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

Background: Bcl- 2 family proteins are key regulators of mitochondrial integrity and comprise both proand 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 (alpha 5-alpha 6 helices in the core and alpha 9 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 alpha 5-alpha 6 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(L) and 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 alpha 5-alpha 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.