Cytoskeletal arrangements and remodeling control many biological functions of the cell, e.g. motility and adhesion. Mechanical properties of cytoskeletal structures are determined by the dynamics of molecular cross-linkers involved in the formation of actin networks. α-Actinin is a crucial component of important actin assemblies including stress fibers and actin caps since it both cross-link actin filaments and can directly connect them to the focal adhesion sites. α-Actinin is an anti-parallel rod-shaped homodimer with one actin-binding domain (ABD) at each end. The ABD is composed of two calponin homology domains, CH1 and CH2, that can exist in either open or closed conformation, which most likely determines their affinity for actin. It has been suggested that the open conformation is more favorable for actin binding but it is not yet clear how molecular interactions of the open ABD are different from the closed conformation. We compared ABD association to actin in both closed and open states and revealed the key differences between the two using all-atomic molecular dynamics simulations. Furhtermore, the k255E mutation in human α-actinin4 is involved in a kidney disease and located at the interface between CH1 and CH2. It was shown that this mutation alters mechanical properties of the cytoskeleton presumably by increasing the affinity between α-actinin and actin. We investigated the effect of this mutaiton on the the ABD conformation and showed the molecular mechanism by which the K255E mutation changes the binding between α-actinin and actin. Our results provide a valuable insight into developing novel therapeutics for the kidney disease cause by the K255E mutation.