The N-terminal domain of annexin 2 serves as a secondary binding site during membrane bridging

Annexin A2 (AnxA2) is a Ca2+- and acidic phospholipid-binding protein involved in many cellular processes. It undergoes Ca2+-mediated membrane bridging at neutral pH and has been demonstrated to be involved in an H+-mediated mechanism leading to a novel AnxA2-membrane complex structure. We used fluorescence techniques to characterize this H+-dependent mechanism at the molecular level; in particular, the involvement of the AnxA2 N-terminal domain. This domain was labeled at Cys-8 either with acrylodan or pyrene-maleimide fluorescent probes. Steady- state and time-resolved fluorescence analysis for acrylodan and fluorescence quenching by doxyl-labeled phospholipids revealed direct interaction between the N-terminal domain and the membrane. The absence of pyrene excimer suggested that interactions between N termini are not involved in the H+-mediated mechanism. These findings differ from those previously observed for the Ca2+-mediated mechanism. Protein titration experiments showed that the protein concentration for half-maximal membrane aggregation was twice for Ca2+-mediated compared with H+-mediated aggregation, suggesting that AnxA2 was able to bridge membranes either as a dimer or as a monomer, respectively. An N-terminally deleted AnxA2 was 2 - 3 times less efficient than the wild-type protein for H+-mediated membrane aggregation. We propose a model of AnxA2-membrane assemblies, highlighting the different roles of the N-terminal domain in the H+- and Ca2+-mediated membrane bridging mechanisms.