Structure assembly of Bcl-XL through α5-α5 and α6-α6 interhelix interactions in lipid membranes

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 alpha 5-helix and Asn185 on alpha 6-helix of two neighboring Bcl-XL 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. (C) 2009 Elsevier B.V. All rights reserved.