Biochemical and Biophysical Investigation of a Calmodulin-Like Protein From Glycine max Delineates Its Role as a Calcium Sensor During Herbivory.

The environment surrounding plants is far from stable, compelling the plant to perceive and adapt to numerous biotic and abiotic constraints, including insect attacks. The perception of a feeding insect typically entails the identification of herbivore-associated molecular patterns causing a sequential increase in cytosolic Ca²⁺ levels. Calmodulin-like proteins (CMLs) are Ca²⁺ sensor proteins with conserved EF-hands, which decode Ca²⁺ signals to generate a stress-specific response. Although few of the CMLs have been investigated and their role in plant defence has been deciphered in model plants, the role of CMLs as a Ca²⁺ sensor protein and their interaction mechanism with calcium and downstream targets remains poorly understood in leguminous crop plants. Herein, we demonstrated the presence of a Ca²⁺ signature in Glycine max during herbivory. Gene expression analysis of GmCML77 (a member of the CML family) indicated its upregulation during S. litura infestation. Phylogenetic analysis and in silico studies predicted GmCML77 as a Ca²⁺ binding protein. Employing computational modelling and MD simulations, we showed that GmCML77 has predominantly α-helical conformation with 3 functional Ca²⁺ binding loops. Also, Ca²⁺ binding initiates an expansion of tertiary structure, leading to the exposure of hydrophobic residues that may be implicated in its interaction with target proteins. Moreover, gel shift assay and CD spectroscopy results confirmed the Ca²⁺ binding ability of GmCML77. Our study demonstrated that GmCML77 is a functional calcium-binding protein, which exhibits conformational changes on Ca²⁺ binding and acts as a Ca²⁺ sensor during insect infestation.