α-actinin-4 is a cytoskeletal protein from the spectrin family. It consists of three domains, an actin-binding domain, a rod and calmodulin-like domain. The active form of α-actinin-4 is represented by antiparallel homodimer, whose central role is the cross-linking of actin filaments, through which it indirectly participates in the formation of focal contacts and adherent bands. Unlike related α-actinin-1, it is also present in the nucleoplasm, where it is thought to act as a transcription factor for many genes implicated in tumor cell changes. We conclude that isoform 4, like isoform 1, is regulated via calcium ion binding. For α-actinin-1, Ca2+-ion binding has been shown to induce conformational changes in the CaMD domain, resulting in a change in affinity for actin and subsequent reorganization of the actin cytoskeleton. In order to determine the binding affinity of Ca2+-ions and to characterize the protein in more detail, two protein constructs were prepared: a calmodulin-like domain and a construct shortened at the N-terminus by 45 amino acid residues that are structurally disordered according to the forecast and, as such, have a negative effect on crystallization. The number of binding sites for Ca2+-ions per domain (n = 1.19) and the binding affinity (Kd = 474 µM) were determined by isothermal titration calorimetry (ITC). For the purpose of crystallization, a methylated (methylation of lysine amino acid residues) form of α-actinin-4 was also prepared and, via size exclusion chromatography coupled with light scattering, revealed that it still forms a stable homodimer. Our results provide a good basis for more detailed structural and functional studies of α-actinin-4.
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