Structure and calcium-binding studies of calmodulin-like domain of human non-muscle α-actinin-1.
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.
Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, SI-1000 Ljubljana, Slovenia.
Sara
S
Drmota Prebil
Slovenian NMR Centre, National Institute of Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Slovenia.
Urška
U
Slapšak
Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, SI-1000 Ljubljana, Slovenia.
Miha
M
Pavšič
Slovenian NMR Centre, National Institute of Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Slovenia.|EN-FIST Centre of Excellence, Trg Osvobodilne fronte 13, SI-1000 Ljubljana, Slovenia.
Gregor
G
Ilc
Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, SI-1000 Ljubljana, Slovenia.
Vid
V
Puž
Department of Structural and Computational Biology, Max F. Perutz Laboratories (MFPL), University of Vienna, Campus Vienna Biocenter 5, A-1030 Vienna, Austria.
Euripedes
E
de Almeida Ribeiro
Mass Spectrometry Service Facility, Max F. Perutz Laboratories (MFPL), University of Vienna, Vienna Biocenter (VBC), Dr. Bohr-Gasse 3, A-1030 Vienna, Austria.
Dorothea
D
Anrather
Mass Spectrometry Service Facility, Max F. Perutz Laboratories (MFPL), University of Vienna, Vienna Biocenter (VBC), Dr. Bohr-Gasse 3, A-1030 Vienna, Austria.
Markus
M
Hartl
Department of Chemistry, Umeå University, Linnaeus väg 10, SE-90187 Umeå, Sweden.
Lars
L
Backman
Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, SI-1000 Ljubljana, Slovenia.|Slovenian NMR Centre, National Institute of Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Slovenia.|EN-FIST Centre of Excellence, Trg Osvobodilne fronte 13, SI-1000 Ljubljana, Slovenia.
Janez
J
Plavec
Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, SI-1000 Ljubljana, Slovenia.|Department of Biochemistry, Molecular Biology and Structural Biology, Jožef Stefan Institute, Jamova 39,SI-1000 Ljubljana, Slovenia.
Brigita
B
Lenarčič
Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, SI-1000 Ljubljana, Slovenia.|Department of Structural and Computational Biology, Max F. Perutz Laboratories (MFPL), University of Vienna, Campus Vienna Biocenter 5, A-1030 Vienna, Austria.
Kristina
K
Djinović-Carugo
D000185:Actinin / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid
C526905:ACTN1 protein, human; D002147:Calmodulin; D000185:Actinin; D002118:Calcium