α-actinin-1 is a major actin filament cross-linking protein in stress fibers and focal adhesions. It contains 4 EF-hand motifs (together they form the calmodulin-like domain – CaMD – of α-actinin-1), but only EF1 is able to bind calcium. Research suggests that Ca2+ binding to α-actinin-1 weakens its ability to cross-link actin filaments and thus acts as a regulator of its function. Recently, the model of regulation of actin-binding activity of α-actinin-1 was proposed, whereupon calcium binding α-actinin-1 obtains a more closed conformation, in which CaMD interacts with the neck region between actin-binding domain and spectrin region. This prevents α-actinin-1 from cross-linking actin filaments into parallel or anti-parallel bundles, but the exact molecular details have not yet been determined. To evaluate the proposed model, we performed limited proteolysis assays of full-length wild type α-actinin-1 (closed form) and a full-length α-actinin-1 mutant with impaired Ca2+ binding ability (open form). Proteolysis with trypsin showed that α-actinin-1 is more resistant to digestion in the presence of Ca2+ (closed form), which is in agreement with the proposed model. Proteolysis with thermolysin also revealed some differences in the cleavage pattern between Ca2+-bound and -free α-actinin-1, but we were unable to characterize the binding of CaMD to the neck region, therefore further experiments are needed to validate the proposed model. To further characterize the conformational changes at the molecular level, we prepared half-dimer of α-actinin-1 and its mutants with reduced surface entropy for crystallization. We have produced protein crystals of the holo (with Ca2+) and apo (without Ca2+) forms of the reduced surface entropy half-dimer construct (mutations K79A and E81A), which will be used to determine the crystal structure of Ca2+-bound and -free α-actinin-1 by X-ray diffraction.
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