lipid headgroups is exaggerated (solvent is not shown). (A) Two apposed DOPC bilayers. A preformed stalk is not stable (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038302#pone-0038302-g004" target="_blank">Fig. 4</a>). (B) A stable ‘hour-glass shaped’ stalk structure formed between a DOPE and DOPC bilayer (4 µs). (C) Elongation of a stalk formed between two DOPE bilayers (4 µs). (I-III) Evolution of a stalk formed between two DOPC bilayers in the vicinity of a pore (stalk-pore complex). Elongation of the stalk, which circumvents the pore, results in the formation of a -shaped hemifusion diaphragm (-HD).</p

Evolution of the stalk in the presence of the peptide bundle.

<p>For sake of clarity the size of the lipid headgroups is exaggerated (solvent is not shown). (A) The elongated stalk (wild-type peptides) after 0.4 s. The bundle has opened up and the stalk and has partly surrounded the formed hole. Notice the readily adopted banana-shape. The stalk forces the peptides to the remaining rim portion. At this stage mixing occurs between both the <i>cis</i>-leaflets and the <i>trans</i>-leaflet of the target membrane (colored gray), while the <i>cis</i>-leaflet of the host cell (colored yellow) does not contribute to lipid mixing. (B) Mutating a single residue in the peptides, Gly1 to Ser1 (colored green), known as the terminal hemifusion mutant G1S <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038302#pone.0038302-Qiao1" target="_blank">[14]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038302#pone.0038302-Lai2" target="_blank">[25]</a>, stabilizes both bundle and stalk but inhibits elongation of the stalk (10 s). Consequentially, the fusion reaction becomes trapped. Note that lipid head-groups are excluded from the pore interior and the <i>trans</i>-leaflets (colored yellow) are hindered from participating in the lipid mixing.</p

Stalk evolution in response of removing the pore (top view on porated bilayer).

<p>(I,II,III) Sudden removal of the pore <i>before</i> completion of the -HD reverses the stalk elongation process, and the stalk completely disappears (DOPC at 310 K, with water molecules per lipid between the membranes). Hydrophobic lipid tails are colored grey, polar-headgroups (DOPC) tan.</p

Detailed view of the ‘super’ bundle (10 s) formed by the leaky fusion mutant G13A (Top view, cross-section through the bilayer center).

<p>Notice that residue 13 (colored green) directly faces the hydrophobic lipid rim around the bundle. The solvent (colored blue) in the center of the bundle suggests the occurrence of leakage prior to lipid mixing <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038302#pone.0038302-Lai1" target="_blank">[11]</a>.</p