Evidence for an Induced-Fit Process Underlying the Activation of Apoptotic BAX by an Intrinsically Disordered BimBH3 Peptide

Abstract

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.

Unified understanding of folding and binding mechanisms of globular and intrinsically disordered proteins

“…NMR relaxation dispersion experiments showed that intrinsically disordered pKID binds KIX by the induced-fit mechanism with accumulation of an intermediate (Sugase et al 2007 ). Coupled folding and binding reactions by the induced-fit mechanism have also been reported for many other IDPs, including the transactivation domains (TADs) of c-Myc, Gal4, and VP16 (Ferreira et al 2005 ), CBD of WASP binding to Cdc42 (Lu et al 2007 ), IA 3 binding with YPrA (Narayanan et al 2008 ), S-peptide binding with S-protein (Kiefhaber et al 2012 ), N TAIL domain from Sendai virus nucleoprotein binding to phosphoprotein PX (Wang et al 2013b ; Dosnon et al 2015 ; Schneider et al 2015 ), PUMA binding with MCL-1 (Rogers et al 2014 ), BimBH3 binding with BAX (Jhong et al 2016 ), and STAT2 TAD binding with the TAZ1 domain of CBP (Lindstrom and Dogan 2017 ).…”

Anti-apoptotic BCL-2 regulation by changes in dynamics of its long unstructured loop

“…This study used a 26-mer-long BimBH3-domain-based peptide (see Fig. 1d for sequence) to induce the activation of pro-apoptotic BAX protein and MOMP 18,[20][21][22] . Interactions of several BH3-only-based peptides with pro-survival proteins have been previously reported, showing that they would vary substantially in affinity with different peptide lengths and truncated forms of the proteins 14,[23][24][25] .…”

“…BAX expression and purification. Full-length BAX and variants were prepared as previously described 18,22,35 . Mouse BAX was cloned into pTYB1 vector (New England Biolabs, Inc.), resulting pTYB1-BAX construct.…”

Identifying Protein Conformational Dynamics Using Spin‐label ESR

“…[21-24, 51, 52] This Gaussian-baseda pproach has also been utilized to capture intermediate conformationso fa ni ntrinsically disordered protein/peptide (IDP). [53] BH3 motif of Bim protein (BimBH3) is an IDP,b ut it is knownt ot rigger the apoptotic activation of BAX by binding to BAX at surface groove between helices 1 and 6. [54] To study the structuralc hanges of BimBH3 peptide, BAX and double-labeled BimBH3 were incubated together and snap-frozen in liquid nitrogen during the activation reaction, followed by DEER measurements on the samples trapped at different time points.…”

“…[54] To study the structuralc hanges of BimBH3 peptide, BAX and double-labeled BimBH3 were incubated together and snap-frozen in liquid nitrogen during the activation reaction, followed by DEER measurements on the samples trapped at different time points. [53] The resultant distance distributions are highly heterogeneous for the mixtures of BimBH3a nd BAX, but they can be decomposed into individual components using the Gaussian-based approach( Figure 3B). The analysis revealed the change in the populations of structured and unstructured BimBH3p eptides at differentl engths of incubation time during the activation.Itwas shown that helical propensity of BimBH3i si ncreased upon binding to BAX but is then reduced after being released from the activated BAX due to large conformational changes between inactive anda ctive BAX.…”