| Annexin A2 is a soluble protein with a number of important membrane-related physiological functions. At present, however, little is known about the conformation of annexin A2 under physiological conditions, especially that of the N-terminus; also little is known about the physiologically important membrane-bound forms of annexin A2. In this study we applied site-directed spin labeling (SDSL) together with electron paramagnetic resonance (EPR) to investigate the structure of annexin A2 in aqueous solution and in its different membrane-bound forms. We found that, in solution, the core domain region of the annexin A2 adopts a structure similar to that of the X-ray crystal structure (Rosengarth et al., 2004). In contrast to the recent crystallographic data on annexin A1, the N-terminus of annexin A2 is highly mobile and structurally unordered. In the calcium-dependent membrane-bound form, most of the annexin A2 core domain remains similar in conformation to that in solution. Loop residues on the convex side of annexin A2 become ordered upon membrane binding. Significant differences exist, however, when compared to the immobilization observed in the corresponding loop residues in annexin B12, suggesting that annexin A2 might adopt a membrane binding mechanism different from that of other annexins. Upon this calcium-induced membrane-interaction, primarily little change can be observed for the N-terminus. However, there is a small amount of order in the N-terminus seen at certain sites under some more aggregating conditions. In the pH-dependent membrane-bound form, lipid binding induced a global change in annexin A2 at mildly acidic pH, as previously described for annexin B12. It is not yet clear whether it is transmembrane or not, but we did find that residues in the core domain and the N-terminus refold and insert into the membrane. We also investigated the conformation of annexin A2 in the complex with S100A10. Upon the Ca2+-induced membrane interaction, we observed neither conformational changes nor membrane interaction at the N-terminus, consistent with a previous model where a S100A10 dimer is sandwiched between two annexin A2 subunits (Lambert, O. et al., 1997). |
