The N-terminus and alpha-5, alpha-6 helices of the pro-apoptotic protein Bax, modulate functional interactions with the anti-apoptotic protein Bcl-x_L

Abstract

Background: Bcl-2 family proteins are key regulators of mitochondrial integrity and comprise both pro- and anti-apoptotic proteins. Bax a pro-apoptotic member localizes as monomers in the cytosol of healthy cells and accumulates as oligomers in mitochondria of apoptotic cells. The Bcl-2 homology-3 (BH3) domain regulates interactions within the family, but regions other than BH3 are also critical for Bax function. Thus, the N-terminus has been variously implicated in targeting to mitochondria, interactions with BH3-only proteins as well as conformational changes linked to Bax activation. The transmembrane (TM) domains (α5-α 6 helices in the core and α9 helix in the C-terminus) in Bax are implicated in localization to mitochondria and triggering cytotoxicity. Here we have investigated N-terminus modulation of TM function in the context of regulation by the anti-apoptotic protein Bcl-x_L. Results: Deletion of 29 amino acids in the Bax N-terminus (Bax 30-192) caused constitutive accumulation at mitochondria and triggered high levels of cytotoxicity, not inhibited by Bcl-x_L. Removal of the TM domains (Bax 30-105) abrogated mitochondrial localization but resulted in Bcl-x_L regulated activation of endogenous Bax and Bax-Bak dependent apoptosis. Inclusion of the a5-a6 helices/TMI domain (Bax 30-146) phenocopied Bax 30-192 as it restored mitochondrial localization, Bcl-x_L independent cytotoxicity and was not dependent on endogenous Bax-Bak. Inhibition of function and localization by Bcl-x_L was restored in Bax 1-146, which included the TM1 domain. Regardless of regulation by Bcl-x_L, all N-terminal deleted constructs immunoprecipitated Bcl-x_Land converged on caspase-9 dependent apoptosis consistent with mitochondrial involvement in the apoptotic cascade. Sub-optimal sequence alignments of Bax and Bcl-x_L indicated a sequence similarity between the α5-α 6 helices of Bax and Bcl-x_L. Alanine substitutions of three residues (T14A-S15A-S16A) in the N-terminus (Bax-Ala3) attenuated regulation by the serine-threonine kinase Akt/PKB but not by Bcl-x_L indicative of distinct regulatory mechanisms. Conclusion: Collectively, the analysis of Bax deletion constructs indicates that the N-terminus drives conformational changes facilitating inhibition of cytotoxicity by Bcl-x_L. We speculate that the TM1 helices may serve as 'structural antagonists' for BH3-Bcl-x_L interactions, with this function being regulated by the N-terminus in the intact protein.