Distinct Ca2+-binding thermodynamics reveal a paralogue-specific structural response in the calmodulin-like domain of α-actinin-4.

The actin cytoskeleton, a complex and dynamic network, is precisely organized into structurally and functionally diverse assemblies by actin-binding proteins. A major group of actin crosslinking proteins are α-actinins. The non-muscle forms, α-actinin-1 and -4, are ubiquitous proteins whose actin crosslinking activity is regulated by Ca2+ ions. The molecular mechanism of regulation is unknown, but it has been suggested to be driven by structural changes triggered by the binding of Ca2+ ions to their calmodulin-like domain (CaMD). While the structure and Ca2+-binding properties of human α-actinin-1 CaMD have already been investigated, no such data were available for the α-actinin-4 CaMD. To address this, we used structurally informed mutagenesis and isothermal titration calorimetry (ITC) to confirm a homologous single Ca2+-binding site in the N-terminal lobe of the α-actinin-4 CaMD. In stark contrast to the α-actinin-1 CaMD, where binding is endothermic, binding to the α-actinin-4 CaMD is exothermic and displays a 6-fold lower affinity towards Ca2+. These distinct thermodynamic signatures suggest a paralogue-specific conformational change. Indeed, protein NMR spectroscopy revealed a unique Ca2+-induced conformational change of the C-terminal lobe, providing new insights into the molecular mechanisms that modulate α-actinin-mediated actin crosslinking.