Membrane Insertion Pathway of the Apoptotic Repressor Bcl-xL: How (DIS)Similar is it to that of Diphtheria Toxin T-Domain?

Abstract

The anti-apoptotic repressor Bcl-xL inserts into the mitochondrial membrane as a supposed part of its physiological action. While the exact molecular mechanism of this transition is poorly understood, the structural similarity of the water-soluble state of Bcl-xL with that of the diphtheria toxin T-domain led to the suggestion that their membrane-insertion pathways will be similar as well. Here we test this hypothesis by applying an array of spectroscopic methods to characterize and compare conformational switching and membrane insertion of the two proteins. CD spectroscopy and thermal denaturation measurements indicate that, unlike the T-domain, Bcl-xL is resistant to acid-induced destabilization in solution. FRET measurements between donor-labeled protein and acceptor-labeled vesicles demonstrate that Bcl-xL undergoes reversible membrane association strongly modulated by the presence of anionic lipids. In contrast, initial stages of membrane action of the T-domain are largely lipid-independent, with anionic lipids playing a role only on the later stages of a multi-step insertion pathway. Site-selective attachment of environment-sensitive fluorophore NBD to the helical hairpin of Bcl-xL (α5-α6) or the T-domain (TH8-TH9) reveals similarities in the topology of the inserted state, but not in the lipid-dependent kinetic regulation of the insertion transition. Taken together our results indicate that while Bcl-xL and the T-domain share structural similarities, their mode of conformational switching and membrane insertion pathways are distinctly different. We suggest that these variations reflect underlying physiological differences: while cellular entry of the toxin via endosomal pathway requires robust insertion of the T-domain, the apoptotic control through the action of Bcl-xL and other members of the Bcl-2 protein family involves multiple levels of regulation, including those modulated by changes in mitochondrial lipid composition. Supported by NIH GM-069783 (A.S.L.) and Fulbright-CONICYT (M.V.U.).