Walensky, Loren DBloch, Noah Benjamin2022-03-1820222022-03-172022-03Bloch, Noah Benjamin. 2021. Mechanistic Insights into the Conformational Regulation of Pro-Apoptotic BAX. Doctoral dissertation, Harvard University Graduate School of Arts and Sciences.28963878https://nrs.harvard.edu/URN-3:HUL.INSTREPOS:37371142BCL-2 family proteins are apoptotic regulators that control the critical balance between cellular life and death at the level of the mitochondrion. BAX is a principal executioner of apoptosis, transforming from a latent cytosolic monomer into a lethal mitochondrial oligomer in response to cell stress. Because renegade BAX activation poses a grave risk to the cell, the architecture of BAX must ensure monomeric stability yet enable conformational transformation. The specific structural features that afford both stability and dynamic flexibility remain ill-defined and represent a critical control point of BAX regulation. In this dissertation, I identified a nexus of interactions involving discrete residues of BAX’s core a5 helix, specifically amino acids 113-116, which are individually essential to maintaining the structural stability of monomeric BAX and are collectively required for higher order BAX assembly. Single alanine mutagenesis of residues 113-116 resulted in autoactive proteins that were capable of membrane permeabilization even in the absence of BH3-only ligand stimulation, which otherwise induces the activation of wild-type BAX. Analysis of conformational dynamics by hydrogen deuterium exchange mass spectrometry revealed regiospecific changes in BAX structure, providing a mechanism for mutant autoactivity. Further, combinatorial mutagenesis of the BAX 113-116 nexus revealed the collective role of these residues in stabilizing the dimeric form of BAX, which represents a key step of oligomeric self-assembly. The dual yet distinct roles of BAX residues 113-116 highlight the intricacy of BAX conformational regulation and opportunities for modulating BAX conformational dynamics for therapeutic benefit in diseases of deregulated apoptosis.application/pdfenApoptosisBAXCell DeathBiochemistryBiologyMolecular biologyThesis or Dissertation2022-03-180000-0002-9121-2783