Interdependence of phospholipid specificity and calcium binding in annexin I as shown by site-directed mutagenesis.

We have mutated the lysine 128 of domain II of annexin I, which flanks a putative calcium-binding loop, into a glutamic acid residue. The properties of the mutated recombinant protein were compared to those of the wild-type recombinant protein. A change in the isotherm of calcium binding in the presence of lipids was observed. A slight decrease in the affinity for lipids was evident. When tested for the vesicle aggregation property, the mutation induced a change in lipid specificity; unlike the wild-type protein, the mutant protein aggregates vesicles containing phosphatidylserine plus phosphatidylethanolamine better than vesicles containing only phosphatidylserine. These experiments are in agreement with a model which suggests that a lipid molecule is inserted into the calcium-binding loop of annexin I and that the conserved lysine residue is involved in the specificity of annexins for anionic phospholipids.