Annexin XII Forms Calcium-Dependent Multimers in Solution and on Phospholipid Bilayers: A Chemical Cross-Linking Study

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Abstract
The annexins are a family of proteins that bind in a Ca2+-dependent manner to phospholipids that are preferentially located on the intracellular face of plasma membranes. Recent X-ray studies of hydra annexin XII showed that it crystallized as a homohexamer with an intermolecular Ca2+ binding site separate from the type II Ca2+-dependent phospholipid binding site. On the basis of this hexamer structure, a novel mechanism was proposed to explain how annexins interact with membranes. The first step toward evaluating this proposal is to determine whether the annexin XII hexamer exists when the protein is not in a crystalline form. We now report that annexin XII in solution can be cross-linked with dimethyl suberimidate into multimers with apparent Mr's corresponding to trimers and hexamers as determined by SDS−polyacrylamide gel electrophoresisthe trimer band may correspond to incompletely cross-linked hexamers. Multimer formation was dependent on Ca2+ and was enhanced when the protein first was bound to phospholipid vesicles. To evaluate the role of the intermolecular Ca2+ site in annexin XII hexamer formation, one of the residues used to coordinate Ca2+, glutamate 105, was replaced with lysine (E105K). In solution, the E105K mutation inhibited hexamer formation in the presence of moderate (3 mM) but not high (25 mM) Ca2+. No inhibition of E105K annexin XII hexamer formation was observed in the presence of phospholipid, thereby suggesting that either (i) other interactions are capable of stabilizing the hexamer when bound to bilayers or (ii) only trimers form on bilayers and the observed hexamer bands were due to cross-linking of closely packed trimers. In summary, this study shows for the first time that annexin XII can form hexamers in solution and implicates the intermolecular Ca2+ site in hexamer formation. This study also shows that multimers form on bilayers but does not clearly establish whether the multimers are trimers or hexamers.