Calorimetric study of the interaction of the C2 domains of classical protein kinase C isoenzymes with Ca2+ and phospholipids.

The affinities of Ca(2+) and anionic lipid vesicles from the C2 domains of classical protein kinase C subfamily (alpha, betaII, and gamma) were studied using isothermal titration calorimetry (ITC). In addition, the thermal stability of these C2 domains in the presence of different ligand concentrations was analyzed using differential scanning calorimetry (DSC). These three closely related C2 domains bind Ca(2+) in a similar way, demonstrating the presence of two sets of sites. The first set of sites binds one Ca(2+) ion exothermically with similar high affinity for the three proteins (K(d) around 1 microM), while the second set of sites binds endothermically approximately two Ca(2+) ions with lower affinity, which varies for each C2 domain: 22.2 microM for the PKCalpha-C2 domain, 17.2 microM for the PKCbetaII-C2 domain, and 4.3 microM for the PKCgamma-C2 domain. In the absence of Ca(2+), the three C2 domains showed a weak interaction with vesicles containing anionic phospholipids. However, in the presence of a saturating Ca(2+) concentration, the C2 domains increased their affinities for the anionic lipid vesicles. In all cases, the C2 domains bound the vesicles exothermically and with similar affinities. A DSC thermal stability study of the C2 domains in the presence of Ca(2+) and anionic lipids provided further information about this protein-ligand interaction. The presence of increasing Ca(2+) concentrations was matched by an increase in the T(m) in all cases, which was even greater in the presence of anionic lipid vesicles. The extent of the change in T(m) differed for each C2 domain, reflecting the differing effect of the ligands bound during the protein stabilization. Denaturation of the C2 domains was irreversible both in the absence and in the presence of ligands, although the thermograms were not kinetically controlled. The dependence of the T(m) on the Ca(2+) concentration indicates that the protein stabilization observed by DSC primarily reflects the saturation by the cation of the low-affinity set of sites.