Structure of transmembrane pore induced by Bax-derived peptide: evidence for lipidic pores

Abstract

The structures of transmembrane pores formed by a large family of pore-forming proteins and peptides are unknown. These proteins, whose secondary structures are predominantly alpha-helical segments, and many peptides form pores in membranes without a crystallizable protein assembly, contrary to the family of beta-pore-forming proteins, which form crystallizable beta-barrel pores. Nevertheless, a protein-induced pore in membranes is commonly assumed to be a protein channel. Here, we show a type of peptide-induced pore that is not framed by a peptide structure. Peptide-induced pores in multiple bilayers were long-range correlated into a periodically ordered lattice and analyzed by X-ray diffraction. We found the pores induced by Bax-derived helical peptides were at least partially framed by a lipid monolayer. Evidence suggests that the formation of such lipidic pores is a major mechanism for alpha-pore-forming proteins, including apoptosis-regulator Bax.

Fig. 1.

Two models of peptide-induced pores in membranes and the experimental proof for each. (A and B) Schematics of the barrel-stave model (A) and the toroidal model (B) are shown in a 3D view cut through the pores, showing only the layers representing the lipid headgroups (silver) and the Br atoms (red). Blue-gray cylinders represent peptide segments; the actual distribution and orientation of peptides are expected to vary with peptide and lipid. Schematic lipid molecules [1,2-distearoyl(9–10dibromo)-sn-glycero-3-phosphocholine] are shown in A, where the red dots represent the Br atoms on the chains. (C and D) The normalized electron density distributions of Br atoms in a unit cell of the R phase containing alamethicin and Bax-α5, respectively. (The data for C were reproduced from ref. .) Br atoms are distributed in the high-density (yellow-red-black) region. The nonuniformity in the low-density region (light blue to dark blue, which average to ≈0 e/ų) is due to the limited resolution (28).

Fig. 2.

Grazing-angle diffraction pattern from the R phase of a Bax-α5 and di18:0(9,10Br)PC mixture in the molar ratio of 1:30 at 60% RH, 25 °C. The vertical (q_z) is normal to the substrate. To show all peaks clearly, intensity attenuators were applied to the peaks on q_z and on the 2 columns next to q_z.

Fig. 3.

Multiwavelength anomalous diffraction analyses for the detected peaks. For each independent peak, the square root of the integrated intensity, ∣F_λ∣, is plotted as a function of ∣f′λ∣/fⁿ. The data are fit with a straight line, from which ∣F₀∣, ∣F₂∣, and the ratio ∣F₀∣/∣F₂∣ are obtained. The results are in Table 1.

Fig. 4.

Three-dimensional swelling method for phase determination applied to the rhombohedral phase (42). Shown is the swelling method applied to different q_r series using the diffraction amplitudes obtained at 50% RH (open circles) and 60% RH (filled circles). (A) q_r = 0 nm⁻¹ series. (B) q_r = 1.11 nm⁻¹ series. (C) q_r = 1.92 nm⁻¹ series. The vertical scales are relative. The solid curves are F(q_H,K,q_z) of equation 12 in ref. constructed from 1 set of data measured at 1 relative humidity (see details in ref. 42).