Dissecting the Activation of BAX Protein Oligomerization by BimBH3 Activator Using Spin-label ESR

• Main Authors: Jhong, Siao-Ru, 鐘筱茹
• Other Authors: Chiang, Yun-Wei
• Format: Others
• Language: zh-TW
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/452dfb

碩士 === 國立清華大學 === 化學系 === 103 === Bcl-2 associated X (BAX) protein is a pro-apoptotic Bcl-2 family member that functions as a critical gateway to mitochondrial apoptosis. BAX is a cytosolic protein in healthy cells. Upon receipt of apoptotic signaling, BAX is activated to form a mitochondria-associated complex, leading mitochondrial outer membrane permeabilization (MOMP) and then cell death. A diversity of stimuli have been implicated in initiating BAX activation, including direct involvement by select BH3 domains and BH3-only proteins. To explore the elusive initiating event for BAX activation, here we study the interaction of BAX with a 26-residue-long BH3-derived peptide, BimBH3, which we previously demonstrated to be effective to initiate the BAX oligomerization and MOMP, using ESR techniques including time-resolved (TR) ESR and double electron-electron resonance (DEER) techniques. Basically, TR-ESR is carried out to monitor the time-dependent interactions between BimBH3 and BAX protein. The process of BAX oligomerization is monitored through the spectral changes of spin-labeled BAX proteins, while the binding kinetics of BimBH3 is obtained by observing the changes in the spectral intensity of spin-labeled BimBH3 peptides over the incubation time. We show that the BAX activation to form oligomers can be induced by BimBH3 peptide through a hit-and-run mechanism, which is coupled with an induced-fit transformation concerning the BimBH3 secondary structure. BimBH3 is structurally disordered prior to the engagement with BAX. Upon incubation with inactive BAX monomers, BimBH3 binds to BAX at two different binding sites to initiate the conformational changes of BAX, which in turn promotes the formation of BAX oligomer. Our DEER results show that the helical propensity of the disordered BimBH3 is clearly increased upon binding to BAX, but is reduced after being released from the activated BAX due to the subsequent conformational changes of BAX that are necessary for the BimBH3-induced BAX oligomerization. The release and binding rates between the BimBH3 and BAX are quantitatively depicted by the spectra from TR-ESR measurements, providing kinetic insights into the hit-and-run process. Our results suggest that the engagements of BimBH3 to both of the binding sites on BAX are required for the oligomerization to proceed in a regular manner. Disruption by mutation in either site would largely reduce the rate of the oligomerization. Taken together, this study shows that BAX activation is a highly regulated, multi-step process involving an interaction-triggered conformational change from the BAX side, an induced-fit conformational change from the BimBH3 activator side, mitochondrial translocation, and oligomerization that ultimately leads to mitochondrial dysfunction and apoptosis.