Allosteric Sensitization of Pro-Apoptotic BAX
Pritz, Jonathan R.
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CitationPritz, Jonathan R. 2018. Allosteric Sensitization of Pro-Apoptotic BAX. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences.
AbstractBCL-2 family proteins are critical regulators of mitochondrial apoptosis and thus serve as prime targets for therapeutic modulation in diseases of deregulated cell death. Whereas a 20-year effort to inhibit anti-apoptotic proteins has yielded a clinically-approved small molecule inhibitor of BCL-2, drug strategies to directly manipulate pro-apoptotic BCL-2 members, such as BAX, remain underdeveloped. BAX is a critical executioner protein of the apoptotic pathway, transforming from a monomer into a mitochondrial membrane-embedded oligomer in response to stress stimuli. Indeed, the relatively unstable and aggregation-prone nature of recombinant BAX has been a major limitation for conducting large-scale screens for drug discovery. Here, we overcame prior challenges and, to our knowledge, performed the first NMR-based molecular fragment screen of full-length BAX. We identified a class of compounds that sensitizes BH3-mediated direct BAX activation. Applying a series of structure-function analyses, spanning HSQC NMR, hydrogen-deuterium exchange mass spectrometry (HXMS), and correlative biochemical testing, we interrogated both the site of interaction and mechanism of BAX sensitization. Intriguingly, the binding site for this sensitizing molecule localized to a region implicated in BAX inhibition by the cytomegalovirus vMIA protein, rather than to one of the established BH3 activation sites. HXMS studies revealed that BIF-44 induces conformational deprotection of the α1–α2 loop and BAX BH3 (α2), two regions mechanistically linked to the conformational activation of BAX. As these induced structural changes occur at the opposite side of protein from the BIF-44 interaction site, our studies identified a novel allosteric mechanism of BAX sensitization. Taken together, the molecular screening results and structure-function analyses inform fundamental mechanisms for conformational regulation of BAX and provide a new opportunity to reduce the apoptotic threshold for potential therapeutic benefit in cancer and other diseases of pathologic cell survival.
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