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

In primary cardiomyocyte cell cultures, α-actinin-2 phosphorylation at the actin-binding domain (ABD) increases with mechanical stress, facilitating adaptation to varying forces. Nevertheless, it is unknown whether these phosphorylation sites in α-actinin-2 have structural consequences that could explain differential binding to F-actin and influence sarcomere stabilization and assembly. This study aims to understand the mechanisms by which α-actinin-2 regulates the assembly dynamics of sarcomeres in the heart. To investigate phosphorylation-specific modifications at the α-actinin-2 ABD, structural modeling was conducted on phosphorylation sites T43, S50, S147, and T237 alongside their phosphomimetic counterparts T43D, S50D, S147D, and T237D. We quantified conformational changes at the ABD using complementary AlphaFold3-generated models and molecular dynamics (MD) simulations of the phosphomimetic variants. AlphaFold3 modeling of phosphorylation and pseudophosphorylation sites showed an increase in the distance between the centers of mass of the two calponin homology domains (CH1 and CH2), and a decrease in torsion angles, opening the α-actinin-2 ABD. These structural changes correlated with more favorable electrostatic interaction energies (ΔH_elec) toward actin. C_α RMSD values for the superimposed α-actinin-2 WT and phosphorylated, as well as pseudophosphorylated variants, demonstrate the alignment of the CH1 domains, but untangling of the loop region, separating the CH1 and CH2 domains. 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. MD simulations show that all the phosphomimetic mutations increased the spread in CH1-CH2 torsional and distance-dependent conformations, with S147D exhibiting the largest spread followed by T237D and T43D. These results imply that α-actinin-2 phosphorylation affects the structural stability of the closed conformation of the ABD, suggesting that phosphorylation could promote the open state of α-actinin-2 ABD and facilitate its engagement with actin.