In silico conformational dynamics of the α-actinin-2 actin-binding domain upon phosphorylation

Figures

• 

  Figure 1. Schematic structure of human α-actinin-2 monomer. (A) The domain structure of human α-actinin-2 and their positions in the full-length α-actinin-2 monomer. The numbers indicate the amino acid residues. The ABD contains two calponin homology domains 1 and 2 (CH1 and CH2) shown in orange and blue, respectively. The residues T43, S50, S147, and T237, which are located at the CH1-CH2 interface and may be phosphorylated, are shown in magenta. The calmodulin-like domain (CaM) contains two pairs of EF-hand motifs (EF1-2 and EF3-4) shown in purple. The rod domain contains four spectrin repeats (SR1-4) shown in yellow. (B) The human α-actinin-2 resolved structure 4D1E is by Ribeiro et al. (4). The above-mentioned colors represent the same α-actinin-2 domains. The dashed black box shows the ABD. Magenta-colored spheres in the ABD depict the four phosphorylatable amino acid residues studied in the paper. The α-actinin-2 crystal structure is missing the first 33 N-terminal residues, starting with residue number 34, and the three last C-terminal amino acid residues, ending with residue number 892. (C) The α-actinin-2 ABD in the closed conformation. CH1 domain is orange, CH2 domain blue, and…

• 

  Figure 2. Phosphorylation and pseudophosphorylation increase the distances between the CH1 and CH2 domain centers of mass to open the α-actinin-2 ABD. (A–C) ChimeraX measured distances between calponin homology 1 (CH1) and calponin homology 2 (CH2) domains for ΑlphaFold3-predicted phosphorylated (A), pseudophosphorylated (B), and neutrally substituted (C) α-actinin-2 (ACTN2) ABD. Coloration changes from light to dark represent the phosphorylation/or substitution increase from one position to four positions. The red bar represents the WT α-actinin-2 predicted structure and the red dashed line shows the baseline for the WT structure. Two-tailed paired t-test showed statistical significance between WT α-actinin-2 and phosphorylated or pseudophosphorylated α-actinin-2 ΑlphaFold3-predicted structures at ∗∗p < 0.01 and ∗∗∗p < 0.001 (α = 0.001) levels, and WT α-actinin-2 and neutral substitution AlphaFold3-predicted structures at ∗∗p < 0.01 and ∗p < 0.05 (α = 0.001) levels. One-way ANOVA showed statistical significance between groups at p < 0.001 (α = 0.01). (C) The human α-actinin-2 ABD sequence. Italic amino acid residues (residues 1–35) illustrate the N-terminal residues of the ABD that show…

• 

  Figure 3. Phosphorylation and pseudophosphorylation increase the Cα RMSD values in superimposed structures of α-actinin-2 ABD in a dose-dependent manner. ChimeraX measured distances between calponin homology 1 (CH1) and calponin homology 2 (CH2) domains for ΑlphaFold3-predicted phosphorylated (A), pseudophosphorylated (B), and neutrally substituted (C) α-actinin-2 ABD. (1–3) The Cα RMSD values for each amino acid residue in the ABD separately. (1) The monophosphorylated, monophosphomimetic, and monosubstituted α-actinin-2 ABD for residues T43, S50, S147, and T237 separately. (2) The biphosphorylated, biphosphomimetic, and bisubstituted α-actinin-2 ABD, using different combinations of the aforementioned four residues. (3) The tri- and tetraphosphorylated/phosphomimetic and substituted α-actinin-2 ABD, where different combinations of the four previously mentioned amino acid residues were used. Solid lines represent the mean Cα RMSD values for each amino acid residue, and the dashed line represents the error bars between each measured prediction. (4) The mean Cα RMSD value for all the pruned amino acid residues. The structures show the superimposed ΑlphaFold3 α-actinin-2 WT to modified…

• 

  Figure 4. Phosphorylation and pseudophosphorylation of all the studied residues increase the net negative electrostatic potential at the CH2 domain while causing a net positive to net negative transition at the CH1 domain. (A–C) ChimeraX measured Coulombic potential separately for ABD (residues 36–256), calponin homology domain 1 (CH1, residues 38–142), calponin homology domain 2 (CH2, residues 151–256), actin-binding site 1 (ABS1, residues 40–49), and actin-binding site 2 (ABS2, residues 114–138) in ΑlphaFold3-predicted α-actinin-2 phosphorylated (A), pseudophosphorylated (B), and neutrally substituted (C) structures, in comparison with the WT α-actinin-2. Indication by double gradient colormap of GraphPad with the red color refers to the most negative Coulombic potential value at −3.5, and the blue color refers to the most positive Coulombic potential value at +2.5. Coulombic potential is shown in kcal/(mol·e). (D and E) The Coulombic electrostatic potential (ESP) maps of α-actinin-2 WT and phosphorylated, pseudophosphorylated, and neutrally substituted versions. (D) Dashed black rectangles indicate the interface between the CH1 and CH2 domains, with the CH1 domain being located to the…

• 

  Figure 5. MD simulations show that all the phosphomimetic mutations increased the spread in CH1-CH2 torsional and distance-dependent conformations. We calculated the CH1-CH2 interdomain distance (x axes) and relative rotation (y axes) for the WT and phosphomimetic MD simulations. Data from all 6 simulations (3× actin-bound-like and 3× actin-free-like) were combined. These data are presented as 2D landscapes where free energies were calculated from normalized population histograms according to the Boltzmann relation. The data from all WT (A), T43D (B), S147D (C), and T237D (D) cMD simulations are shown here, and results for the separate actin-bound-like and actin-unbound-like simulations are shown in Fig. S8. The histogram bins are colored according to the apparent free energy within each basin and all FE values are expressed relative to the maximally populated bin among all simulated sequences. Cyan circles denote the conformation of the initial actin-bound-like model and pink squares denote the conformation of the initial actin-free-like model. USMD-derived FE profiles of the open-closed transition were calculated using the WHAM method for all sequences and the T43D (E, pink), S147D (F,…

• 

  Figure 6. Working hypothesis of the redistribution of the open-closed equilibrium state of the α-actinin-2 ABD by phosphorylation. (A) The location of the α-actinin-2 ABD is highlighted by a dashed box and the respective closed (left) and open (right) sates are highlighted unbound and bound to F-actin, respectively. The open state depicts the phosphorylation sites at the ABD that possibly enhance such a state. (B) The energy landscape for the equilibrium state between the closed and open states is highlighted for the WT (light blue) and the phosphomimetic α-actinin-2 ABD (magenta), showing that the phosphomimetic variants redistribute the equilibrium from overweight to marginally overweight for the closed state relative to the open state.