The N-terminal domain of annexin 2 serves as a secondary binding site during membrane bridging.
Annexin A2 (AnxA2) is a Ca(2+)- and acidic phospholipid-binding protein involved in many cellular processes. It undergoes Ca(2+)-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 Ca(2+)-mediated mechanism. Protein titration experiments showed that the protein concentration for half-maximal membrane aggregation was twice for Ca(2+)-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 Ca(2+)-mediated membrane bridging mechanisms.
Zibouche
Malik
M
INSERM UMRS893, CdR Saint-Antoine, Paris, F-75012.
Vincent
Michel
M
Illien
Françoise
F
Gallay
Jacques
J
Ayala-Sanmartin
Jesus
J
D015081:2-Naphthylamine / Q000031:analogs & derivatives / Q000378:metabolism; D017306:Annexin A2 / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D006863:Hydrogen-Ion Concentration; D007365:Intercellular Junctions / Q000378:metabolism*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence
D017306:Annexin A2; C038518:acrylodan; D015081:2-Naphthylamine