Calcium-dependent protein kinases (CDPKs) are structurally unique Ser/Thr kinases found in plants and certain protozoa. They are distinguished by a calmodulin-like regulatory apparatus (calmodulin-like domain (CaM-LD)) that is joined via a junction (J) region to the C-terminal end of the kinase catalytic domain. Like CaM, the CaM-LD is composed of two globular EF structural domains (N-lobe, C-lobe), each containing a pair of Ca2+binding sites. Spectroscopic analysis shows that the CaM-LD is comprised of helical elements, but the isolated CaM-LD does not form a conformationally homogeneous tertiary structure in the absence of Ca2+. The addition of substoichiometric amounts of Ca2+is sufficient to stabilize the C-terminal lobe in a construct containing J and CaM-LD (JC) but not in the CaM-LD alone. Moreover, as J is titrated into Ca2+-saturated CaM-LD, interactions are stronger with the C-lobe than the N-lobe of the CAM-LD. Measurements of Ca2+affinity for JC reveal two cooperatively interacting high affinity binding sites (Kd,mean= 5.6 nM at 20 nM KCl) in the C-lobe and two weaker sites in the N-lobe (Kd,mean= 110 nM at 20 mM KCl). The corresponding Ca2+binding constants in the isolated CaM-LD are lower by more than 2 orders of magnitude, which indicates that the J region has an essential role in stabilizing the structure of the CDPK regulatory apparatus. The large differential affinity between the two domains together with previous studies on a plasmodium CDPK (Zhao, Y., Pokutta, S., Maurer, P., Lindt, M., Franklin, R. M., and Kappes, B. (1994) Biochemistry 33, 3714-3721) suggests a model whereby even at normally low cytosolic levels of Ca2+, the C-lobe interacts with the junction, but the kinase remains in an autoinhibited state. Activation then occurs when Ca2+levels rise to fill the two weaker affinity binding sites in the N-lobe, thereby triggering a conformational change that leads to release of the autoinhibitory region.