BAX and BAK are essential mediators of intrinsic apoptosis that permeabilise the mitochondrial outer membrane (MOM). BAX activation requires its translocation from cytosol to mitochondria where conformational changes cause its oligomerisation. Translocation is dependent on the accessibility of a transmembrane domain (α9) that is either exposed or protected (folded in a hydrophobic groove at the surface of the protein). To better understand the critical step of translocation, we examined its blockade by several mutations or by antibody-binding near the N-terminus.
Structural comparisons between the wild type BAX, its mutants or the antibody bound BAX do not explain the translocation defect. Complementary biophysical and biochemical studies, including hydrogen-deuterium exchange (HDX) by mass spectrometry or limited proteolysis revealed evidence of allosteric mechanisms controlling BAX targeting to the MOM. Cytosolic BAX exists as an ensemble of conformers. This ensemble is shifted to conformations in which α9 is sequestered in inhibitory conditions.
Interestingly, the same antibody that blocks cytosolic BAX translocation can activate BAX anchored at the MOM through its α9. In that case, the unfolding of certain helices seen upon binding of the antibody to BAX is able to active BAX as the α9 lock is no more present.
Our data suggest that BAX displays allosteric properties that derive not from differences in equilibrium structure but rather from differences in its ensemble of structures, from which ensues allostery without apparent conformational change 1.