In:
European Journal of Biochemistry, Wiley, Vol. 207, No. 3 ( 1992-08), p. 923-930
Abstract:
Site‐directed mutagenesis was employed to map and characterize Ca 2+ ‐binding sites in annexin II, a member of the annexin family of Ca 2+ – and phospholipid‐binding proteins which serves as a major cellular substrate for the tyrosine kinase encoded by the src oncogene. Several single amino acid substitutions were introduced in the human annexin II and the various mutant proteins were scored for their affinity towards Ca 2+ in different assays. The data support our previous finding [Thiel, C., Weber, K. and Gerke V. (1991) J. Biol. Chem. 266 , 14732–14739] that a Ca 2+ –binding site is present in the third of the four repeat segments which comprise the 33‐kDa protein core of annexin II. In addition to Gly206 and Thr207, which are localized in the highly conserved endonexin fold of the third repeat, Glu246 is involved in the formation of this site. Thus the architecture of this Ca 2+ –binding site in solution is very similar, if not identical, to that of Ca 2+ sites identified recently in annexin V crystals [Huber, R., Schneider, M., Mayr, I., Römisch, J. and Paques, E.‐P. (1990) FEBS Lett. 275 , 15–21]. In addition to the site in repeat 3, we have mapped sites of presumably similar architecture in repeats 2 and 4 of annexin II. Again, an acidic amino acid which is located 40 residues C‐terminal to the conserved glycine at position 4 of the endonexin fold is indispensable for high‐affinity Ca 2+ binding: Asp161 in the second and Asp321 in the fourth repeat. In contrast, repeat 1 does not contain an acidic amino acid at a corresponding position and also shows deviations from the other repeats in the sequence surrounding the conserved glycine. These results on annexin II together with the crystallographic information on annexin V reveal that annexins can differ in the position of the Ca 2+ sites. Ca 2+ –binding sites of similar structure are present in repeats 2, 3, and 4 of annexin II while in annexin V they occur in repeats 1, 2, and 4. We also synthesized an annexin II derivative with mutations in all three Ca 2+ sites. This molecule shows a greatly reduced affinity for the divalent cation. However, it is still able to bind Ca 2+ , indicating the presence of (an) additional Ca 2+ site(s) of presumably different architecture.
Type of Medium:
Online Resource
ISSN:
0014-2956
,
1432-1033
DOI:
10.1111/ejb.1992.207.issue-3
DOI:
10.1111/j.1432-1033.1992.tb17125.x
Language:
English
Publisher:
Wiley
Publication Date:
1992
detail.hit.zdb_id:
1398347-7
detail.hit.zdb_id:
1464377-7
SSG:
12
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