Structural Superimposition

Abstact

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 alpha-actinins. The non-muscle forms, alpha-actinin-1 and -4, are ubiquitous proteins whose actin crosslinking activity is regulated by Ca(2+) ions. The molecular mechanism of regulation is unknown, but it has been suggested to be driven by structural changes triggered by the binding of Ca(2+) ions to their calmodulin-like domain (CaMD). While the structure and Ca(2+)-binding properties of human alpha-actinin-1 CaMD have already been investigated, no such data were available for the alpha-actinin-4 CaMD. To address this, we used structurally informed mutagenesis and isothermal titration calorimetry (ITC) to confirm a homologous single Ca(2+)-binding site in the N-terminal lobe of the alpha-actinin-4 CaMD. In stark contrast to the alpha-actinin-1 CaMD, where binding is endothermic, binding to the alpha-actinin-4 CaMD is exothermic and displays a 6-fold lower affinity towards Ca(2+). These distinct thermodynamic signatures suggest a paralogue-specific conformational change. Indeed, protein NMR spectroscopy revealed a unique Ca(2+)-induced conformational change of the C-terminal lobe, providing new insights into the molecular mechanisms that modulate alpha-actinin-mediated actin crosslinking.