Change in the N-terminal domain conformation of annexin I that correlates with liposome aggregation is impaired by Ser-27 to Glu mutation that mimics phosphorylation.
Annexin I is a member of the annexin family of calcium-dependent membrane binding proteins. The core domain of these proteins is similar in all annexins but the N-terminal domain is specific for each member. This domain is thought to regulate annexin function through phosphorylation. In annexin I, Ser-27 is one of the amino acids that can be phosphorylated by protein kinase C. Phosphorylations are thought to regulate some annexin I functions by increasing calcium requirement. Two mutants were prepared in this study: S27E and S27A proteins. The first mimics phosphorylation while the second prevents phosphorylation at residue 27. Wild-type annexin I and S27A mutant protein showed the same calcium dependence for phospholipid vesicles aggregation, while S27E mutant protein showed a higher calcium requirement and a low maximal extent of aggregation. By contrast, liposome binding and self-association required identical calcium concentrations for the wild-type and the two mutant proteins. To examine whether the regulation observed is due to modification of the N-terminal structure, we investigated conformational changes by using two approaches. Firstly we analysed proteinase sensibility. Limited proteolysis of the N-terminal tail showed similar patterns for the three proteins. Using drastic conditions of proteolysis, we observed strong resistance of the core domain to digestion in the presence of calcium. Secondly, since Ser-27 is located on the N-terminal domain that contains a tryptophan located at position 12, the fluorescence of this residue was analysed during Ca2+-binding of wild-type annexin I and S27E mutant protein. The results demonstrated that Ca2+ induces a slight change in the Trp environment of wild-type annexin I, corresponding to a burying of the residue. No changes in fluorescence features were observed with S27E mutant protein. The results obtained show that phosphorylation of the N-terminal tail plays a regulatory role in phospholipid vesicle aggregation, which is based on a mechanism distinct from protein self-association. This phosphorylation induces a conformational change in the tail probably related to aggregation property.
Porte
F
F
INSERM U-431, Département Biologie-Santé, Université Monpellier II, France.
de Santa Barbara
P
P
Phalipou
S
S
Liautard
J P
JP
Widada
J S
JS
D017305:Annexin A1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008081:Liposomes / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D012694:Serine / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D014357:Trypsin / Q000378:metabolism; D014364:Tryptophan / Q000378:metabolism
D017305:Annexin A1; D008081:Liposomes; D011994:Recombinant Proteins; D012694:Serine; D014364:Tryptophan; D014357:Trypsin; D002118:Calcium