BCL-2-associated X (BAX) protein acts as a gatekeeper in regulating mitochondria-dependent apoptosis. Under cellular stress, BAX becomes activated and transforms into a lethal oligomer that causes mitochondrial outer membrane permeabilization (MOMP). Previous studies have identified several structural features of the membrane-associated BAX oligomer; they include the formation of the BH3-in-groove dimer, the collapse of the helical hairpin α5-α6, and the membrane insertion of α9 helix. However, it remains unclear as to the role of lipid environment in determining the conformation and the pore-forming activity of the BAX oligomers. Here we study molecular details of the membrane-associated BAX in various lipid environments using fluorescence and ESR techniques. We identify the inactive versus active forms of membrane-associated BAX, only the latter of which can induce stable and large membrane pores that are sufficient in size to pass apoptogenic factors. We reveal that the presence of CL is crucial to promoting the association between BAX dimers, hence the active oligomers. Without the presence of CL, BAX dimers assemble into an inactive oligomer that lacks the ability to form stable pores in the membrane. This study suggests an important role of CL in determining the formation of active BAX oligomers.

While activation of BAX is required for initiating mitochondria-mediated apoptosis, the underlying mechanisms remain unsettled. We studied conformations of BAX protein using pressure- and temperature-resolved ESR techniques and obtained the thermodynamic properties of the conformations. We show that inactive BAX is structurally heterogeneous and exists in equilibrium between two major populations of the conformations, UM and UM', of which the former is thermodynamically favored at room temperature. An increase in the population of UM', induced by either pressure or point mutations of BAX, renders BAX susceptible to oligomerization, which leads to cell death. This study uncovers the biological significance of BAX conformations and shows that the pro-apoptotic activity of BAX can be triggered by altering the equilibrium between the two states. It suggests that therapeutic intervention may focus on shifting the balance in the conformational heterogeneity.

A non-aggregation colorimetric sensing method for Pb(ii) ions using S2O32−/Au NR probes.

BCL-2, a key protein in inhibiting apoptosis, has a 65-residue-long highly flexible loop domain (FLD) located on the opposite side of its ligand-binding groove. In vivo phosphorylation of the FLD enhances the affinity of BCL-2 for pro-apoptotic ligands, and consequently anti-apoptotic activity. However, it remains unknown as to how the faraway, unstructured FLD modulates the affinity. Here we investigate the protein-ligand interactions by fluorescence techniques and monitor protein dynamics by DEER and NMR spectroscopy tools. We show that phosphomimetic mutations on the FLD lead to a reduction in structural flexibility, hence promoting ligand access to the groove. The bound pro-apoptotic ligands can be displaced by the BCL-2-selective inhibitor ABT-199 efficiently, and thus released to trigger apoptosis. We show that changes in structural flexibility on an unstructured loop can activate an allosteric protein that is otherwise structurally inactive.

Apoptotic BAX protein functions as a critical gateway to mitochondria-mediated apoptosis. A diversity of stimuli has been implicated in initiating BAX activation, but the triggering mechanism remains elusive. Here we study the interaction of BAX with an intrinsically disordered BH3 motif of Bim protein (BimBH3) using ESR techniques. Upon incubation with BAX, BimBH3 binds to BAX at helices 1/6 trigger site to initiate conformational changes of BAX, which in turn promotes the formation of BAX oligomers. The study strategy is twofold: while BAX oligomerization was monitored through spectral changes of spin-labeled BAX, the binding kinetics was studied by observing time-dependent changes of spin-labeled BimBH3. Meanwhile, conformational transition between the unstructured and structured BimBH3 was measured. We show that helical propensity of the BimBH3 is increased upon binding to BAX but is then reduced after being released from the activated BAX; the release is due to the BimBH3-induced conformational change of BAX that is a prerequisite for the oligomer assembling. Intermediate states are identified, offering a key snapshot of the coupled folding and binding process. Our results provide a quantitative mechanistic description of the BAX activation and reveal new insights into the mechanism underlying the interactions between BAX and BH3-mimetic peptide.