Fusion peptides promote formation of bilayer cubic phases in lipid dispersions. An x-ray diffraction study

Abstract

Small angle x-ray diffraction revealed a strong influence of the N-terminal influenza hemagglutinin fusion peptide on the formation of nonlamellar lipid phases. Comparative measurements were made on a series of three peptides, a 20-residue wild-type X-31 influenza virus fusion peptide, GLFGAIAGFIENGWEGMIDG, and its two point-mutant, fusion-incompetent peptides G1E and G13L, in mixtures with hydrated phospholipids, either dipalmitoleoylphosphatidylethanolamine (DPoPE), or monomethylated dioleoyl phosphatidylethanolamine (DOPE-Me), at lipid/peptide molar ratios of 200:1 and 50:1. All three peptides suppressed the HII phase and shifted the L(α)-H(II) transition to higher temperatures, simultaneously promoting formation of inverted bicontinuous cubic phases, Q(II), which becomes inserted between the L(α) and H(II) phases on the temperature scale. Peptide-induced Q(II) had strongly reduced lattice constants in comparison to the Q(II) phases that form in pure lipids. Q(II) formation was favored at the expense of both L(α) and H(II) phases. The wild-type fusion peptide, WT-20, was distinguished from G1E and G13L by the markedly greater magnitude of its effect. WT-20 disordered the L(α) phase and completely abolished the HII phase in DOPE-Me/WT-20 50:1 dispersions, converted the Q(II) phase type from Im3m to Pn3m and reduced the unit cell size from ∼38 nm for the Im3m phase of DOPE-Me dispersions to ∼15 nm for the Pn3m phase in DOPE-Me/WT-20 peptide mixtures. The strong reduction of the cubic phase lattice parameter suggests that the fusion-promoting WT-20 peptide may function by favoring bilayer states of more negative gaussian curvature and promoting fusion along pathways involving Pn3m phase-like fusion pore intermediates rather than pathways involving H(II) phase-like intermediates.

Figure 1

The modified stalk mechanism of membrane fusion and inverted phase formation. (A) Planar lamellar (L_α) phase bilayers; (B) the stalk intermediate; the stalk is cylindrically symmetrical about the dashed vertical axis; (C) the TMC (trans monolayer contact) or hemifusion structure; the TMC can rupture to form a fusion pore, referred to as interlamellar attachment, ILA (D); (E) If ILAs accumulate in large numbers, they can rearrange to form Q_II phases. (F) For systems close to the L_α/H_II phase boundary, TMCs can also aggregate to form H_II precursors and assemble into H_II domains (reproduced from (20) with permission).

Figure 2

Sequences of XRD patterns recorded during heating-cooling scans of DPoPE/fusion peptide mixtures (heating 1°C/min, cooling 3°C/min). (A) DPoPE control (no peptide); (B) DPoPE/WT-20; (C) DPoPE/G1E; (D) DPoPE/G13L. Lipid/peptide molar ratios 50:1.

Figure 3

Comparison of the XRD patterns recorded at 20°C from DPoPE/fusion peptide mixtures after heating-cooling scans shown in Fig. 2. The lattice parameters of the phases are summarized in Table 1.

Figure 4

Evolution of the Pn3m phase lattice parameter of DPoPE/fusion peptide dispersions during heating-cooling cycles.

Figure 5

Upward shift of the L_α → H_II transition in a 50:1 DPoPE/WT-20 50:1 mixture (solid symbols) with respect to the pure DPoPE dispersions (open symbols).

Figure 6

L_α and H_II spacings of DPoPE (solid symbols) and 50:1 DPoPE/fusion peptide mixtures: WT-20 (squares), G1E (triangles), G13L (circles).

Figure 7

Phase conversions during heating (1°C/min) and cooling (5°C/min) of 10 wt % DOPE-Me dispersions (A) and DOPE-Me/WT-20 mixtures at molar ratios 200:1 (B) and 50:1 (A). PBS, pH 7.2. The cubic phase lattice constants are (A) Im3m 38 nm; (B) Im3m 24.2 nm/Pn3m 19.0 nm mixture; (C) Pn3m 14.8 nm (Table 1).

Figure 8

Phase transformations during heating (1°C/min) and cooling (5°C/min) of 10 wt % DOPE-Me/G1E (A) and DOPE-Me/G13L (B) mixtures at lipid/peptide molar ratio 50:1. The lattice constants of the final cubic phases are given in Table 1.

Figure 9

Comparison of XRD patterns recorded at 20°C from DOPE-Me/fusion peptide (50:1) mixtures after heating-cooling scans. (A) pH 7.2; (B) pH 5.0. The lattice constants of the cubic phases are summarized in Table 1.