Energetics of stalk intermediates in membrane fusion are controlled by lipid composition

Abstract

We have used X-ray diffraction on the rhombohedral phospholipid phase to reconstruct stalk structures in different pure lipids and lipid mixtures with unprecedented resolution, enabling a quantitative analysis of geometry, as well as curvature and hydration energies. Electron density isosurfaces are used to study shape and curvature properties of the bent lipid monolayers. We observe that the stalk structure is highly universal in different lipid systems. The associated curvatures change in a subtle, but systematic fashion upon changes in lipid composition. In addition, we have studied the hydration interaction prior to the transition from the lamellar to the stalk phase. The results indicate that facilitating dehydration is the key to promote stalk formation, which becomes favorable at an approximately constant interbilayer separation of 9.0 ± 0.5 Å for the investigated lipid compositions.

Fig. 1.

(A) Cartoon of the cross-section of a stalk. (B) Arrangement of stalks in the nonprimitive hexagonal unit cell of the stalk phase spanned by vectors , , . Shown is the electron density isosurface Δρ = 0.3 (DOPC/DOPE 1∶1, RH = 74%). (C) Grazing-incidence X-ray diffraction pattern of the stalk phase (DPhPC, RH = 70%, recorded at the MS beamline, Swiss Light Source) composed of four overlapping Pilatus images. An attenuator with a transmission of 10^-3 was used in case of the detector position covering primary and specular beam.

Fig. 2.

Bilayer structure and interactions as determined from electron density profiles: (A) Typical X-ray reflectivity data indicating eight clearly resolved orders of diffraction, (B) corresponding electron density profiles Δρ(z) (shifted vertically for clarity) reconstructed by aid of the swelling method, (C) structural data d,d_hh,d_w for DOPC/DOPE mixtures and (D) pressure-distance curves of all investigated lipids. The hydration properties of the branched-chain lipid DPhPC clearly deviate from those of di-monounsaturated PC lipids. Addition of DOPE or cholesterol facilitates dehydration and therefore close bilayer contact. For all investigated samples, stalk phase formation becomes favorable at d_w < 9.0 ± 0.5 Å. In case of DOPC, curves Π(d_w) from two independent measurements are shown.

Fig. 3.

Side-by-side comparison of different stalks: (A) Slices through the xz plane of a stalk in different lipid systems. The colorbar applies to all four density maps. (B) The density map obtained by a recent MD simulation using POPC (46) shows a very similar stalk architecture. Shown is the lipid headgroup number density. Fig. 3B adapted with permission from ref. . Copyright (2010) American Chemical Society.

Fig. 4.

Quantitative analysis of the stalk phase: (A) Definition of structural parameters by local electron density maxima: Electron density contrast in the xy plane and corresponding stalk waist diameter d_s and maximum electron density contrast (left), slices through a stalk in the xz and yz plane (center) and slice through the stalk phase in the yz plane and electron density along the white line in vertical direction (right) (DOPC/DOPE 1∶1, RH = 74%). (B) Summary of results for eight different datasets. In the bottom box, the dashed line indicates the value , at which the transition from the lamellar to the rhombohedral phase starts. For most lipid compositions, formation of the stalk phase is associated with an increase of d_w.

Fig. 5.

Evaluation of lipid monolayer curvature by electron density isosurface analysis: (A) Electron density isosurface for DOPC/DOPE 1∶1 (RH = 70%). (B) Isocontours Δρ = 0.3 at different hydration levels for stalks formed by pure DOPC and DOPC/DOPE 1∶1 indicating a very similar structure. (C) Principle curvatures c_1,2, mean curvature H and Gaussian curvature K for (top view) and (D) results of the integrals Σ₁, Σ₂, A, ∫KdA.

Fig. 6.

Combining our structural results with values for bending modulus κ and spontaneous curvature c₀ allows to estimate the bending energy of a stalk: (A) Bending energy term κ(Σ₁ + c₀Σ₂) for the case of DOPC (κ = 9 k_BT, c₀ = -0.0115 Å^-1) and an equimolar DOPC/DOPE mixture (κ = 9 k_BT, c₀ = -0.024 Å^-1) as a function of the isodensity value ρ_iso The values of κ and c₀ were obtained from (16), in case of the lipid mixture, molar fraction-weighted values are used. (B) The same energy for a fixed isosurface (DOPC/DOPE 1∶1, ρ_iso = 0.34) as a function of κ and c₀. If c₀ becomes slightly more negative upon dehydration, as explained in the main text, κ(Σ₁ + c₀Σ₂) may approach negative values.

Fig. P1.

(A) 2D Electron density representations (DOPC/DOPE 1∶1) of two lipid bilayers (left) and of a slice through a stalk (right). Regardless of the molar fraction of the nonbilayer lipids DOPE or cholesterol, stalk phase formation occurred at a critical interbilayer distance . The dashed black lines indicate contours of constant electron density contrast Δρ = 0.3. (B) Corresponding 3D electron density isosurface and the local distribution of mean curvature H. (C) Energy landscape of the bending energy (κ/2)∫(2H - c₀)²dA (left). Published values of the elastic coefficients lead to about 15 k_BT. Pressure-distance curves P(d_w) in the lamellar phase (right) allow determination of the energy required for dehydration to .