ANNEXIN-XII FORMS CALCIUM-DEPENDENT MULTIMERS IN SOLUTION AND ON PHOSPHOLIPID-BILAYERS - A CHEMICAL CROSS-LINKING STUDY

The annexins are a family of proteins that bind in a Ca2+-dependent ma nner to phospholipids that are preferentially located on the intracell ular face of plasma membranes, Recent X-ray studies of hydra annexin X II showed that it crystallized as a homohexamer with an intermolecular Ca2+ binding site separate from the type II Ca2+-dependent phospholip id binding site. On the basis of this hexamer structure, a novel mecha nism was proposed to explain how annexins interact with membranes. The first step toward evaluating this proposal is to determine whether th e 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 w ith dimethyl suberimidate into multimers with apparent M-r's correspon ding to trimers and hexamers as determined by SDS-polyacrylamide gel e lectrophoresis-the trimer band may correspond to incompletely cross-li nked hexamers. Multimer formation was dependent on Ca2+ and was enhanc ed when the protein first was bound to phospholipid vesicles. To evalu ate the role of the intermolecular Ca2+ site in annexin XII hexamer fo rmation, one of the residues used to coordinate Ca2+, glutamate 105, w as replaced with lysine (E105K). In solution, the E105K mutation inhib ited hexamer formation in the presence of moderate (3 mM) but not high (25 mM) Ca2+. No inhibition of E105K annexin XII hexamer formation wa s observed in the presence of phospholipid, thereby suggesting that ei ther (i) other interactions are capable of stabilizing the hexamer whe n bound to bilayers or (ii) only trimers form on bilayers and the obse rved 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 bi layers but does not clearly establish whether the multimers are trimer s or hexamers.