Structure assembly of Bcl-x(L) through alpha5-alpha5 and alpha6-alpha6 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 alpha5-helix and Asn185 on alpha6-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.
Feng
Yu
Y
Department of Molecular Pharmacology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu-Chong-Zhi Road, Shanghai 201203, China.
Liu
Dongxiang
D
Shen
Xu
X
Chen
Kaixian
K
Jiang
Hualiang
H
D006801:Humans; D008051:Lipid Bilayers / Q000378:metabolism*; D008563:Membrane Lipids / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000378:metabolism*; D011487:Protein Conformation; D051020:bcl-X Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D008051:Lipid Bilayers; D008563:Membrane Lipids; D010446:Peptide Fragments; D051020:bcl-X Protein