Figure 1.
Bax undergoes several conformational changes enabling it to form pores in the mitochondrial outer membrane.
(A) Bax activation leads to mitochondrial outer membrane permeabilization (MOMP). Inactive Bax is a cytosolic monomer. Activator-induced opening of loop 1–2 allows the activator to bind. Subsequently, the C-domain of the now active Bax vacates the BH groove and inserts into the mitochondrial membrane. Additionally, the Bax BH3 domain gets exposed. Bax oligomerization ensues via the BH groove and the BH3 domain, eventually leading to the formation of pores which permeabilize the membrane. (B) Location of the BH3 domain (cyan), the C-domain (yellow), loop 1–2 (pink) in inactive Bax (the rest of the protein in gray). (C) Location of above domains (same color coding) in active Bax (the rest of the protein in orange). The activator peptide Bim-SAHB is shown in purple.
Figure 2.
Flowchart of calibration of an EEM model for calculating partial atomic charges in proteins.
(A) The reference data used in this study consisted of QM atomic charges for protein fragments in two reference sets (RS1, RS2) and one test set (T1–T5). (B) Two atom type definitions were used. The atomic electronegativity equations were grouped together based on the atom type. The EEM model parameters for each atom type were then obtained by least squares fitting to reference QM charges. (C) Each EEM model was subjected to internal and external validation by comparing the EEM charges with reference QM charges for all available data sets (RS1, RS2, T1–T5).
Figure 3.
Validation of EEM models by comparing EEM atomic charges against QM atomic charges.
Statistical descriptors comprising the average correlation coefficient (Ravg), the average root mean square deviation (RMSDavg) and the average absolute difference (Davg) are given. These descriptors quantify the agreement between EEM model charges and QM charges for molecules belonging to the reference sets RS1 and RS2, and for five further test molecules T1–T5. All quantities are given in elementary charges (1 e∼1.602×10−19 coulombs). The names of the parameter sets encode the reference set and atom classification scheme based on which they were developed (RS1-E, RS1-EX, RS2-E, RS2-EX). Good agreement between QM and EEM charges was found for all data sets, as Ravg is close to 1, and RMSDavg and Davg are minimal. Calibrations that used the coarse atom type classification ‘E’ gave a similarly good agreement as those where the more detailed classification scheme ‘EX’ was used.
Figure 4.
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.
Figure 5.
Proposed charge transfer network in Bax, indicated by net changes in residue charges.
The information of the Bim-SAHB induced activation of Bax is transmitted from the Bax activation site via a charge transfer network through the core of the Bax protein, up to the Bax C- and BH3-domains. Inside the hydrophobic core of Bax, the central helix, helix 5, acts as a hub which collects and distributes charge density, mainly through residues Trp107, Arg109 and Lys119. The color coding from Figure 1 is maintained. Additionally, helix 5 is highlighted in green. The Bax residues which transfer an amount of charge of one standard deviation higher than average (Table S1) are explicitly displayed and color coded according to whether they become more positive (blue) or negative (red) upon activation. (A) The residues which transfer a significant amount of charge were found at the Bax activation site, on the loop 1–2, inside the BH groove holding the Bax C-domain, and at one end of the C-domain (see also Figure S1). Additionally, several such residues were found on helix 5, the central helix in Bax, and on the Bax BH3 domain, suggesting that the interaction at the Bax activation site is transmitted via a network of charges from the activation site, through the protein core, to the C- and BH3-domains. (B) Top view of helix 5 is given. The organization of residues Trp107, Arg109 and Lys119 inside the hydrophobic core of Bax suggests that helix 5 acts as a charge transfer hub, which integrates and distributes charge density.
Figure 6.
Proposed Charge Transfer Network in Bak.
(A) Sequence alignment of central helices of Bak and Bax. An asterisk indicates a single, fully conserved residue. A colon indicates conservation between groups of strongly similar biochemical properties. A period indicates conservation between groups of weakly similar biochemical properties. The residues involved in the charge transfer network in Bax are conserved in Bak as Trp125, Arg127 and Arg137. (B) Bak structure (ochre) is displayed according to Figure 5B, with the same top view of the central helix, and the same color coding for C-domain (yellow), BH3-domain (cyan), central helix (green) and hub residues (red and blue). Residues Trp125, Arg127 and Arg137 are organized in a similar manner to their Bax homologues, suggesting that they may also play an essential role during Bak activation.
Table 1.
Summary of the atomic composition of all the protein fragments used for EEM model calibration.