Abstract
Proapoptotic Bcl-2 associated X (BAX) protein, which comprises 9 helical domains with a molecular weight of 21 kDa, is largely cytosolic in healthy cells, but it oligomerizes and translocates to mitochondria upon receiving apoptotic stimuli, which eventually leads to mitochondrial outer membrane permeabilization (MOMP) and cell death. It is not known whether the formation of BAX oligomers occurs prior to or at the moment of its interaction with mitochondria. A longstanding challenge has been the inability to capture any structural information beyond the onset of activation. A complete view about the activation mechanism is yet to be explored. The goal of this study is to reveal the structure of BAX protein oligomer so as to unravel the molecular details of the apoptotic activation process. This dissertation is organized as follows. In Chapter 1, we present general background information about the central topic of this dissertation, Bcl-2 mediated apoptosis. In Chapter 2, we give general introduction to principles of the experimental tools used in the dissertation, and also sample preparation of the BAX protein, a gatekeeper protein in the Bcl-2 family. Details of the expression, purification, and functional tests of BAX protein, which are important to successful BAX protein preparations but may be too much detail to be put into a regular journal article, are given in Chapter 3. In Chapter 4, we describe our preliminary results for the study of local dynamics of the BH3 domain of BAX oligomer using cw-ESR and theoretical approach based on ESR lineshape theory. The local ordering and dynamics along the BH3 domain can thus be quantitatively described by the results of the theoretical analysis. Then we present the main finding of this dissertation in Chapter 5, wherein a brief introduction to the main finding and experimental/supplemental results specific for this chapter are all included. Briefly, we report a comprehensive study that attempts to resolve the structures of soluble forms of BAX oligomer using spin-label ESR techniques including cw-ESR and DEER, as well as small-angle X-ray scattering (SAXS) technique. Various BAX mutants were prepared to carry single or double spin probes for the ESR study. The determined structure reveals details of the assemblies of BH3-in-groove dimer and supports the opening of the hairpin formed by helices 5 and 6 in the oligomer (> 440 kDa) state. Besides, this study reveals that helix 1 is highly flexible and solvent-exposed in the oligomeric state while the tail-anchor (helix 9) is buried within the assembly, providing details about the monomer-monomer interfaces in the oligomer and how the oligomer is assembled from homodimers. We show that this soluble oligomer undergoes a direct conversion into membrane-inserted oligomer, which has the ability of inducing apoptosis and structurally resembles a membrane-embedded oligomer formed from BAX monomers in lipid environment. Structural differences between the soluble and the membrane-inserted oligomers are manifested in the C-terminal helices. Our data suggests an alternative pathway of apoptosis in which BAX oligomer formation occurs prior to membrane insertion.