Lipid-modulation of membrane insertion and refolding of the apoptotic inhibitor Bcl-xL

Abstract

Bcl-xL is a member of the Bcl-2 family of apoptotic regulators, responsible for inhibiting the permeabilization of the mitochondrial outer membrane, and a promising anti-cancer target. Bcl-xL exists in the following conformations, each believed to play a role in the inhibition of apoptosis: (a) a soluble folded conformation, (b) a membrane-anchored (by its C-terminal α8 helix) form, which retains the same fold as in solution and (c) refolded membrane-inserted conformations, for which no structural data are available. Previous studies established that in the cell Bcl-xL exists in a dynamic equilibrium between soluble and membranous states, however, no direct evidence exists in support of either anchored or inserted conformation of the membranous state in vivo. In this in vitro study, we employed a combination of fluorescence and EPR spectroscopy to characterize structural features of the bilayer-inserted conformation of Bcl-xL and the lipid modulation of its membrane insertion transition. Our results indicate that the core hydrophobic helix α6 inserts into the bilayer without adopting a transmembrane orientation. This insertion disrupts the packing of Bcl-xL and releases the regulatory N-terminal BH4 domain (α1) from the rest of the protein structure. Our data demonstrate that both insertion and refolding of Bcl-xL are modulated by lipid composition, which brings the apparent pK_a of insertion to the threshold of physiological pH. We hypothesize that conformational rearrangements associated with the bilayer insertion of Bcl-xL result in its switching to a so-called non-canonical mode of apoptotic inhibition. Presented results suggest that the alteration in lipid composition before and during apoptosis can serve as an additional factor regulating the permeabilization of the mitochondrial outer membrane.

FIGURE 1.. Bcl-xL membrane targeting/insertion and topology of membrane inserted form.

(a) The inactive form of the anti-apoptotic protein Bcl-xL exists in a soluble state that must interact with membranes to transition to its active conformation. (b) The targeting of Bcl-xL to the membrane leads to its anchoring through its C-terminal α8 helix. (c) The conformation of membrane inserted Bcl-xL, however, has not yet been determined. Here we characterize the lipid determinants that regulate the insertion of Bcl-xL into membranes. Additionally, we report the release of its N-terminal BH4 domain and its link to Bcl-xL insertion and the topology of its hydrophobic α6 helix.

FIGURE 2.. Protonation-dependent membrane insertion and refolding of Bcl-xL.

(a) Representative spectra of Bcl-xL ΔTM N175C-NBD inserting into cardiolipin containing LUV (1TOCL:2POPC). Increase in fluorescence intensity and blue shift in spectral position of maxima observed upon acidification in the presence of membranes are indicative of the membrane insertion of the NBD-labeled site. (b) Changes in fluorescence emission spectra of donor- and acceptor-labeled Bcl-xL ΔTM upon acidification in the presence of 1TOCL:2POPC LUV. The release of BH4 domain was determined by the loss of FRET between Alexa 488 (donor), attached to single-Cys mutant D189C, and mCherry (acceptor) fused to the N-terminus next to the BH4 domain. (c) Comparison of protonation-dependent insertion and refolding of Bcl-xL (see text for details). Both datasets can be accurately described by a single global fitting curve (blue).

FIGURE 3.. Membrane insertion of Bcl-xL is modulated by cardiolipin content.

Insertion of Bcl-xL was measured as a function of pH into membranes with increasing cardiolipin molar contents. Measurements were performed as in Fig. 2 using either Bcl-xL ΔTM (a) or full-length Bcl-xL (b). Increase in content of anionic cardiolipin shifts membrane insertion towards more neutral pH. This leads to a shift in the insertion pK_a by 1.0 pH unit between low cardiolipin (1TOCL:6POPC) and high cardiolipin (3TOCL:2POPC) membranes. The presence of the C-terminal α8 helix in the full-length protein does not affect this lipid-dependent modulation of Bcl-xL insertion.

FIGURE 4.. Effect of membrane surface potential (Ψ_o) on the insertion of α6 and release of Bcl-xL BH4 Domain.

Bcl-xL membrane insertion and the release of its N-terminal BH4 domain were characterized in several anionic lipid compositions. (a) Protonation-dependent free energy of transmembrane insertion (ΔG_TM^H+) was calculated from the measured transition pK_a and plotted as a function of membrane surface potential (Ψ₀). The membrane insertion of α6 is modulated by the bilayer anionic content and lipid geometry, giving rise to two different slopes. Closed symbols represent measurements performed with the Bcl-xL ΔTM variant, while open symbols denote the results using full-length Bcl-xL. (b) Release of the N-terminal BH4 domain was modulated in the same fashion as membrane insertion. The calculated protonation-dependent free energy for the release of the BH4 domain (ΔG_BH4^H+) overlapped with the previously observed membrane insertion slopes in Fig. 4a. These results confirm that both, the insertion of α6 and the release of the N-terminal BH4 domain correlate in all lipid compositions.

FIGURE 5.. EPR O₂/NiEDDA accessibility of MTSL labeled helix α6 in membrane inserted Bcl-xL.

Accessibility of spin labeled Bcl-xL ΔTM single-Cys mutants inserted into 75POPG:25POPC (orange) LUV at pH 4.5. Residues that were also measured in 3TOCL:2POPC LUV at pH 4.5 are indicated in green. Results are plotted as the ratio in quenching between membrane accessible O₂ and water-soluble Ni-EDDA for each residue tested. Accessibility was determined as described in the methods section. A Φ > 0 represents membrane-protected spin probes and a Φ < 0 denotes a more solvent exposed probe. The black trace represents a cosine fit of the quenching data, which yielded a periodicity of 3.6 residues, consistent with an α-helix. The asymmetric protection of spin-labeled residues along α6 from O₂/NiEDDA indicates an asymmetrically solvated α-helix.

FIGURE 6.. Depth-dependent fluorescence quenching of NBD selectively attached along the α6 helix in membrane inserted Bcl-xL.

The following positions along α6 were selectively labeled (top to bottom): 177, 169, 168 and 161. Insertion of Bcl-xL ΔTM NBD-labeled mutants was initiated by mixing the samples incubated with 3TOCL:2POPC LUV (no quencher composition) at pH 4.5. The vesicles that contained quenchers had 30% of one of the five spin-labeled lipids used substituting an equimolar concentration of POPC. Left panels: Differential Quenching Profiles for each tested mutant were obtained by subtracting the dynamic quenching component from steady-state quenching measurements (original data are presented in Fig. S2). The most probable depth (h_m) of each labeled residue and the width of the transverse distribution (σ) were obtained by fitting the data to Eq. 5. Panels on the right: Support-plane analysis of the robustness of the fits used in the determination of the bilayer depth (see text for details). (i) All labeling sites along α6 were 8–16 Å away from the center of the bilayer, eliminating the possibility of a transmembrane orientation. The interfacial orientation for α6 is also consistent with EPR O₂/NiEDDA accessibility measurements (Fig. 5).