Acidic pH promotes oligomerization and membrane insertion of the BclXL apoptotic repressor

Abstract

Solution pH is believed to serve as an intricate regulatory switch in the induction of apoptosis central to embryonic development and cellular homeostasis. Herein, using an array of biophysical techniques, we provide evidence that acidic pH promotes the assembly of BclXL apoptotic repressor into a megadalton oligomer with a plume-like appearance and harboring structural features characteristic of a molten globule. Strikingly, our data reveal that pH tightly modulates not only oligomerization but also ligand binding and membrane insertion of BclXL in a highly subtle manner. Thus, while oligomerization and the accompanying molten globular content of BclXL is least favorable at pH 6, both of these structural features become more pronounced under acidic and alkaline conditions. However, membrane insertion of BclXL appears to be predominantly favored under acidic conditions. In a remarkable contrast, while ligand binding to BclXL optimally occurs at pH 6, it is diminished by an order of magnitude at lower and higher pH. This reciprocal relationship between BclXL oligomerization and ligand binding lends new insights into how pH modulates functional versatility of a key apoptotic regulator and strongly argues that the molten globule may serve as an intermediate primed for membrane insertion in response to apoptotic cues.

Introduction

The Bcl2 family of proteins plays a central role in coupling apoptotic stimuli to the removal of damaged and unwanted cells during physiological processes such as embryonic development and cellular homeostasis [1], [2], [3], [4], [5], [6], [7], [8]. The Bcl2 proteins can be divided into three major groups: activators, effectors and repressors. Activators such as Bid and Bad belong to the BH3-only proteins, where BH3 is the Bcl2 homology 3 domain. Effectors such as Bax and Bak contain the BH3–BH1–BH2–TM modular architecture, where TM is the transmembrane domain located C-terminal to Bcl2 homology domains BH3, BH1 and BH2. Repressors such as Bcl2, BclXL and BclW are characterized by the BH4–BH3–BH1–BH2–TM modular organization, with an additional N-terminal Bcl2 homology 4 domain. According to one school of thought, the apoptotic fate, or the decision of a cell to continue to live or pull the trigger to commit suicide, is determined by the cellular ratio of activator, effector and repressor molecules [9], [10]. In quiescent and healthy cells, the effectors are maintained in an inactive state via complexation with repressors. Upon receiving apoptotic cues, in the form of DNA damage and cellular stress, the activators are stimulated and compete with effectors for binding to the repressors and, in so doing, not only do they neutralize the anti-apoptotic action of repressors but also unleash the pro-apoptogenicity of effectors. The effectors subsequently initiate apoptotic cell death by virtue of their ability to insert into the mitochondrial outer membrane (MOM)¹ resulting in the formation of mitochondrial pores in a manner akin to the insertion of bacterial toxins such as colicins and diphtheria [11], [12], [13], [14], [15]. This leads to the release of apoptogenic factors such as cytochrome c and Smac/Diablo from mitochondria into the cytosol. Subsequently, rising levels of apoptogenic factors in the cytosol switch on aspartate-specific proteases termed caspases, which in turn, demolish the cellular architecture by cleavage of proteins culminating in total cellular destruction.

Despite their low sequence convergence, all members of Bcl2 family share a remarkably conserved 3D topological fold characterized by a central predominantly hydrophobic α-helical hairpin “dagger” (α5 and α6) surrounded by a “cloak” comprised of six amphipathic α-helices (α1–α4 and α7–α8) of varying lengths [16]. A prominent feature of repressors is that they contain what has come to be known as the “canonical hydrophobic groove”, formed by the juxtaposition of α2–α5 helices, that serves as the docking site for the BH3 domain (α2 helix) of activators and effectors. Additionally, the effectors and repressors also contain a C-terminal hydrophobic α-helix termed α9, or more commonly the TM domain, because it allows these members of the Bcl2 family to localize to MOM upon apoptotic induction [17], [18], [19]. The “cloak and dagger” structural topology of Bcl2 members is the hallmark of their functional duality in that they are able to co-exist as “soluble factors” under quiescent cellular state and as “membrane channels” upon apoptotic induction. Notably, the hydrophobic dagger not only provides the bulk of the thermodynamic force in driving the water–membrane transition of various Bcl2 members upon apoptotic induction but also directly participates in the formation of mitochondrial pores that provide a smooth channel for the exit of apoptogenic factors. In particular, the water–membrane transition of effectors and repressors is believed to be driven by acidic pH, and optimally occurs at around pH 4, in a manner akin to pore formation by the bacterial toxins [20], [21], [22], [23], [24], [14], [25], [26]. The acidic pH destabilizes the solution conformation of these proteins while at the same time inducing the formation of molten globule, which is believed to serve as an intermediate for subsequent insertion into membranes [27], [11], [28], [29]. It should be noted that the molten globule is a partially disordered conformation which contains a native-like secondary structure but without the tightly-packed hydrophobic core comprised of nonpolar residues [30], [31], [32], [33]. Importantly, several lines of evidence suggest the formation of a pH gradient across the mitochondria, accompanied by the alkalinization of mitochondrial matrix and acidification of the cytosol, upon the induction of apoptosis [34], [35], [36], [37], [38], [39]. This observation further corroborates the role of acidic pH in driving apoptotic machinery.

We have previously shown that BclXL displays the propensity to oligomerize in solution and that such oligomerization is driven by the intermolecular binding of its C-terminal TM domain to the canonical hydrophobic groove in a domain-swapped trans-fashion, whereby the TM domain of one monomer occupies the canonical hydrophobic groove within the other monomer and vice versa [40]. We postulated that such oligomerization serves as a regulatory switch to turn the anti-apoptotic action of BclXL “off” in quiescent cells but “on” in response to apoptotic cues. In an effort to understand how solution pH modulates oligomerization of BclXL and the effect of such oligomerization on subsequent binding of BH3 ligands in the form of activators and effectors and membrane insertion in the context of apoptosis, we undertook the present study. Herein, we provide evidence that acidic pH promotes the assembly of BclXL apoptotic repressor into a megadalton oligomer with a plume-like appearance and harboring structural features characteristic of a molten globule. Strikingly, our data reveal that pH tightly modulates not only oligomerization but also ligand binding and membrane insertion of BclXL in a highly subtle manner. Thus, while oligomerization and the accompanying molten globular content of BclXL is least favorable at pH 6, both of these structural features become more pronounced under acidic and alkaline conditions. However, membrane insertion of BclXL appears to be predominantly favored under acidic conditions. In a remarkable contrast, while ligand binding to BclXL optimally occurs at pH 6, it is diminished by an order of magnitude at lower and higher pH. This reciprocal relationship between BclXL oligomerization and ligand binding lends new insights into how pH modulates functional versatility of a key apoptotic regulator and strongly argues that the molten globule may serve as an intermediate primed for membrane insertion in response to apoptotic cues.