Fig. 5: Collective rather than singular mutagenesis of the BAX 113-116 nexus is required to disrupt the conformational stability of the BAX α2–α5 dimer.

a Crystal structure of dimeric GFP-BAX α2–α5 (PDB: 4BDU) with the N-terminal GFP-tag removed for clarity. The positions of residues 113-116 are shown in blue. b SEC of the indicated wild-type and single alanine and quadruple alanine (AAAA) mutant forms of GFP-BAX α2–α5. The dimeric complex of GFP-BAX α2–α5 is observed as a dimer/oligomer of dimers (e.g. tetramerization mediated by GFP). The experiment was performed twice using independent preparations of proteins with similar results. (c–d) Deuterium difference plots showing the relative deuterium incorporation of (c) BAX α2–α5 F116A and (d) BAX α2–α5 L113A/F114A/Y115A/F116A minus the relative deuterium incorporation of wild-type BAX α2–α5, as measured at 10 sec, 1 min, and 10 min of deuterium labeling. Regions of deprotection (orange) and protection (green) above the 0.5 Da significance threshold at 1 min labeling are mapped onto the crystal structure (PDB: 4BDU) of the BAX α2–α5 dimer for (c) and a monomeric subunit for (d), with the respective mutated residue(s) colored in blue. HDX MS experiments were performed at least twice using independent preparations of BAX proteins. All peptides display EX2 kinetics (uncorrelated local dynamics producing a binomial exchange profile). The HDX MS data used to create this figure can be found in Supplementary Data 2. e SEC profiles of the indicated double and triple alanine mutant forms of GFP-BAX α2–α5. Elution positions of wild-type dimer and quadruple-mutant monomer are shaded in grey and gold, respectively, as references. The experiment was performed twice using independent preparations of proteins with similar results. Source data are provided as a Source Data file.