BPS2026 - 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) is found to increase 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 phospho-mimetic counterparts T43D, S50D, S147D, and T237D utilizing AlphaFold3, complemented by molecular dynamics (MD) simulations of these phospho-mimetic variants to quantify conformational changes at the ABD. AlphaFold3 modeling of phosphorylation and pseudo-phosphorylation sites showed an increase in the distances between the CH1 and CH2 domain’s centers of masses, opening the α-actinin-2 ABD. The CαRMSD values for the superimposed α-actinin-2 WT and phosphorylated, as well as pseudo-phosphorylated variants, demonstrate the alignment of the CH1 domains, but straightening of the loop region, separating the CH1 and CH2 domains. Phosphorylation and pseudo-phosphorylation 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 phospho-mimetic mutations increased the spread in CH1-CH2 torsional and distance-dependent conformations with S147D exhibiting the largest spread followed by T23D and T43D. These results suggest 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.