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Open AccessArticle

Conformational Switching in Bcl-xL: Enabling Non-Canonic Inhibition of Apoptosis Involves Multiple Intermediates and Lipid Interactions

1
Department of Biochemistry and Molecular Biology, The University of Kansas Medical Center, Kansas City, KS 66160, USA
2
Institute of Chemistry and School of Chemistry, V. N. Karazin Kharkiv National University, 4 Svobody Square, 61022 Kharkiv, Ukraine
*
Author to whom correspondence should be addressed.
Cells 2020, 9(3), 539; https://doi.org/10.3390/cells9030539
Received: 27 January 2020 / Revised: 13 February 2020 / Accepted: 17 February 2020 / Published: 26 February 2020
(This article belongs to the Special Issue Regulation of Apoptosis by the Bcl-2 Family of Proteins)
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. View Full-Text
Keywords: Bcl-2 proteins; BH4 domain; apoptotic regulation; conformational switching; protein-membrane interactions; Fluorescence Spectroscopy; Fluorescence Correlation Spectroscopy (FCS); Single Molecule FRET Bcl-2 proteins; BH4 domain; apoptotic regulation; conformational switching; protein-membrane interactions; Fluorescence Spectroscopy; Fluorescence Correlation Spectroscopy (FCS); Single Molecule FRET
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MDPI and ACS Style

Vasquez-Montes, V.; Kyrychenko, A.; Vargas-Uribe, M.; Rodnin, M.V.; Ladokhin, A.S. Conformational Switching in Bcl-xL: Enabling Non-Canonic Inhibition of Apoptosis Involves Multiple Intermediates and Lipid Interactions. Cells 2020, 9, 539.

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