Calcium-Induced Conformational Switching of Paramecium Calmodulin Provides Evidence for Domain Coupling^†

Calmodulin (CaM) is an intracellular calcium-binding protein essential for many pathways in eukaryotic signal transduction. Although a structure of Ca²⁺-saturated Paramecium CaM at 1.0 Å resolution (1EXR.pdb) provides the highest level of detail about side-chain orientations in CaM, information about an end state alone cannot explain driving forces for the transitions that occur during Ca²⁺-induced conformational switching and why the two domains of CaM are saturated sequentially rather than simultaneously. Recent studies focus attention on the contributions of interdomain linker residues. Electron paramagnetic resonance showed that Ca²⁺-induced structural stabilization of residues 76−81 modulates domain coupling [Qin and Squier (2001) Biophys. J. 81, 2908−2918]. Studies of N-domain fragments of Paramecium CaM showed that residues 76−80 increased thermostability of the N-domain but lowered the Ca²⁺ affinity of sites I and II [Sorensen et al. (2002) Biochemistry 41, 15−20]. To probe domain coupling during Ca²⁺ binding, we have used ¹H−¹⁵N HSQC NMR to monitor more than 40 residues in Paramecium CaM. The titrations demonstrated that residues Glu78 to Glu84 (in the linker and cap of helix E) underwent sequential phases of conformational change. Initially, they changed in volume (slow exchange) as sites III and IV titrated, and subsequently, they changed in frequency (fast exchange) as sites I and II titrated. These studies provide evidence for Ca²⁺-dependent communication between the domains, demonstrating that spatially distant residues respond to Ca²⁺ binding at sites I and II in the N-domain of CaM.