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

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. Author Summary: Apoptosis is a physiological form of cell death that is fundamental for development, growth and homeostasis in multi-cellular organisms. Deviations in the apoptosis machinery are known to be involved in cancer, neurodegenerative disorders, and autoimmune diseases. The proteins Bax and Bak are essential for executing apoptosis, yet the mechanism of their activation is not properly understood at the structural level. To understand this mechanism, we investigated how the electronic density is reorganized (i.e., how charge is transferred) inside the Bax molecule when Bax binds a functional peptide of its natural activator protein. We identified the specific interactions responsible for the exposure of the functional sites of Bax, rendering Bax active. Furthermore, we found a network of charge transfer that conveys activation information from the Bax activation site, through the hydrophobic core of Bax, to the well-distanced functional sites of Bax. This network consists of three residues inside the hydrophobic core of Bax, which are present also in the hydrophobic core of Bak, suggesting that these residues are functionally important and thus potential drug targets. We provide a straightforward and accessible methodology to identify the key residues involved in the fast activation of proteins during signal transduction.