Calmodulin-like (CML) proteins are major EF hand-containing, calcium (Ca(2+))-binding proteins with crucial roles in plant development and in coordinating plant stress tolerance. Given their abundance in plants, the properties of Ca(2+) 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 Ca(2+) and Mg(2+) binding to the individual EF hands, observed metal-induced conformational changes, and identified a physiologically relevant target. CML36 possesses two high affinity Ca(2+)/Mg(2+) mixed binding sites and two low affinity Ca(2+)-specific sites. Binding of Ca(2+) 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 Ca(2+) or Mg(2+), 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 Ca(2+)-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 Ca(2+) sensor that binds to and modulates ACA8, uncovering a possible involvement of CML protein family in the modulation of plant auto-inhibited Ca(2+)-pumps.

Arabidopsis calmodulin-like protein CML36 is a calcium (Ca(2+)) sensor that interacts with the plasma membrane Ca(2+)-ATPase Isoform ACA8 and stimulates its activity

ASTEGNO, Alessandra;Vallone, Rosario;LA VERDE, Valentina;D'ONOFRIO, Mariapina;MOLESINI, Barbara;DOMINICI, Paola
2017

Abstract

Calmodulin-like (CML) proteins are major EF hand-containing, calcium (Ca(2+))-binding proteins with crucial roles in plant development and in coordinating plant stress tolerance. Given their abundance in plants, the properties of Ca(2+) 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 Ca(2+) and Mg(2+) binding to the individual EF hands, observed metal-induced conformational changes, and identified a physiologically relevant target. CML36 possesses two high affinity Ca(2+)/Mg(2+) mixed binding sites and two low affinity Ca(2+)-specific sites. Binding of Ca(2+) 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 Ca(2+) or Mg(2+), 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 Ca(2+)-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 Ca(2+) sensor that binds to and modulates ACA8, uncovering a possible involvement of CML protein family in the modulation of plant auto-inhibited Ca(2+)-pumps.
Arabidopsis thaliana; calcium; calcium sensor; calmodulin-like proteins; metal ion-protein interaction; plasma membrane Ca2+-ATPase; protein conformation; protein-protein interaction
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11562/968512
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