Volume 23, Issue 1, Pages 139-148 (January 2015) BAX-Induced Apoptosis Can Be Initiated through a Conformational Selection Mechanism  Chia-Jung Tsai, Sophia Liu, Chien-Lun Hung, Siao-Ru Jhong, Tai-Ching Sung, Yun-Wei Chiang  Structure  Volume 23, Issue 1, Pages 139-148 (January 2015) DOI: 10.1016/j.str.2014.10.016 Copyright © 2015 Elsevier Ltd Terms and Conditions

Structure 2015 23, 139-148DOI: (10.1016/j.str.2014.10.016) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 1 Cw-ESR of BAX (A) SEC results for WT BAX after the activation by BimBH3 shows that (black) the activated BAX exists as oligomeric and monomeric forms and that (blue trace) the monomeric fractions would not oligomerize even reactivated with excess BimBH3. [BAX] is approximately 0.002 mM in the SEC fractions. The sequences of the BimBH3 variants are shown. (B) Raw experimental cw-ESR spectra (300 K) of BimBH3-R1 corresponding to the monomeric and oligomeric fractions eluted from the SEC analysis of the overnight incubation of WT BAX with BimBH3-R1. BimBH3-R1 is confirmed to bind to both BAX monomer and oligomer. (C) A proposed conformational selection mechanism of BAX upon activation by BimBH3. (D) A time course (10 hr) collection of cw-ESR spectra of WT BAX-R1 upon incubation with BimBH3 at 300 K. The displayed width of the spectra is 100 G. The Initial was recorded 5 min after adding BimBH3. (E) The height of the central ESR line plotted against incubation time is represented by the blue markers. An exponential fit (broken red line) to the blue data points yields a mean lifetime of 121.9 min for the observed kinetics of BAX activation. (F) A good fit to the Final can be achieved by a linear combination of the BM and O states of normalized spectra at a ratio of 21:79, respectively. (G) The spectra of the four species that could possibly exist during the activation reaction. The broken lines highlight the characteristic peaks of the Final. The spectra Final and Initial were taken from the time-course collection. The spectra BM and O were from the fractions of the SEC results. All were collected under the same condition. (H) A population plot of the three most likely components as a function of incubation time. The SD of the populations is approximately 4. See also Figure S1. Structure 2015 23, 139-148DOI: (10.1016/j.str.2014.10.016) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 2 Conformations of Inactive BAX (A) Six sites were selected for spin labeling study. They correspond to different helices (as indicated by colors) and cover the vast majority of the entire protein. (B) DEER measurements and analysis results of spin-labeled BAX monomers. It shows the normalized time-domain (background-removed) experimental data (left) and the P(r) results of Tikhonov analysis (right). Distances used to reconstruct the UM and UM′ BAX conformations are denoted by ↓ and ▾, respectively. The reconstructed UM BAX is consistent with the NMR-derived structure. (C) A ribbon cartoon model illustrating the conformational differences between the UM BAX and the UM′ BAX under the assumptions that helical segments are rigid and linker regions are flexible (see details in Experimental Procedures). Zoomed areas are some major differences between the two states. They include the differences in the segment of α5-α6 and the C-terminal region α9. The structures were derived using the DEER distances as constraints in the MtsslWizard program. (D) The local environment of the BH3 domain becomes more readily accessible for interactions with substrates in the UM′ than the UM. See also Figure S2. Structure 2015 23, 139-148DOI: (10.1016/j.str.2014.10.016) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 3 Structural Conformation of the Bound Monomer BM (A) Normalized time-domain (background removed) DEER data and the corresponding P(r) results of Tikhonov analysis for various BAX mutants in the BM state. Distances denoted by ↓ are used to reconstruct the BM conformation. Distances denoted by ∗ are excluded from the analysis (see Experimental Procedures for details). (B) An illustration of conformational differences between the UM and the BM states. The differences are small but distinct in the P(r) results. (C) Differences in the local environment of BH3 domain between the three states of BAX. The largest difference is observed between the UM and the UM′ states. See also Figure S3. Structure 2015 23, 139-148DOI: (10.1016/j.str.2014.10.016) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 4 Cytochrome c Release Essays Cytochrome c release from mitochondria on incubation of BAX variants as a function of the incubation time. Inactive WT BAX monomer is denoted by N. BAX in the O state is most potent for the release activity. The intensity is normalized to 100% efficiency for the result of O. The estimation of errors is based on three independent measurements. The data were quantified using ImageJ. See also Figure S5. Structure 2015 23, 139-148DOI: (10.1016/j.str.2014.10.016) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 5 A Schematic View of the Dynamic Energy Landscapes Inactive BAX exists in two distinct conformations, UM and UM′. Upon ligand-induced activation, BAX converts into two ligand-bound forms, monomer (BM) and oligomer (O), and the dominant population shifts from the UM to the UM′, leading to the product O being dominant in the end of the activation. See also Figure S6. Structure 2015 23, 139-148DOI: (10.1016/j.str.2014.10.016) Copyright © 2015 Elsevier Ltd Terms and Conditions