Structure assembly of Bcl-x L through α5–α5 and α6–α6 interhelix interactions in lipid membranes

Abstract

Lipid bilayer membrane is the main site where Bcl-x L executes its anti-apoptotic function. Here we used site-directed mutagenesis and cysteine-directed cross-linking to trap the structure of Bcl-x L upon membrane insertion. Cys151 on α5-helix and Asn185 on α6-helix of two neighboring Bcl-x L are found in close positions, respectively. The FRET based binding assay indicated that the BH3-peptide binding pocket in Bcl-x L is disrupted after its membrane insertion. Co-immunoprecipitation experiments showed that the membrane-bound Bcl-x L sequestered tBid by direct interaction at physiological pH. If Bcl-x L behaves similarly at low pH as it does at physiological pH, the membrane-bound Bcl-x L should bind to tBid through protein regions other than the BH3 domain of tBid and the hydrophobic pocket of Bcl-x L. Previously, a crystallography study demonstrated that Bcl-x L formed homodimers through domain swapping in water, where Cys151 and Asn185 of two monomeric subunits are far apart from each other and the BH3-peptide binding pocket is intact. Our results indicated that Bcl-x L dimer trapped by cross-linking in lipids is distinct from the domain swapped dimer, suggesting that Bcl-x L transits through a structural change from the water-soluble state to the membrane-bound state and there are multiple possibilities for structural reorganization of Bcl-x L protein.