Annexin XII forms calcium-dependent multimers in solution and on phospholipid bilayers: A chemical cross-linking study
Annexin XII forms calcium-dependent multimers in solution and on phospholipid bilayers: A chemical cross-linking study
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DOI：
10.1021/bi970749v
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发表时间：
1997-07-22
期刊：
影响因子：
2.9
通讯作者：
Haigler, HT
中科院分区：
文献类型：
--
作者：
Mailliard, WS;Luecke, H;Haigler, HT
膜联蛋白是一类以钙离子依赖的方式与磷脂结合的蛋白质家族，最近对Heda Annin XII的X射线研究表明，它是以六聚体的形式结晶的，其分子间钙结合部位与II型钙依赖的磷脂结合部位分开。基于这种六聚体结构，提出了一种新的机制来解释膜联蛋白与膜的相互作用。评估这一提议的第一步是确定当蛋白质不是以晶体形式存在时，膜联蛋白XII六聚体是否存在。我们现在报道，溶液中的Annexin XII可以与亚氨酸二甲酯交联成多聚体，通过SDS-聚丙烯酰胺凝胶电泳法确定明显的M-r对应于三聚体和六聚体--三聚体带可能对应于不完全交联的六聚体。多聚体的形成依赖于钙离子，当蛋白质第一次结合到磷脂小泡上时，多聚体的形成被促进。为了评价分子间钙离子位点在膜联蛋白XII六聚体形成中的作用，用赖氨酸(E105K)取代了用于协调钙离子的残基之一谷氨酸105。在溶液中，在中等(3 MM)但不高(25 MM)的钙离子存在下，E105K突变抑制六聚体的形成。在磷脂存在下，没有观察到抑制E105K Annexin XII六聚体的形成，从而表明：(I)当与双分子层结合时，其他相互作用能够稳定六聚体，或(Ii)双分子层上只形成三聚体，观察到的六聚体条带是由于紧密堆积的三聚体的交联所致。综上所述，本研究首次表明Annexin XII可以在溶液中形成六聚体，并涉及六聚体形成中的分子间钙离子位点。这项研究还表明，多聚体是在双层上形成的，但不清楚地确定多聚体是三聚体还是六聚体。
The annexins are a family of proteins that bind in a Ca2+-dependent manner to phospholipids that are preferentially located on the intracellular face of plasma membranes, Recent X-ray studies of hydra annexin XII showed that it crystallized as a homohexamer with an intermolecular Ca2+ binding site separate from the type II Ca2+-dependent phospholipid binding site. On the basis of this hexamer structure, a novel mechanism was proposed to explain how annexins interact with membranes. The first step toward evaluating this proposal is to determine whether the annexin XII hexamer exists when the protein is not in a crystalline form. We now report that annexin XII in solution can be cross-linked with dimethyl suberimidate into multimers with apparent M-r's corresponding to trimers and hexamers as determined by SDS-polyacrylamide gel electrophoresis-the trimer band may correspond to incompletely cross-linked hexamers. Multimer formation was dependent on Ca2+ and was enhanced when the protein first was bound to phospholipid vesicles. To evaluate the role of the intermolecular Ca2+ site in annexin XII hexamer formation, one of the residues used to coordinate Ca2+, glutamate 105, was replaced with lysine (E105K). In solution, the E105K mutation inhibited hexamer formation in the presence of moderate (3 mM) but not high (25 mM) Ca2+. No inhibition of E105K annexin XII hexamer formation was observed in the presence of phospholipid, thereby suggesting that either (i) other interactions are capable of stabilizing the hexamer when bound to bilayers or (ii) only trimers form on bilayers and the observed hexamer bands were due to cross-linking of closely packed trimers. In summary, this study shows for the first time that annexin XII can form hexamers in solution and implicates the intermolecular Ca2+ site in hexamer formation. This study also shows that multimers form on bilayers but does not clearly establish whether the multimers are trimers or hexamers.