Cation and peptide binding properties of CML7, a calmodulin-like protein from Arabidopsis thaliana

• Published in: Journal of inorganic biochemistry 2019-10, Vol.199, p.110796-110796, Article 110796
• Main Authors: Trande, Matteo, Pedretti, Marco, Bonza, Maria Cristina, Di Matteo, Adele, D'Onofrio, Mariapina, Dominici, Paola, Astegno, Alessandra
• Format: Article
• Language: English
• Subjects: Arabidopsis thaliana; Calcium sensor; Calmodulin-like proteins; EF-hand; Melittin
• ISSN: 0162-0134; 1873-3344
• DOI: 10.1016/j.jinorgbio.2019.110796

Plants contain a large family of so-called calmodulin-like proteins (CMLs) which differ from canonical calmodulin in that they show greater variability in sequence, length, and number of EF-hand domains. The presence of this extended CML family has raised questions regarding the role of these proteins: are they functionally redundant or do they play specific functions in physiological plant processes? To answer these questions, comprehensive biochemical and structural information on CML proteins is fundamental. Among the 50 CMLs from Arabidopsis thaliana, herein we described the ability of CML7 to bind metal ions focusing on the Ca2+ and Mg2+ sensing properties, as well as on metal-induced conformational changes. Circular dichroism and nuclear magnetic resonance (NMR) studies indicated that both Ca2+ and Mg2+ stabilize CML7, as reflected in conformational rearrangements in secondary and tertiary structure and in increases in thermal stability of the protein. However, the conformational changes that binding induces differ between the two metal ions, and only Ca2+ binding controls a structural transition that leads to hydrophobic exposure, as suggested by 8-anilino-1-naphthalenesulfonic acid fluorescence. Isothermal titration calorimetry data coupled with NMR experiments revealed the presence of two high affinity Ca2+-binding sites in the C-lobe of CML7 and two weaker sites in the N-lobe. The paired nature of these CML7 EF-hands enables them to bind Ca2+ with positive cooperativity within each globular domain. Our results clearly place CML7 in the category of Ca2+ sensors. Along with this, the protein can bind to a model target peptide (melittin) in a Ca2+-dependent manner. Calmodulin-like protein 7 from Arabidopsis thaliana displays properties consistent with a role as Ca2+ sensor, including those associated with Ca2+ affinity, Ca2+-induced conformational changes and binding to the model target peptide melittin. Mg2+ binds and stabilizes the protein but it is not expected to substitute for Ca2+ in target interaction. [Display omitted] •Arabidopsis calmodulin-like protein 7 (CML7) behaves like a Ca2+ sensor in vitro.•Apo-, Mg2+- and Ca2+-bound CML7 exist in distinct structural conformations.•CML7 has two high affinity Ca2+ sites in the C-lobe and two weaker in the N-lobe.•CML7 binds to the model target peptide melittin in a Ca2+-dependent manner.•CML7 is localized in the cytoplasm.