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Charge profile analysis reveals that activation of pro-apoptotic regulators Bax and Bak relies on charge transfer mediated allosteric regulation.

Ionescu CM, Svobodová Vařeková R, Prehn JH, Huber HJ, Koča J - PLoS Comput. Biol. (2012)

Bottom Line: The affinity of the Bax C-domain for its binding groove is decreased due to the Arg94-mediated abrogation of the Ser184-Asp98 interaction.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.

View Article: PubMed Central - PubMed

Affiliation: CEITEC - Central European Institute of Technology, Masaryk University, Brno, Czech Republic.

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.

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In active Bax, Arg94 recruits Asp98, destabilizing the C-domain inside the BH groove.Upon activation, Arg94 becomes more positive, leading to the recruitment of Asp98, abrogation of the Asp98-Ser184 interaction, and ultimately destabilization of the C-domain inside the BH groove [16], [40]. The color coding from Figure 1 is maintained. Additionally, the atoms in residues Arg94, Asp98 and Ser184 are displayed explicitly. Colors are coded according to their EEM charges, where the color scale ranges from red, through green, to blue, as values of atomic charges go from negative to positive. The EEM charges were computed using parameter set RS2-E (see Figures 2 and 3). (A) In inactive Bax, Asp98 is engaged in an interaction with Ser184, which keeps the C-domain in its binding pocket. (B) In active Bax, the now more positively charged Arg94 (see also Table S1) has sequestered Asp98, which no longer contributes to the stabilization of the Bax C-domain in its BH groove.
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pcbi-1002565-g004: In active Bax, Arg94 recruits Asp98, destabilizing the C-domain inside the BH groove.Upon activation, Arg94 becomes more positive, leading to the recruitment of Asp98, abrogation of the Asp98-Ser184 interaction, and ultimately destabilization of the C-domain inside the BH groove [16], [40]. The color coding from Figure 1 is maintained. Additionally, the atoms in residues Arg94, Asp98 and Ser184 are displayed explicitly. Colors are coded according to their EEM charges, where the color scale ranges from red, through green, to blue, as values of atomic charges go from negative to positive. The EEM charges were computed using parameter set RS2-E (see Figures 2 and 3). (A) In inactive Bax, Asp98 is engaged in an interaction with Ser184, which keeps the C-domain in its binding pocket. (B) In active Bax, the now more positively charged Arg94 (see also Table S1) has sequestered Asp98, which no longer contributes to the stabilization of the Bax C-domain in its BH groove.

Mentions: Experimental evidence suggests that, in inactive Bax, the C-terminal helix is bound tightly to its hydrophobic pocket (‘BH-groove’). During activation, this binding gets destabilized, causing the C-domain to subsequently vacate the BH-groove and insert into the mitochondrial outer membrane. Early mutagenesis studies revealed a critical interaction between residues Ser184 and Asp98 at the C-domain-BH-groove interface, whose abrogation is sufficient to immediately activate Bax [16], [40]. We therefore focused on the changes in charge density distribution in the vicinity of this interaction. While our calculations did not show any change in the charge profile of Ser184, they indicated that any interaction that might have taken place between Asp98 and Ser184 in the inactive structure has been replaced by an Asp98-Arg94 salt bridge in the active structure (Figure 4). Upon activation, Arg94 becomes more positive (see also Table S1), which is suggested to lead to the recruitment of Asp98, the abrogation of the Asp98-Ser184 interaction, and ultimately the destabilization of the C-domain. This demonstrates that the binding of Bim-SAHB to Bax can activate Bax by destabilizing the interaction between the Bax C-domain and its binding groove.


Charge profile analysis reveals that activation of pro-apoptotic regulators Bax and Bak relies on charge transfer mediated allosteric regulation.

Ionescu CM, Svobodová Vařeková R, Prehn JH, Huber HJ, Koča J - PLoS Comput. Biol. (2012)

In active Bax, Arg94 recruits Asp98, destabilizing the C-domain inside the BH groove.Upon activation, Arg94 becomes more positive, leading to the recruitment of Asp98, abrogation of the Asp98-Ser184 interaction, and ultimately destabilization of the C-domain inside the BH groove [16], [40]. The color coding from Figure 1 is maintained. Additionally, the atoms in residues Arg94, Asp98 and Ser184 are displayed explicitly. Colors are coded according to their EEM charges, where the color scale ranges from red, through green, to blue, as values of atomic charges go from negative to positive. The EEM charges were computed using parameter set RS2-E (see Figures 2 and 3). (A) In inactive Bax, Asp98 is engaged in an interaction with Ser184, which keeps the C-domain in its binding pocket. (B) In active Bax, the now more positively charged Arg94 (see also Table S1) has sequestered Asp98, which no longer contributes to the stabilization of the Bax C-domain in its BH groove.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3375244&req=5

pcbi-1002565-g004: In active Bax, Arg94 recruits Asp98, destabilizing the C-domain inside the BH groove.Upon activation, Arg94 becomes more positive, leading to the recruitment of Asp98, abrogation of the Asp98-Ser184 interaction, and ultimately destabilization of the C-domain inside the BH groove [16], [40]. The color coding from Figure 1 is maintained. Additionally, the atoms in residues Arg94, Asp98 and Ser184 are displayed explicitly. Colors are coded according to their EEM charges, where the color scale ranges from red, through green, to blue, as values of atomic charges go from negative to positive. The EEM charges were computed using parameter set RS2-E (see Figures 2 and 3). (A) In inactive Bax, Asp98 is engaged in an interaction with Ser184, which keeps the C-domain in its binding pocket. (B) In active Bax, the now more positively charged Arg94 (see also Table S1) has sequestered Asp98, which no longer contributes to the stabilization of the Bax C-domain in its BH groove.
Mentions: Experimental evidence suggests that, in inactive Bax, the C-terminal helix is bound tightly to its hydrophobic pocket (‘BH-groove’). During activation, this binding gets destabilized, causing the C-domain to subsequently vacate the BH-groove and insert into the mitochondrial outer membrane. Early mutagenesis studies revealed a critical interaction between residues Ser184 and Asp98 at the C-domain-BH-groove interface, whose abrogation is sufficient to immediately activate Bax [16], [40]. We therefore focused on the changes in charge density distribution in the vicinity of this interaction. While our calculations did not show any change in the charge profile of Ser184, they indicated that any interaction that might have taken place between Asp98 and Ser184 in the inactive structure has been replaced by an Asp98-Arg94 salt bridge in the active structure (Figure 4). Upon activation, Arg94 becomes more positive (see also Table S1), which is suggested to lead to the recruitment of Asp98, the abrogation of the Asp98-Ser184 interaction, and ultimately the destabilization of the C-domain. This demonstrates that the binding of Bim-SAHB to Bax can activate Bax by destabilizing the interaction between the Bax C-domain and its binding groove.

Bottom Line: The affinity of the Bax C-domain for its binding groove is decreased due to the Arg94-mediated abrogation of the Ser184-Asp98 interaction.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.

View Article: PubMed Central - PubMed

Affiliation: CEITEC - Central European Institute of Technology, Masaryk University, Brno, Czech Republic.

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.

Show MeSH
Related in: MedlinePlus