Jost M, Thiel C, Weber K, Gerke V
Max Planck Institute for Biophysical Chemistry, Department of Biochemistry, Göttingen, Federal Republic of Germany.
Eur J Biochem. 1992 Aug 1;207(3):923-30. doi: 10.1111/j.1432-1033.1992.tb17125.x.
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 Ca2+ in different assays. The data support our previous finding [Thiel, C., Weber, K. and Gerke V. (1991) J. Biol. Chem. 266, 14,732-14,739] 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 Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ sites. This molecule shows a greatly reduced affinity for the divalent cation. However, it is still able to bind Ca2+, indicating the presence of (an) additional Ca2+ site(s) of presumably different architecture.
采用定点诱变技术来定位和表征膜联蛋白II中的钙离子结合位点。膜联蛋白II是钙离子和磷脂结合蛋白家族的成员，是src癌基因编码的酪氨酸激酶的主要细胞底物。在人膜联蛋白II中引入了几个单氨基酸替换，并在不同的测定中对各种突变蛋白对钙离子的亲和力进行了评分。这些数据支持了我们之前的发现[蒂尔，C.，韦伯，K.和格克，V.（1991年）《生物化学杂志》266，14732 - 14739]，即在构成膜联蛋白II 33 kDa蛋白核心的四个重复片段中的第三个片段中存在一个钙离子结合位点。除了位于第三个重复片段高度保守的内联蛋白折叠区的甘氨酸206和苏氨酸207外，谷氨酸246也参与了该位点的形成。因此，溶液中这个钙离子结合位点的结构与最近在膜联蛋白V晶体中鉴定出的钙离子位点的结构非常相似（如果不是完全相同的话）[胡伯，R.，施奈德，M.，迈尔，I.，罗米施，J.和帕克斯，E.-P.（1990年）《欧洲生物化学学会联合会快报》275，15 - 21]。除了重复片段3中的位点外，我们还在膜联蛋白II的重复片段2和4中定位了结构可能相似的位点。同样，在内联蛋白折叠区第4位保守甘氨酸C端40个残基处的一个酸性氨基酸对于高亲和力钙离子结合是必不可少的：第二个重复片段中的天冬氨酸161和第四个重复片段中的天冬氨酸321。相比之下，重复片段1在相应位置不包含酸性氨基酸，并且在保守甘氨酸周围的序列中也与其他重复片段存在差异。这些关于膜联蛋白II的结果以及关于膜联蛋白V的晶体学信息表明，膜联蛋白的钙离子位点位置可能不同。膜联蛋白II的重复片段2、3和4中存在结构相似的钙离子结合位点，而在膜联蛋白V中它们出现在重复片段1、2和4中。我们还合成了一种在所有三个钙离子位点都有突变的膜联蛋白II衍生物。该分子对二价阳离子的亲和力大大降低。然而，它仍然能够结合钙离子，这表明可能存在结构不同的其他钙离子位点。
