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

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 the α-helical content and a conformational change that lead to the exposure of hydrophobic regions responsible for target protein recognition. Cation binding, either Ca ²⁺ or Mg ²⁺, stabilized the secondary and tertiary structures of CML36, guiding a large structural transition from a molten globule apo-state to a compact holoconformation. Importantly, through in vitro binding and activity assays, we showed that CML36 interacts directly 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 the CML protein family in the modulation of plant-autoinhibited Ca ²⁺ pumps.