Biochemical and biophysical characterization of a plant calmodulin: Role of the N- and C-lobes in calcium binding, conformational change, and target interaction

• Plant GAD is activated in response to abiotic stress via association with CaM.
• AtCaM1 binds four Ca2 + ions with an apparent Kd of 20.0 ± 1.2 μM.
• The C-lobe of AtCaM1 has a ~ 6-fold stronger Ca2 + binding affinity than the N-lobe.
• N- and C-lobes of AtCaM1 differ in their conformational dynamics.
• Only intact AtCaM1, and not the form with perturbed N- or C-lobe, is able to fully activate GAD1.

In plants, transient elevation of intracellular Ca2 + concentration in response to abiotic stress is responsible for glutamate decarboxylase (GAD) activation via association with calmodulin (CaM), an EF-hand protein consisting of two homologous domains (N and C). An unusual 1:2 binding mode of CaM to CaM-binding domains of GAD has long been known, however the contribution of the two CaM domains in target recognition and activation remains to be clarified. Here, we explored the coupling between physicochemical properties of Arabidopsis CaM1 (AtCaM1) and Arabidopsis GAD1 activation, focusing on each AtCaM1 lobe. We found that the four EF-loops of AtCaM1 differently contribute to the ~ 20 μM apparent affinity for Ca2 + and the C-lobe shows a ~ 6-fold higher affinity than N-lobe (Kdapp 5.6 μM and 32 μM for C- and N-lobes, respectively). AtCaM1 responds structurally to Ca2 + in a manner similar to vertebrate CaM based on comparison of Ca2 +-induced changes in hydrophobicity exposure, secondary structure, and hydrodynamic behavior. Molecular dynamics simulations of AtCaM1 apo and Ca2 +-bound reveal that the latter state is significantly less flexible, although regions of the N-lobe remain quite flexible; this suggests the importance of N-lobe for completing the transition to the extended structure of holoprotein, consistent with data from ANS fluorescence, CD spectroscopy, and SEC analysis. Moreover, enzymatic analysis reveal that mutations in the two lobes affect GAD1 activation in similar ways and only intact AtCaM1 can fully activate GAD1. Taken together, our data provide new insights into the CaM lobes role in interactions between CaM and plant GAD.