1 Scientific RepoRts | 6:27383 | DOI: 10.1038/srep27383 www.nature.com/scientificreports structure and calcium-binding studies of calmodulin-like domain of human non-muscle α-actinin-1 sara Drmota prebil 1 , Urška slapšak 2 , Miha Pavšič 1 , Gregor Ilc 2,3 , Vid Puž 1 , euripedes de Almeida Ribeiro 4 , Dorothea Anrather 5 , Markus Hartl 5 , Lars Backman 6 , Janez plavec 1,2,3 , Brigita Lenarčič 1,7 & Kristina Djinović-Carugo 1,4 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 fbers. 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 frst 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 fexible 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. Te actin cytoskeleton plays an essential role in many important cellular processes, such as muscle cell contrac- tion, motility, signalling, intracellular trafc, and maintenance of cell shape and stability. Te central components of the actin cytoskeleton are actin flaments, which are further equipped with diferent proteins giving them var- ious functionalities. α-actinins are one of the major actin cross-linking proteins found in virtually all cell types and are the ancestral proteins of a larger family that includes spectrin, dystrophin and utrophin 1 . Four isoforms of human α-actinins have been identifed: the calcium-insensitive muscle actinins (isoforms 2 and 3), which crosslink actin flaments in sarcomere-delimiting Z-disk complexes, and calcium-sensitive non-muscle isoforms (isoforms 1 and 4). Te non-muscle isoforms are found in all types of cells where they crosslink and organ- ize actin flaments into two general types of structures: three-dimensional networks, where the actin flaments are oriented in various directions, and bundles of tightly packed parallel actin flaments 2,3 . Bundles function as scafolds that support or stabilize cellular structures such as focal adhesion contacts, cell protrusions, and stress fbers 2,4,5 . For example, it was shown that dorsal stress fbres require α-actinin-1 and that their loss in α-actinin-1 depleted cells results in defective maturation of the leading edge in focal adhesions 6 . Te actin crosslinking function of all α-actinin isoforms is determined by its overall domain organization (Fig. 1), where the two actin-binding domains (ABD, composed of calponin homology domains CH1 and CH2) 1 Department of chemistry and Biochemistry, faculty of chemistry and chemical technology, University of Ljubljana, Večna pot 113, SI-1000 Ljubljana, Slovenia. 2 Slovenian NMR Centre, National Institute of Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Slovenia. 3 EN-FIST Centre of Excellence, Trg Osvobodilne fronte 13, SI-1000 Ljubljana, Slovenia. 4 Department of Structural and Computational Biology, Max F. Perutz Laboratories (MFPL), University of Vienna, Campus Vienna Biocenter 5, A-1030 Vienna, Austria. 5 Mass Spectrometry Service Facility, Max F. Perutz Laboratories (MFPL), University of Vienna, Vienna Biocenter (VBC), Dr. Bohr-Gasse 3, A-1030 Vienna, Austria. 6 Department of Chemistry, Umeå University, Linnaeus väg 10, SE-90187 Umeå, Sweden. 7 Department of Biochemistry, Molecular Biology and Structural Biology, Jožef Stefan Institute, Jamova 39,SI-1000 Ljubljana, Slovenia. Correspondence and requests for materials should be addressed to B.L. (email: brigita.lenarcic@fkt.uni-lj.si) or K.D-C. (email: kristina. djinovic@univie.ac.at) Received: 02 March 2016 Accepted: 16 May 2016 Published: 07 June 2016 OPEN