Isas J Mario, Patel Darshana R, Jao Christine, Jayasinghe Sajith, Cartailler Jean-Philippe, Haigler Harry T, Langen Ralf
Department of Biochemistry and Molecular Biology, Keck School of Medicine, Zilkha Neurogenetic Institute and Arnold and Mabel Beckman Macular Research Center, University of Southern California, Los Angeles, California 90033, USA.
J Biol Chem. 2003 Aug 8;278(32):30227-34. doi: 10.1074/jbc.M301228200. Epub 2003 May 19.
Ca2+-dependent membrane interaction has long been recognized as a general property of the annexin (ANX) family of proteins. More recently, it has become clear that ANXs can also undergo Ca2+-independent membrane interactions at mildly acidic pH. Here we use site-directed spin labeling in combination with circular dichroism and biochemical labeling methods to compare the structure and membrane topography of these two different membrane-bound forms of ANX12. Our results reveal strong similarities between the solution structure and the structure of the Ca2+-dependent membrane-bound form at neutral pH. In contrast, all Ca2+-independent membrane interactions tested resulted in large scale conformational changes and membrane insertion. Pairs of spin labels that were in close proximity across the interface of different domains of the protein in both the soluble and Ca2+-dependent membrane form were >25 A apart in the Ca2+-independent membrane-bound form. Despite these major conformational changes, the overall secondary structure content did not appear to be strongly altered and ANX12 remained largely helical. Thus, Ca2+-independent membrane interaction leads to massive refolding but not unfolding. Refolding did not occur at low pH in the absence of membranes but occurred within a few seconds after phospholipid vesicles were added. The phospholipid composition of the vesicles was an important modulator of Ca2+-independent membrane interaction. For example, cardiolipin-containing vesicles induced Ca2+-independent membrane interaction even at near neutral pH, thereby raising the possibility that lipid composition could induce relatively rapid Ca2+-independent membrane interaction in vivo.
长期以来，钙离子依赖性膜相互作用一直被认为是膜联蛋白（ANX）家族蛋白质的一个普遍特性。最近，人们清楚地认识到，在轻度酸性pH条件下，膜联蛋白也能发生非钙离子依赖性膜相互作用。在这里，我们结合定点自旋标记、圆二色性和生化标记方法，比较了膜联蛋白12这两种不同膜结合形式的结构和膜拓扑结构。我们的结果揭示了溶液结构与中性pH条件下钙离子依赖性膜结合形式的结构之间有很强的相似性。相比之下，所有测试的非钙离子依赖性膜相互作用都导致了大规模的构象变化和膜插入。在可溶性和钙离子依赖性膜形式中，跨越蛋白质不同结构域界面紧密相邻的自旋标记对，在非钙离子依赖性膜结合形式中相距超过25埃。尽管发生了这些主要的构象变化，但整体二级结构含量似乎没有受到强烈改变，膜联蛋白12在很大程度上仍保持螺旋结构。因此，非钙离子依赖性膜相互作用导致大量重折叠而非解折叠。在没有膜的情况下，低pH时不会发生重折叠，但加入磷脂囊泡后几秒钟内就会发生。囊泡的磷脂组成是非钙离子依赖性膜相互作用的一个重要调节因素。例如，含心磷脂的囊泡即使在接近中性pH时也能诱导非钙离子依赖性膜相互作用，从而增加了脂质组成在体内可能诱导相对快速的非钙离子依赖性膜相互作用的可能性。
