Functional characterization of orchardgrass cytosolic Hsp70 (DgHsp70) and the negative regulation by Ca2+/AtCaM2 binding

When plants are exposed to extreme temperature, stress-inducible proteins are highly induced and involved in subcellular defence mechanisms. Hsp70, one of stress-inducible proteins, functions as an ATP-dependent molecular chaperone in broad organisms to process such as the inhibition of protein denaturation, promotion of protein folding, and renaturation of denatured proteins. In this study, we isolated a heat-inducible orchardgrass Hsp70 (DgHsp70) that is a homolog of cytosolic Hsp70 that possesses a CaM-binding domain. Purified DgHsp70 protein displayed dose-dependent ATPase, holdase, and ATP-dependent foldase activities. To investigate functional roles of DgHsp70 by the association of Arabidopsis calmodulin-2 (AtCaM2), showing heat-sensitive reduction on transcription, we first characterized the binding activity by gel-overlay assay. DgHsp70 binds to AtCaM2 in the presence of Ca2+ via a conserved CaM-binding domain. Ca2+/AtCaM2 binding decreased ATPase activity of DgHsp70, and concomitantly, reduced foldase activity. Based on the protein structure of bovine Hsc70, which is the closest structural homolog of DgHsp70, a CaM-binding domain is located near the ATP-binding site and CaM may span the ATP-binding pocket of Hsp70. Its decreased functional foldase activity may be caused by blocking ATP hydrolysis after Ca2+/AtCaM2 binding. It may associate with inhibition of functional activity of DgHsp70 in the absence of stress and/or de novo protein synthesis of DgHsp70 in the presence of thermal stress condition.