The activity of many cytosolic proteins depends on calcium concentration. One of the important intracellular proteins that respond to calcium ions is α-actinin-4. α actinins are cytoskeletal proteins of the spectrin family that play a crosslinking role between different cellular structures. They consist of four regions, namely an actin binding domain, a rod region with four spectrin repeats, linker region “neck” and a calmodulin-like domain. α actinins are divided into two groups according to their tissue specificity. The muscle isoforms, α-actinin-2 and -3, do not bind Ca2+-ions. On the other hand, the non-muscle isoforms, α-actinin-1 and -4, bind Ca2+-ions, which affects their structure and consequently the spatial arrangement of the actin filaments connected to them. The active form of α-actinin-4 is an antiparallel homodimer with an actin-binding domain at both ends, the central role of which is the cross-linking of actin filaments into bundles and networks. Unlike the related α-actinin-1, it is also present in the nucleoplasm, where it is thought to act as a transcription co-activator for many genes involved in tumorigenic cell changes.
Research so far has shown that the binding of Ca2+-ion in the calmodulin-like domain of α-actinin-1 triggers conformational changes in this part of the molecule, which are predicted to be transferred to the actin-binding domain and the resulting reorganization of the actin cytoskeleton. To compare the Ca2+-binding properties of non-muscle α actinins, we focused on the yet uncharacterized isoform 4.
As part of the master's thesis, we prepared protein crystals of the dimeric form of α-actinin-4, which contains all the domains involved in the interaction described above, and recorded a set of diffraction data up to a resolution of 2.9 Å.
By bioinformatic analysis, where we compared the sequences of the calmodulin-like domains of different isoforms, we determined those amino acid residues in the calmodulin-like domain of α-actinin-4, which are most likely to be crucial in the coordination of the Ca2+-ion. Like α-actinin-1, only one of the four EF1-hands is active in binding Ca2+-ions.
In order to test this hypothesis, we introduced various point mutations in the calmodulin-like domain of α-actinin-4, and the mutant forms were then characterized using isothermal titration calorimetry. We confirmed that only one of the potential binding sites for Ca2+ is indeed active, and the binding affinity is 4 times lower than that of α-actinin-1. We assume that the observed difference is one of the factors influencing the different behavior of these two isoforms and consequently, to their different preferential involvement in cytoskeletal structures such as focal junctions and curved membrane structures.
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