Biology subjects

Kervadec, J.

Publications and source records attributed to Kervadec, J..

2 recordsLinked to original sources

bioRxiv2026

Production of membrane-embedded Bcl-2 proteins - Use of cell-free synthesis in continuous exchange for co-translational insertion of Bcl-2 proteins in lipid bilayer nanodiscs

The BCL-2 family proteins are key regulators of apoptosis, functionally divided in pro- and anti-apoptotic proteins, with a third group acting as regulators. Their ability to partition between the cytosol and intra-cellular membranes (essentially the mitochondrial outer membrane) is a primary regulator of their functions. A second contributor is their ability to form homotypic complexes (pro-pro or anti-anti) or heterotypic complexes (pro-anti). If the structures of monomeric cytosolic members have largely been characterized, the functional and structural study of membrane-embedded proteins remains incomplete. Unlocking this knowledge is expected to enable evaluating new therapeutic strategies to either activate pro-apoptotic members, or inactivate anti-apoptotic ones. Lipid bilayer nanodiscs and improved cell-free protein synthesis have provided the technical breakthrough to achieve the description at the atomic level of conformations and higher order assemblies of these proteins in their membrane-associated states. Here we describe detailed and straightforward protocols for generating nanodisc-inserted members of the Bcl-2 family, through the example of anti-apoptotic Bcl-xL, and pro-apoptotic Bax and Bak. Full-length, untagged proteins are expressed from bacterial extracts in the presence of pre-assembled nanodiscs to allow co/post-translational insertion in lipid bilayer, followed by affinity chromatography purification. A more detailed characterization is presented for Bak, to exemplify structural and mechanistic studies enabled by these methods. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/745005v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@5da1d1org.highwire.dtl.DTLVardef@12aca96org.highwire.dtl.DTLVardef@5a3e73org.highwire.dtl.DTLVardef@ba009d_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

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.

biochemistry↗