Folding energetics of ligand binding proteins II. Cooperative binding of Ca2+ to annexin I

The calcium binding properties of annexin I as observed by thermodynamic DSC studies have been compared to the structural information obtained from X-ray investigation. The calorimetric experiment permitted to evaluate both the reaction scheme - including binding of ligand and conformational changes - and the energetics of each reaction step. According to published X-ray data Annexin I has six calcium binding sites, three medium-affinity type II and three low-affinity type III sites. The present study shows that at 37 degrees C annexin I binds in a Hill type fashion simultaneously two calcium ions in a first step with medium affinity at a concentration of 0.6 mM and another three Ca(2+) ions again cooperatively at 30 mM with low affinity. Therefore it can be concluded that only two medium-affinity type II binding sites are available. The third site, that should be accessible in principle appears to be masked presumably due to the presence of the N terminus. In view of the large calcium concentration needed for saturation of the binding sites, annexin I may be expected to be Ca(2+) free in vivo unless other processes such as membrane interaction occur simultaneously. This assumption is consistent with the finding, that the affinity of annexins to calcium is usually markedly increased by the presence of lipids.