STRUCTURAL TRANSITIONS OF BAX AFTER THE APOPTOTIC ACTIVATION
Orion Shih1*, Yi-Qi Yeh1, Tai-Ching Sung2, Yun-Hsuan Kuo2, Yun-Wei Chiang2, U-ser Jeng1
1X-ray scattering group, NSRRC, Hsinchu, Taiwan
2Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan
* presenting author:Orion Shih, email:shih.orion@gmail.com
In stressed cells, apoptosis ensues when BAX proteins oligomerize and permeabilize the mitochondrial outer membrane [1]. The mechanism utilized by BAX still remains elusive because BAX changes its functional structures along the apoptotic signaling pathway from cytosolic monomer, oligomer, to membrane-associated complex. This polymorphism of BAX poses a great challenge in tracing its structural transitions in functioning. Here we use a combination of electron spin resonance (ESR) and small angle X-ray scattering (SAXS) to study the conformational changes of BAX before and after the apoptotic activation. Particularly, an on-line HPLC is used to separate the BAX monomers, dimers, and oligomers in the eluted sample solution along the flow path for in-situ SAXS data collection at the 23A SWAXS endstation of National Synchrotron Radiation Research Center, Hsinchu, Taiwan [2].
The HPLC elution profile and previous ESR studies [3] indicate that there exist two forms of BAX monomer, UM and UM’. Upon the engagement of BimBH3, UM and UM’ are transformed into a ligand-bound monomer and a oligomer, respectively. We construct the solution structures of BAX along the oligomerization pathway and prove that BAX oligomers are assembled in solution via linear polymerization-like α6 association. Our results specifically establish oligomerization interfaces and intermediates, which are important structural basis for manipulating BAX activity pharmacologically. This study also suggests an alternative pathway of apoptosis in which BAX oligomer formation occurs prior to membrane insertion.

References
[1] Antonsson, B., Montessuit, S., Sanchez, B., and Martinou, J. C. (2001) J. Biol. Chem. 276, 11615-11623.
[2] Jeng, U., Su, C., Su, C., Liao, K., Chuang, W., Lai, Y., Chang, J., Chen, Y., Huang, Y., Lee, M., Yu, K., Lin, J., Liu, D., Chang, C., Liu, C., Chang, C., and Liang, K. S. (2010) J. Appl. Cryst. 43, 110–121.
[3] Tsai, C., Liu, S., Hung, C., Jhong, S., Sung, T. and Chiang, Y. (2015) Structure 23, 139-148.


Keywords: SAXS, ESR, HPLC