Abstract
Bax and Bid are pro-apoptotic members of the Bcl-2 protein family. Upon cleavage by caspase-8, Bid activates Bax. Activated Bax inserts into the mitochondrial outer membrane forming oligomers which lead to membrane poration, release of cytochrome c, and apoptosis. The detailed mechanism of Bax activation and the topology and composition of the oligomers are still under debate. Here molecular details of Bax activation and oligomerization were obtained by application of several biophysical techniques, including atomic force microscopy, cryoelectron microscopy, and particularly electron paramagnetic resonance (EPR) spectroscopy performed on spin-labeled Bax. Incubation with detergents, reconstitution, and Bid-triggered insertion into liposomes were found to be effective in inducing Bax oligomerization. Bid was shown to activate Bax independently of the stoichiometric ratio, suggesting that Bid has a catalytic function and that the interaction with Bax is transient. The formation of a stable dimerization interface involving two Bcl-2 homology 3 (BH3) domains was found to be the nucleation event for Bax homo-oligomerization. Based on intermolecular distance determined by EPR, a model of six adjacent Bax molecules in the oligomer is presented where the hydrophobic hairpins (helices alpha5 and alpha6) are equally spaced in the membrane and the two BH3 domains are in close vicinity in the dimer interface, separated by >5 nm from the next BH3 pairs.
Highlights
Members of the Bcl-2 protein family are essential players in the complex regulation of apoptosis [1, 2]
Bax has a globular fold composed of nine ␣-helices (␣1 to ␣9), with ␣2 representing the Bcl-2 homology 3 (BH3) domain and ␣5/␣6 the hydrophobic hairpin
A number of models have been proposed to describe interactions between Bcl-2 proteins. (i) an NMR study reports an activation mechanism based on the interaction of a stabilized helix of the BH3-only protein Bim with ␣1 and ␣6 of Bax, initiating conformational changes in Bax [22]. (ii) The Bax homolog Bak is reported to homodimerize by an interaction of a fused ␣2/␣3 helix with the ␣2/␣3 helix of a second Bak monomer (Fig. 1B) [23]. (iii) A heterodimerization-based survival mechanism has been proposed in which the BH1–3 domains of anti-apoptotic Bcl-2 proteins form a hydrophobic groove to sequester the BH3 domain of pro-apoptotic family members (24 –26)
Summary
To a concentration of 0.5 mg/ml in the presence or absence of Additional details regarding cell culture, protein preparation, and biophysical techniques used are provided under the supplemental “Experimental Procedures.”. The extruded liposomes containing cytochrome or 4-phosphonooxy-TEMPO only in their lumen were mixed to a final concentration of 10 mg/ml E. coli polar lipid (ECL) extract with 0.1– 0.3 mg/ml Bax and Bid-derivatives (1:1 stoichiometric ratio) and incubated at 37 °C for 3 h. For the cryo-EM experiments, large unilamellar vescicles were extruded through a 100-nm filter, mixed to a final concentration of 13 mg/ml ECL with BaxC62R1 (10 M) and p15/7-Bid in a 1:1 stoichiometric ratio, and incubated at 37 °C for 1 h. For each sample (absence and presence of Bax and Bid derivative) 3.5-l aliquots were applied to a lacy carbon grids (Plano, Germany) supported on a copper grid These grids were blotted for 3 s and plunge-frozen in ethane at liquid nitrogen temperatures in a humidity (95%)- and temperature (25 °C)-controlled chamber using a Vitrobot automated blotting device (FEI Co., Eindhoven, The Netherlands). Data analysis was performed with the software DeerAnalysis 2008 [31]
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