Characterization of a Ca(2+)-binding site in human annexin II by site-directed mutagenesis.
Annexin II, a major cytoplasmic substrate of the src tyrosine kinase, is a member of the annexin family of Ca2+/phospholipid-binding proteins. It is composed of a short N-terminal tail (30 residues) followed by four so-called annexin repeats (each 70-80 residues in length) which share sequence homologies and are thought to form (a) new type(s) of Ca(2+)-binding site(s). We have produced wild-type and site specifically mutated annexin II molecules to compare their structure and biochemistry. The recombinant wild-type annexin II displays biochemical and spectroscopical properties resembling those of the authentic protein purified from mammalian cells. In particular, it shows the Ca(2+)-induced blue shift in fluorescence emission which is typical for this annexin. Replacement of the single tryptophan in annexin II (Trp-212) by a phenylalanine abolishes the fluorescence signal and allows the unambiguous assignment of the Ca(2+)-sensitive spectroscopic properties to Trp-212. This residue is located in the third annexin repeat in a highly conserved stretch of 17 amino acids which are also found in the other repeats and known as the endonexin fold. To study the precise architecture of the Ca2+ site which must reside in close proximity to Trp-212, we changed several residues of the endonexin fold in repeat 3 by site-directed mutagenesis. An analysis of these mutants by fluorescence spectroscopy and Ca(2+)-dependent phospholipid binding reveals that Gly-206 and Thr-207 seem indispensible for a correct folding of this Ca(2+)-binding site.
Thiel
C
C
Department of Biochemistry, Max Planck Institute for Biophysical Chemistry, Goettingen, Federal Republic of Germany.
Weber
K
K
Gerke
V
V
D000595:Amino Acid Sequence; D017302:Annexins; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005454:Fluorescence Polarization; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism
D017302:Annexins; D002135:Calcium-Binding Proteins; D011994:Recombinant Proteins; D014364:Tryptophan; D002118:Calcium