Research Article: Charge Profile Analysis Reveals That Activation of Pro-apoptotic Regulators Bax and Bak Relies on Charge Transfer Mediated Allosteric Regulation

Date Published: June 14, 2012

Publisher: Public Library of Science

Author(s): Crina-Maria Ionescu, Radka Svobodová Vařeková, Jochen H. M. Prehn, Heinrich J. Huber, Jaroslav Koča, James M. Briggs

Abstract: The pro-apoptotic proteins Bax and Bak are essential for executing programmed cell death (apoptosis), yet the mechanism of their activation is not properly understood at the structural level. For the first time in cell death research, we calculated intra-protein charge transfer in order to study the structural alterations and their functional consequences during Bax activation. Using an electronegativity equalization model, we investigated the changes in the Bax charge profile upon activation by a functional peptide of its natural activator protein, Bim. We found that charge reorganizations upon activator binding mediate the exposure of the functional sites of Bax, rendering Bax active. The affinity of the Bax C-domain for its binding groove is decreased due to the Arg94-mediated abrogation of the Ser184-Asp98 interaction. We further identified a network of charge reorganizations that confirms previous speculations of allosteric sensing, whereby the activation information is conveyed from the activation site, through the hydrophobic core of Bax, to the well-distanced functional sites of Bax. The network was mediated by a hub of three residues on helix 5 of the hydrophobic core of Bax. Sequence and structural alignment revealed that this hub was conserved in the Bak amino acid sequence, and in the 3D structure of folded Bak. Our results suggest that allostery mediated by charge transfer is responsible for the activation of both Bax and Bak, and that this might be a prototypical mechanism for a fast activation of proteins during signal transduction. Our method can be applied to any protein or protein complex in order to map the progress of allosteric changes through the proteins’ structure.

Partial Text: Mitochondrial outer membrane permeabilization (MOMP) is a hallmark of programmed cell death (apoptosis). Following MOMP, apoptotic proteins from the mitochondrial inter-membrane space are released, causing the activation of cell death proteases which cleave the cell’s cytoskeleton and genetic material. MOMP is executed by the Bcl-2 family proteins Bak and Bax that, upon activation during apoptosis, oligomerize and form pores in the mitochondrial membrane [1]–[4].

Allosteric proteins are characterized by a regulatory site that is distinct and often well distanced from the protein’s active site. Regulation of the protein’s activity which occurs via this distinct site is termed allosteric regulation. Recent reports indicate that allosteric regulation is particularly important during cell signaling processes, where it has been shown to stabilize receptor proteins, or to be responsible for the rapid, stress induced release of dormant signaling proteins bound to the cytoskeleton [48], [49]. An interesting structure-function analysis of Bax performed by George et al. [41] concluded that monomeric Bax may be held in an inactive conformation by multiple helices in the absence of stress, and that Bax may be activated through perturbation at multiple sites. Nevertheless, later Gavathiotis et al. identified a unique and well defined activation site on Bax [18], and subsequently demonstrated that binding of an activator BH3 peptide induces reverberations in the core of the Bax protein, a phenomenon they named allosteric sensing [19]. The present study found that this allosteric regulation is mediated by a charge transfer network, which conveys the activation information from the Bax activation site to the functional regions of Bax without compromising the structure of the BH groove (essential for pro-apoptotic activity). As charge transfer is significantly faster than domain rearrangements, the charge transfer mediated alosteric regulation in Bax also allows for a swift control of the apoptotic fate [50].

Source:

http://doi.org/10.1371/journal.pcbi.1002565