Insights on Bax pore-forming activity

The Bcl-2 family of proteins regulates apoptosis by mitochondrial outer membrane permeabilization (MOMP). For years, it was believed that effector proteins Bax and Bak do so by making pores in the membrane. However, direct evidence of a pore in the mitochondria outer membrane has never been shown. In the mean time, the interplay of different family members and the mechanism of action that regulate and control MOMP are still not well-understood.

Up until the mid 2000s, the nature of the apoptotic pore has not been characterized. But with the advent of better technologies, and our improved ability to recombinantly produce Bax and Bak, various biophysical studies that characterize the mechanism of pore formation emerged.

In 2013, our lab produced two papers to help understand the mechanism of pore-formation of Bcl-2 proteins.

The first paper [1] distinguished the mechanism of action of Bax and Bcl-xL on model membrane systems. In this paper, we used single-vesicle permeabilization studies to show that Bax and Bcl-xL both permeabilize model membranes. However pores induced by Bcl-xL eventually close in time, while Bax remains open. In short, Bax makes stable pores.

This is quite intriguing in some sense because if one looks at the 3D structures of Bax and Bcl-xL in solution, they are very similar with great overlap. However, Bax acts as a pro-apoptotic affector in apoptosis, while Bcl-xL is anti-apoptotic.

We rationalized this mechanistic difference by Bax’s ability to self-interact and oligomerize in the membrane (as shown by Fluorescence Correlation Spectroscopy). During the oligomerization, Bax undergoes a conformational change, which is able to stabilize the open pore. On the other hand, Bcl-xL does not oligomerize in the membrane, thereby only producing transient pores.

To understand this better, one needs to look at the energetics of pore formation in lipid membranes [2]. An asymmetric insertion of material into a lipid bilayer introduces membrane tension. The reaction of the bilayer is then to rearrange the lipids and in doing so, may stochastically open up a pore. The opening of the pore would expose the hydrophobic tails of the lipids to water, and this is energetically not favored. To prevent exposure of the hydrophobic tails, lipids at the edge of the pore will bend and introduce packing defects in the membrane. And while this is less energetically costly than exposing hydrophobic regions, it is unfavorable. The energetic cost of keeping a pore open is often called line tension. In the absence of agents to stabilize the edge of the pore, the pore will simply close.

In the case of Bax and Bcl-xL, this is what we hypothesized to be happening. The insertion of proteins introduces enough membrane tension to open pores in the membrane. However only Bax is able to stabilize the edge of the pore, supposedly through oligomerization.

In the 2nd paper, Bleicken and Landeta, et al. [3] showed that Bax and Bak pores are not of a particular size. Using similar single vesicle permeabilization studies, we demonstrated that changing the concentration of the proteins changes the total permeabilized area in the membrane. This is akin to saying that the size of the pore is dependent on how many proteins are in the membrane.

While this may be generally be intuitive, pore size mainly depends on the nature of the pore. Pores may either be protein-lined (or proteinaceous) or toroidal (both protein and lipids line the pore)[4]. In protein-lined pores, the proteins serve as a scaffold to stabilize the pore; as such the protein structure defines the size of the pore. In these pores, proteins often form an oligomer with a defined number of units, and therefore the pore size is also definite. In toroidal pores, both protein and lipids line the pore, as such protein size is more flexible. They do not necessarily form a scaffold that holds the pore strongly together, but rather serve as a support so that lipids intercalate around the pore rim. The protein oligomers are believed to alleviate curvature stress of the lipid bending around the pore.

Unfortunately, there is no membrane-inserted structure of Bax to show how Bax stabilizes the pore edge. Putative membrane-inserted structures show that three helices in Bax are membrane-associated [5] , but no real 3D structure for Bax in the membrane has ever been solved to support this.

References

[1] S. Bleicken, C. Wagner, Ana J. García-Sáez, Mechanistic Differences in the Membrane Activity of Bax and Bcl-xL Correlate with Their Opposing Roles in Apoptosis, Biophys. J., 104 (2013) 421-431.

[2] M.-T. Lee, F.-Y. Chen, H.W. Huang, Energetics of Pore Formation Induced by Membrane Active Peptides, Biochemistry-US, 43 (2004) 3590-3599.

[3] S. Bleicken, O. Landeta, A. Landajuela, G. Basanez, A.J. Garcia-Saez, Proapoptotic Bax and Bak Proteins Form Stable Protein-permeable Pores of Tunable Size, J. Biol. Chem., 288 (2013) 33241-33252

[4] K. Cosentino, U. Ros, A.J. Garcia-Saez, Assembling the puzzle: Oligomerization of alpha-pore forming proteins in membranes, Biochim Biophys Acta, 1858 (2016) 457-466

[5] M. G. Annis, E. L. Soucie, P. J. Dlugosz, J. A. Cruz-Aguado, L. Z. Penn, B. Leber, and
D. W. Andrews. Bax forms multispanning monomers that oligomerize to permeabilize
membranes during apoptosis. EMBO J., 24 (2005) 2096–2103 .

Note: This is a five-part piece on our group’s recent contributions to Bax’s structure and function in apoptosis.

Quick links:

Part 1. Insights on Bax pore-forming activity

Part 2. Structural Model of Bax in the Membrane

Part 3. Counting Bax Molecules in the Membrane – A single molecule approach (to be released)

Part 4. Bax forms Rings, Arcs and Lines in Apoptotic Mitochondria (to be released)

Part 5. Membrane effects of Bax (to be released)