Characterization of the Ca2+-binding sites of annexin II tetramer

Annexin II heterotetramer (AIIt) is a multifunctional Ca2+-binding protein composed of two 11-kDa subunits and two annexin II subunits, The annexin II subunit contains three type II and two type III Ca2+-binding sites which are thought to regulate the interaction of AIIt with anionic phospholipid, F-actin, and heparin, In the present study we utilized site-directed mutagenesis to create AIIt mutants with inactive type III (TM AIIt), type II (CM AIIt), and both type II and III Ca2+-binding sites (TCM AIIt), Surprisingly, we found that in the presence of Ca2+, the TM, CM, and TCM AIIt bound phospholipid and F-actin with similar affinity to the wild type AIIt (WT AIIt). Furthermore, the TCM mutant, and to a lesser extent the TM and CM AIIt displayed dose-dependent Ca2+-independent phospholipid aggregation and binding. While the TM and CM AIIt demonstrated Ca2+-dependent binding to F-actin, the binding of the TCM AIIt was Ca2+-independent. These results suggest that the type II or type III Ca2+-binding sites do not directly participate in anionic phospholipid or F-actin binding, We therefore propose that in the absence of Ca2+, the type II and type III Ca2+-binding sites of AIIt stabilize a conformation of AIIt that is unfavorable for binding phospholipid and F-actin, Ca2+ binding to these sites, or the inactivation of these Ca2+-binding sites by site-directed mutagenesis, results in a conformational change that promotes binding to anionic phospholipid and F-actin. Since the TM, CM, and TCM AIIt require Ca2+ for binding to heparin, we also propose that novel Ca2+-binding sites regulate this binding event.