Topology of active, membrane-embedded Bax in the context of a toroidal pore

Abstract

Bax is a Bcl-2 protein critical for apoptosis induction. In healthy cells, Bax is mostly a monomeric, cytosolic protein, while upon apoptosis initiation it inserts into the outer mitochondrial membrane, oligomerizes, and forms pores that release proapoptotic factors like Cytochrome c into the cytosol. The structures of active Bax and its homolog Bak are only partially understood and the topology of the proteins with respect to the membrane bilayer is controversially described in the literature. Here, we systematically review and examine the protein-membrane, protein-water, and protein-protein contacts of the nine helices of active Bax and Bak, and add a new set of topology data obtained by fluorescence and EPR methods. We conclude based on the consistent part of the datasets that the core/dimerization domain of Bax (Bak) is water exposed with only helices 4 and 5 in membrane contact, whereas the piercing/latch domain is in peripheral membrane contact, with helix 9 being transmembrane. Among the available structural models, those considering the dimerization/core domain at the rim of a toroidal pore are the most plausible to describe the active state of the proteins, although the structural flexibility of the piercing/latch domain does not allow unambiguous discrimination between the existing models.

Oleuropein-Induced Apoptosis Is Mediated by Mitochondrial Glyoxalase 2 in NSCLC A549 Cells: A Mechanistic Inside and a Possible Novel Nonenzymatic Role for an Ancient Enzyme

“…Thus, our data provide evidence of a proapoptotic role for mGlo2 after OP exposure as well as a physical association with a known mediator of apoptosis, Bax. It has been demonstrated that during apoptosis, Bax and Bak mediate the release of cytochrome c from the mitochondria by clustering on the outer mitochondrial membrane and thereby increasing its permeability [39]. However, it remains unclear how outer membrane openings form.…”

A new perspective on membrane-embedded Bax oligomers using DEER and bioresistant orthogonal spin labels

“…Point mutations as well as label attachment can affect Bax fold and function 13,18 . Therefore, we analyzed the pore-forming activity of all spin-labeled Bax variants by kinetic experiments following fluorophore release from liposomes (Fig.…”

“…In healthy cells it is mainly a soluble, monomeric, inactive protein whose structure is known 12 , while upon a pro-apoptotic stimulus Bax transforms into the active pore-forming, membrane-embedded oligomer, whose structure is only partially known (recently reviewed in 13 ). The pores formed by active Bax enable mitochondrial outer membrane permeabilization and apoptosis execution 4,13 and the formation of “mega-pores” (Fig. 1a) is likely related to the generation of pro-inflammatory signals 14–17 .…”

“…First, Bax oligomerization creates complex protein systems with multiple intra- and inter- dimer spin distances that are difficult to assign when analyzing singly-labeled proteins. Notably, cross-link studies face the same problem (for a recent review see 13 ). To simplify the spin system and obtain distance constraints within each monomer in the oligomer, we used doubly-labeled proteins spin-diluted with unlabeled wild type partners 18 , which enhanced the intra-monomer distance within the distance distribution.…”

“…To simplify the spin system and obtain distance constraints within each monomer in the oligomer, we used doubly-labeled proteins spin-diluted with unlabeled wild type partners 18 , which enhanced the intra-monomer distance within the distance distribution. Based on these data we could propose a coarse-grained model of active Bax 13 . Additionally, the conventional nitroxide labels were found to be incompatible with cellular extracts or isolated mitochondria, as intra-cellular agents can chemically reduce the nitroxide group to the EPR silent hydroxylamine or can release the labels from the protein after reduction of the S-S linker 18,30 .

Figure 1Bax activation scheme, spin labeling and activity assays.

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