Abstract
The inhibition of mitochondrial permeabilization by the anti-apoptotic protein Bcl-xL is crucial for cell survival and homeostasis. Its inhibitory role requires the partitioning of Bcl-xL to the mitochondrial outer membrane from an inactive state in the cytosol, leading to its extensive refolding. The molecular mechanisms behind these events and the resulting conformations in the bilayer are unclear, and different models have been proposed to explain them. In the most recently proposed non-canonical model, the active form of Bcl-xL employs its N-terminal BH4 helix to bind and block its pro-apoptotic target. Here, we used a combination of various spectroscopic techniques to study the release of the BH4 helix (α1) during the membrane insertion of Bcl-xL. This refolding was characterized by a gradual increase in helicity due to the lipid-dependent partitioning-coupled folding and formation of new helix αX (presumably in the originally disordered loop between helices α1 and α2). Notably, a comparison of various fluorescence and circular dichroism measurements suggested the presence of multiple Bcl-xL conformations in the bilayer. This conclusion was explicitly confirmed by single-molecule measurements of Förster Resonance Energy Transfer from Alexa-Fluor-488-labeled Bcl-xL D189C to a mCherry fluorescent protein attached at the N-terminus. These measurements clearly indicated that the refolding of Bcl-xL in the bilayer is not a two-state transition and involves multiple membranous intermediates of variable compactness.
Highlights
The anti-apoptotic protein Bcl-xL is a member of the Bcl-2 family of apoptotic regulators [1,2].Its role in the cell is to block the mitochondrial outer membrane permeabilization (MOMP) caused by pro-apoptotic Bcl-2 proteins (i.e., BAX) (Figure 1a, green) [3,4]
The addition of large unilamellar vesicles (LUV) composed of the anionic lipid cardiolipin (TOCL) and the zwitterionic lipid phosphatidylcholine (POPC) at a 1:2 molar ratio had no effect at pH 8 (Figure 3b, orange)
Hyperactive apoptosis contributes to neurodegeneration and immunodeficiency, while insufficient apoptosis leads to autoimmunity and cancer, and the ability of cancer cells to avoid apoptosis significantly complicates treatment [36]
Summary
The anti-apoptotic protein Bcl-xL is a member of the Bcl-2 family of apoptotic regulators [1,2]. Its role in the cell is to block the mitochondrial outer membrane permeabilization (MOMP) caused by pro-apoptotic Bcl-2 proteins (i.e., BAX) (Figure 1a, green) [3,4]. The prevailing Embedded Together model of MOMP regulation postulates that membrane interactions are critical for both pro- and anti-apoptotic activities of Bcl-2 proteins [5,6]. Bcl-xL is expressed in the cytosol in an inactive state and must redistribute to the MOM where it refolds to become active. Bcl-xL promotes cell survival by inhibiting the formation of multimeric BAX pores and forming non-productive Bcl-xL/BAX heterodimers at the MOM (Figure 1a, purple) [7,8]. Recent evidence suggests that Bcl-xL is a target of BH3-only apoptotic triggers [9,10]
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