Apoptosis
Programmed cell death
Stepwise activation of the pro-apoptotic protein Bid at mitochondrial membranes
Caspase-8-cleaved Bid (cBid) associates with mitochondria and promotes the activation of BAX, leading to mitochondria outer membrane permeabilization (MOMP) and apoptosis. However, current structural models of cBid are largely based on studies using membrane vesicles and detergent micelles. Here we employ spin-label ESR and site-directed PEGylation methods to identify conformations of cBid at real mitochondrial membranes, revealing stepwise mechanisms in the activation process. Upon the binding of cBid to mitochondria, its structure is reorganized to expose the BH3 domain while leaving the structural integrity only slightly altered. The mitochondria-bound cBid is in association with Mtch2 and it remains in the primed state until interacting with BAX. The interaction subsequently triggers the fragmentation of cBid, causes large conformational changes, and promotes BAX-mediated MOMP. Our results reveal structural differences of cBid between mitochondria and other lipid-like environments and, moreover, highlight the role of the membrane binding in modifying cBid structure and assisting the inactive-to-active transition in function.
Anti-apoptotic BCL-2 regulation by changes in dynamics of its long unstructured loop
BCL-2, a key protein in inhibiting apoptosis, has a 65-residue-long highly flexible loop domain (FLD) located on the opposite side of its ligand-binding groove. In vivo phosphorylation of the FLD enhances the affinity of BCL-2 for pro-apoptotic ligands, and consequently anti-apoptotic activity. However, it remains unknown as to how the faraway, unstructured FLD modulates the affinity. Here we investigate the protein-ligand interactions by fluorescence techniques and monitor protein dynamics by DEER and NMR spectroscopy tools. We show that phosphomimetic mutations on the FLD lead to a reduction in structural flexibility, hence promoting ligand access to the groove. The bound pro-apoptotic ligands can be displaced by the BCL-2-selective inhibitor ABT-199 efficiently, and thus released to trigger apoptosis. We show that changes in structural flexibility on an unstructured loop can activate an allosteric protein that is otherwise structurally inactive.
ESR & Fluorescence Techniques
Membrane pores
Although membrane pores are induced by the formation of BAX complexes at membrane, the pore size depends critically on lipid composition.
Role of Cardiolipin in the BAX Activation
Lipid-dependent BAX oligomers
We identify the inactive versus active forms of membrane-associated BAX, only the latter of which can induce stable and large membrane pores that are sufficient in size to pass apoptogenic factors. We reveal that the presence of CL is crucial to promoting the association between BAX dimers, hence the active oligomers. Without the presence of CL, BAX dimers assemble into an inactive oligomer that lacks the ability to form stable pores in the membrane. This study suggests an important role of CL in determining the formation of active BAX oligomers.
Apoptosis
Mitochondria-mediated apoptosis
One of the main topics in the Chiang Lab is about understanding the molecular details of how the BCL2 protein family regulates the mitochondria-dependent apoptosis. In particular, we are interested in the BAX-mediated cell death pathway.
Protein & Cell Membrane
Membrane protein & protein-membrane associated complexes
Our recent study reveals a complete solution structure of apoptotic BAX protein oligomer. The results suggest an alternative pathway of apoptosis in which BAX oligomer formation occurs prior to membrane insertion.
Conformational Heterogeneity
Allosteric regulation of protein function
The Chiang Lab reports the BAX-induced apoptosis can be initiated through a conformational selection mechanism.
