Mailliard W S, Luecke H, Haigler H T
Department of Physiology and Biophysics, University of California, Irvine, California 92697, USA.
Biochemistry. 1997 Jul 22;36(29):9045-50. doi: 10.1021/bi970749v.
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 Mr'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.
膜联蛋白是一类蛋白质，它们以钙离子依赖的方式与磷脂结合，这些磷脂优先位于质膜的细胞内表面。最近对水螅膜联蛋白XII的X射线研究表明，它结晶为同型六聚体，其分子间钙离子结合位点与II型钙离子依赖的磷脂结合位点不同。基于这种六聚体结构，人们提出了一种新机制来解释膜联蛋白如何与膜相互作用。评估这一假设的第一步是确定膜联蛋白XII六聚体在蛋白质非结晶形式时是否存在。我们现在报告，溶液中的膜联蛋白XII可以用亚胺二甲酯交联成多聚体，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定，其表观分子量对应于三聚体和六聚体——三聚体条带可能对应于未完全交联的六聚体。多聚体的形成依赖于钙离子，并且当蛋白质首先与磷脂囊泡结合时会增强。为了评估分子间钙离子位点在膜联蛋白XII六聚体形成中的作用，将用于配位钙离子的一个残基谷氨酸105替换为赖氨酸（E105K）。在溶液中，E105K突变在中等（3 mM）但不是高（25 mM）钙离子存在下抑制六聚体形成。在磷脂存在下未观察到E105K膜联蛋白XII六聚体形成受到抑制，从而表明要么（i）其他相互作用在与双层膜结合时能够稳定六聚体，要么（ii）在双层膜上仅形成三聚体，观察到的六聚体条带是由于紧密堆积的三聚体交联所致。总之，本研究首次表明膜联蛋白XII在溶液中可以形成六聚体，并表明分子间钙离子位点参与六聚体形成。本研究还表明在双层膜上形成多聚体，但没有明确确定多聚体是三聚体还是六聚体。
