Site-directed mutagenesis of a calcium binding site modifies specifically the different biochemical properties of annexin I.
All the functions of annexins in vitro as well as in vivo are mediated and probably regulated by calcium. We have used recombinant annexin I, synthesized by Escherichia coli, and we have performed site-directed mutagenesis. We have mutated the endonexin fold of domain 2 that binds calcium. Mutations were performed in this domain of the molecule because it perfectly matches the calcium binding consensus sequence. The two glycines of this fold were mutated into glutamic acid. The helix content and the stability of the mutants are identical to those of the wild-type, suggesting that the mutations did not drastically affect the structure of the protein. The two mutants showed modified calcium binding affinities. However, the calcium binding affinity of the G131E mutant was far more altered than that of the G129E mutant. Furthermore, other biochemical properties of these mutants were modified to different extents. The binding to phospholipid was not seriously affected, whereas the self-association was lost by the G131E mutant. In the same way, liposome aggregation is conserved, but modified, while the calcium affinity measured by equilibrium dialysis is dramatically altered.
Travé
G
G
INSERM U-65, Departement Biologie Santé, Université Montpellier II, France.
Cregut
D
D
Lionne
C
C
Quignard
J F
JF
Chiche
L
L
Sri Widada
J
J
Liautard
J P
JP
D017305:Annexin A1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D003956:Dialysis; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics; D005971:Glutamates; D018698:Glutamic Acid; D005998:Glycine; D008081:Liposomes / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D017305:Annexin A1; D005971:Glutamates; D008081:Liposomes; D011994:Recombinant Proteins; D018698:Glutamic Acid; D002118:Calcium; D005998:Glycine