Structure and calcium-binding studies of calmodulin-like domain of human non-muscle α-actinin-1.

Scientific reports 2016 Jun 07

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The activity of several cytosolic proteins critically depends on the concentration of calcium ions. One important intracellular calcium-sensing protein is α-actinin-1, the major actin crosslinking protein in focal adhesions and stress fibers. The actin crosslinking activity of α-actinin-1 has been proposed to be negatively regulated by calcium, but the underlying molecular mechanisms are poorly understood. To address this, we determined the first high-resolution NMR structure of its functional calmodulin-like domain (CaMD) in calcium-bound and calcium-free form. These structures reveal that in the absence of calcium, CaMD displays a conformationally flexible ensemble that undergoes a structural change upon calcium binding, leading to limited rotation of the N- and C-terminal lobes around the connecting linker and consequent stabilization of the calcium-loaded structure. Mutagenesis experiments, coupled with mass-spectrometry and isothermal calorimetry data designed to validate the calcium binding stoichiometry and binding site, showed that human non-muscle α-actinin-1 binds a single calcium ion within the N-terminal lobe. Finally, based on our structural data and analogy with other α-actinins, we provide a structural model of regulation of the actin crosslinking activity of α-actinin-1 where calcium induced structural stabilisation causes fastening of the juxtaposed actin binding domain, leading to impaired capacity to crosslink actin.

Citation

Sara Drmota Prebil, Urška Slapšak, Miha Pavšič, Gregor Ilc, Vid Puž, Euripedes de Almeida Ribeiro, Dorothea Anrather, Markus Hartl, Lars Backman, Janez Plavec, Brigita Lenarčič, Kristina Djinović-Carugo. Structure and calcium-binding studies of calmodulin-like domain of human non-muscle α-actinin-1. Scientific reports. 2016 Jun 07;6:27383