Molecular determinants of Bcl-xL membrane insertion – Structural plasticity exploration in solution and in nanodiscs identifies IsoAsp deamidation as a loss-of function mechanism in vivo

Multi-domain Bcl-2 family proteins share the ability to form dimers and oligomers, regardless of their pro- or anti-apoptotic activity. Homotypic interactions (pro-pro and anti-anti) and heterotypic interactions (pro-anti) are well-documented, but the role of higher-order organization in their survival/death functions and membrane interactions remains largely unresolved. Looking into anti-apoptotic Bcl-xL, essentially engineered/truncated proteoforms lacking the disordered loop and/or the hydrophobic C-terminal helix, have been used as proxies of the full-length (FL) protein, to elaborate on structural transitions and intermediate states between monomers and homooligomers prior to membrane insertion. Using a minimalist approach with recombinant FL-Bcl-xL (aa 1-233) and artificial nano-membranes, we demonstrate that both the loop and the C-terminal helix are potent contributors to Bcl-xL structural plasticity. Unlike 3D domain swapping (3DDS) dimers resolved with the C-terminal truncated protein, FL-Bcl-xL organized in solution as dimers bridging the unique Cys151 from two monomers. This spontaneous fold indicates that the C-terminal helix drives FL-Bcl-xL to explore different conformations than truncated Bcl-xL. Yet, dimerization was not a prerequisite for membrane insertion into nanodiscs and Cys151 did not contribute to Bcl-xL survival functions in cells. These data support monomeric Bcl-xL as the minimal functional unit in membranes. Further exploring the frequently deleted disordered loop, we discovered that deamidation of Asn52 and Asn66 in IsoAsp, but not in Asp, impairs membrane insertion into nanodiscs. Thus, this reductionist biochemical approach clarifies the loss of tumorigenic function we observed for deamidated Bcl-xL in xenograft experiments in vivo .