Peptides derived from apoptotic Bax and Bid reproduce the poration activity of the parent full-length proteins

Abstract

Bax and Bid are proapoptotic proteins of the Bcl-2 family that regulate the release of apoptogenic factors from mitochondria. Although they localize constitutively in the cytoplasm, their apoptotic function is exerted at the mitochondrial outer membrane, and is related to their ability to form transbilayer pores. Here we report the poration activity of fragments from these two proteins, containing the first alpha-helix of a colicinlike hydrophobic hairpin (alpha-helix 5 of Bax and alpha-helix 6 of Bid). Both peptides readily bind to synthetic lipid vesicles, where they adopt predominantly alpha-helical structures and induce the release of entrapped calcein. In planar lipid membranes they form ion conducting channels, which in the case of the Bax-derived peptide are characterized by a two-stage pattern, a large conductivity and lipid-charge-dependent ionic selectivity. These features, together with the influence of intrinsic lipid curvature on the poration activity and the existence of two helical stretches of different orientations for the membrane-bound peptide, suggest that it forms mixed lipidic/peptidic pores of toroidal structure. In contrast, the assayed Bid fragment shows a markedly different behavior, characterized by the formation of discrete, steplike channels in planar lipid bilayers, as expected for a peptidic pore lined by a bundle of helices.

FIGURE 1

Effects of lipidic composition on the permeability of LUVs exerted by Bax^α5 (solid lines) and Bid^α6 (dashed lines). The percentage of calcein release, according to Eq. 1, is represented as a function of the concentration of added peptide. Vesicles were prepared with the following compositions: (▪) PC, (•) PA, and (▴) PC/LPC (80:20).

FIGURE 2

Formation of ion channels by Bax^α5 in planar lipid membranes. Addition of Bax^α5 to the buffer bathing the cis side of asolectin planar lipid membranes increases the ion flux across the bilayer. (A) Just upon addition of the peptide, a microchannel-like increase in the ion current was observed at positive potentials. (B) Step-like current increases corresponding to the opening of one large single channel (0.3–5 nS) were observed at several voltages, as indicated in the traces. The reference scales on the bottom right of the traces indicate 5 pA on the y axis and 2 s on the x axis, except in the last trace, where the thicker scale refers to 50 pA on the y axis and 20 s on the x axis. (C) Current/voltage representation of the different permeabilizing activities corresponding to A (○) and B (▪).

FIGURE 3

Ion channel activity displayed by Bid^α6 in planar lipid membranes. When a solution containing Bid^α6 was added to the buffer bathing the cis side of the membrane, discrete increases of the ionic current through the bilayer were observed. Each step-like current increase corresponded to the opening of one single channel with an average conductance of 13–15 pS. The closed state (C) and the different open levels (O_i) are marked with dotted lines, where i is the number of single channels simultaneously open at the indicated voltages. On the righthand side, the occupation histogram of each level is reported. The membrane composition was PC/PA (1:1).

FIGURE 4

CD spectra of Bax^α5 and Bid^α6 in lipid-mimetic media. A and C show spectra of Bax^α5 and Bid^α6, respectively, recorded in aqueous buffer in the absence (dotted lines) or presence (solid lines) of different amounts of TFE. B and D show spectra of Bax^α5 and Bid^α6, respectively, in pure aqueous buffer (dotted lines) or titrated with increasing amounts of SDS (solid lines). In all samples peptides were at a 30 μM concentration, and the aqueous buffer was 10 mM sodium phosphate, pH 7.0. The amounts of added TFE (in percentage) or SDS (in mmol/L) are indicated in the graphs.

FIGURE 5

Infrared attenuated total reflection spectra of Bid^α6 and Bax^α5 in aqueous buffer or TFE solvent. (A) Analysis of the amide I′ band of deuterated films of Bid^α6 samples deposited from TFE (curve a) and from a buffer solution (curve b). The original spectrum (solid line in b) was deconvoluted and curve fitted to resolve the component frequencies. The corresponding Lorentzian bands are reported as dotted lines and their sum (thick dashed line) was superimposed to the original spectrum. Bands labeled as Agg_I and Agg_II derive from aggregated peptide and were excluded from the secondary structure calculation. The other bands are t (β-turn), α₁ and α₂ (α-helix), and r (random coil). The evaluated percentages of secondary structures are reported in Table 3. (B) Same as in A, but for the Bax^α5 peptide.

FIGURE 6

ATR-FTIR spectra of Bid^α6 and Bax^α5 in PC/PA layers and their analysis with polarizer. (A) Deuterated films of Bid^α6 bound to PC/PA vesicles (a, solid line), Bax^α5 bound to PC/PA vesicles (b, dashed line) and PC/PA vesicles alone (c, dotted line). Indicated are the bands corresponding to CH₃ stretching (asymmetric, as, and symmetric, s, at 2956 cm⁻¹ and 2872 cm⁻¹, respectively); CH₂ stretching (asymmetric, as, and symmetric, s, at 2923 cm⁻¹ and 2853 cm⁻¹, respectively); OD stretching of deuterated water; CO stretching of the phospholipid carbonyl groups; amide I′ and II′ bands. (B) Spectra were taken with either parallel (0°) or perpendicular (90°) polarization. The amide I′ region of Bid^α6 bound to PC/PA vesicles was reported after subtraction of the lipid contribution. The best curve fit with Lorentzian components (dotted lines) was superimposed as a thick dashed line to the 90° polarized trace (solid line). The absorption bands in the parallel and perpendicular configuration were used to calculate the orientation of the corresponding structural element as reported in Table 4. Bands are α, α-helix; r, random coil; and Agg, aggregated peptide.

FIGURE 7

Model representing the interaction of Bax^α5 peptides with lipid membranes and the formation of toroidal pores. (A) The two helices, α₁ and α₂, observed by FTIR can be envisioned as an equilibrium between two structural states (column 1). Both states can interact with membranes (column 2), creating a tension that induces the opening of a lipidic pore. The state α₁, acquiring a tilted orientation, interacts with the pore edge, which reduces the curvature stress and stabilizes the pore. The state α₂ remains oriented flat at the membrane interface. Alternatively, α₁ and α₂ are two stretches of the same peptide molecule with slightly different α-helical structure (B). The pore is formed through the same mechanism as above, but the membrane-inserted state is represented by a kinked peptide, where the part encompassing α₂ remains aligned flat with respect to the membrane plane, whereas the stretch corresponding to α₁ is tilted over the rim of the pore. Helix α₁ can be adscribed to the more hydrophobic, N-terminal half of the peptide (see text).