Title
Arabidopsis calmodulin-like protein CML36 is a calcium (Ca2+) sensor that interacts with the plasma membrane Ca2+-ATPase Isoform ACA8 and stimulates its activity
Author
Alessandra Astegno, Maria Cristina Bonza, Rosario Vallone, Valentina La Verde, Mariapina D'Onofrio, Laura Luoni, Barbara Molesini, Paola Dominici
Year
2017
Journal
Journal of Biological Chemistry
Abstract
Calmodulin-like (CML) proteins are major EF hand-containing, calcium (Ca2+)-binding proteins with crucial roles in plant development and in coordinating plant stress tolerance. Given their abundance in plants, the properties of Ca2+ sensors and identification of novel target proteins of CMLs deserve special attention. To this end, we recombinantly produced and biochemically characterized CML36 from Arabidopsis thaliana. We analyzed Ca2+ and Mg2+ binding to the individual EF hands, observed metal-induced conformational changes, and identified a physiologically relevant target. CML36 possesses two high affinity Ca2+/Mg2+ mixed binding sites and two low affinity Ca2+-specific sites. Binding of Ca2+ induced an increase in α-helical content and a conformational change that leads to the exposure of hydrophobic regions responsible for target protein recognition. Cation binding, either Ca2+ or Mg2+, stabilized the secondary and tertiary structure of CML36, guiding a large structural transition from a molten globule apo-state to a compact holo-conformation. Importantly, through in vitro binding and activity assays, we showed that CML36 directly interacts with the regulative N-terminus of the Arabidopsis plasma membrane Ca2+-ATPase isoform 8 (ACA8) and that this interaction stimulates ACA8 activity. Gene expression analysis revealed that CML36 and ACA8 are co-expressed mainly in inflorescences. Collectively, our results support a role for CML36 as a Ca2+ sensor that binds to and modulates ACA8, uncovering a possible involvement of CML protein family in the modulation of plant auto-inhibited Ca2+-pumps.
Instrument
FP-8200
Keywords
Fluorescence, Protein structure, Ligand binding, Biochemistry, Sensors