The role of conformational heterogeneity in regulating the apoptotic activity of BAX protein

Abstract

While activation of BAX is required for initiating mitochondria-mediated apoptosis, the underlying mechanisms remain unsettled. We studied conformations of BAX protein using pressure- and temperature-resolved ESR techniques and obtained the thermodynamic properties of the conformations. We show that inactive BAX is structurally heterogeneous and exists in equilibrium between two major populations of the conformations, UM and UM', of which the former is thermodynamically favored at room temperature. An increase in the population of UM', induced by either pressure or point mutations of BAX, renders BAX susceptible to oligomerization, which leads to cell death. This study uncovers the biological significance of BAX conformations and shows that the pro-apoptotic activity of BAX can be triggered by altering the equilibrium between the two states. It suggests that therapeutic intervention may focus on shifting the balance in the conformational heterogeneity.

Targeting BAX ubiquitin‐binding sites reveals that BAX activation is essential for its ubiquitin‐dependent degradation

“…In response to apoptosis induction, BAX is activated and undergoes conformation change firstly, and then it is translocated to the mitochondria outer membrane and forms the homoligomerized multimer, which could induce mitochondrial outer membrane permeabilization, followed by release of cytochrome c, Smac/DIABLO, and other apoptogenic factors . In addition, it has been reported that BAX conformational heterogeneity regulates its apoptotic activity . Prosurvival BCL‐2 proteins could bind to BAX and inhibit the formation of BAX multimer …”

Conformational Heterogeneity in the Activation Mechanism of Bax

“…(Renault and Chipuk, 2014; Renault and Manon, 2011; Schellenberg et al, 2013; Suzuki et al, 2000; Youle and Strasser, 2008). Recently, however, it was reported, using the mouse homolog of Bax, that monomeric Bax is in equilibrium between two conformationally distinct states, with the previously unobserved state serving as a transitionary state to Bax's membrane-bound form (Tsai et al, 2015; Kao et al, 2017). Using electron paramagnetic resonance double electron-electron resonance at varying temperatures and pressures, Chiang and coworkers “captured” and provided a structural model of this “new” form of monomeric Bax.…”

Identifying Protein Conformational Dynamics Using Spin‐label ESR

“…[60,61] This pressure-resolved DEER (PR-DEER) approachh as been employed with pressure up to 4k bar to confirm the conformational heterogeneity of inactive BAX monomer. [62] Inactive BAX monomer was confirmed to exist in two conformational states, denoted by UM and UM',w hich exhibit an equilibrium that favors UM at physiological temperature ( Figure 3C). [62] Since then, evidencef rom other studies hasg athered to support the conformational heterogeneityo fi nactive BAX.…”

“…[62] Inactive BAX monomer was confirmed to exist in two conformational states, denoted by UM and UM',w hich exhibit an equilibrium that favors UM at physiological temperature ( Figure 3C). [62] Since then, evidencef rom other studies hasg athered to support the conformational heterogeneityo fi nactive BAX. [63] As the population ratio of UM'/UM can be directly obtained from the change in the respective peaks in distance distributions, the pressure dependenceo ft he corresponding equilibrium constant K eq (P) relative to that at atmospheric (reference) pressure (1 bar) is given to second order in pressure P by, where each substate has ad ifferent partial molarv olume (V) and partial molar isothermal compressibility (b), P 0 is the reference pressure, R is the universal gas constant,a nd T is temperature.…”

“…Moreover, inspired by the development of PR‐DEER, temperature‐resolved DEER (TR‐DEER) approach was also introduced . Protein samples are first pre‐equilibrated at temperatures (200–300 K) using ultra‐low temperature bath, followed by plunge‐freezing in liquid nitrogen for DEER measurement.…”