Structural and dynamic changes in human annexin VI induced by a phosphorylation-mimicking mutation, T356D.
Structural and dynamic changes in human annexin VI induced by a phosphorylation-mimicking mutation, T356D.
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由磷酸化模拟突变 T356D 诱导的人膜联蛋白 VI 的结构和动态变化。
DOI：
10.1021/bi026742h
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
Creutz,CarlE
中科院分区：
文献类型：
--
作者：
Freye-Minks,Caroline;Kretsinger,RobertH;Creutz,CarlE
膜联蛋白家族的一些成员的磷酸化可能在控制它们与膜的钙依赖性相互作用中起重要作用。最近对膜联蛋白VI的电子显微镜研究表明，该蛋白的两个核心结构域表现出很大程度的灵活性，并且能够经历相对的构象变化，这可能会引发膜之间的接触[Avila-Sakar， a.j, et al.(2000)]。结构体。现年54岁的Biol.130−62]。为了评估磷酸化在这种行为中的调节作用的可能性，磷酸化模拟突变体（人类膜联蛋白VI柔性连接区域的T356D）的晶体结构被确定为2.65 Å分辨率。当将突变体与野生型膜联蛋白VI进行比较时，在突变位点可以看到细微的差异，而在一个钙结合环和5个钙离子存在的情况下，更大的变化是明显的。此外，生化研究为T356D和野生型溶液结构之间的其他构象差异提供了证据。荧光发射和丙烯酰胺猝灭表明，在钙存在的情况下，Trp-343在T356D的连接区域的溶剂暴露水平较高。天然凝胶电泳延迟系数的比较显示，T356D具有更延伸的形状，而蛋白水解研究显示，连接区域内胰蛋白酶裂解位点的可及性更大，表明其构象比野生型更开放。这些数据提供了一种可能的调控机制，导致膜联蛋白VI在磷酸化时具有更高程度的灵活性和更高的钙结合亲和力。
Phosphorylation of some members of the annexin family of proteins may play a significant role in controlling their calcium-dependent interactions with membranes. Recent electron microscopic studies of annexin VI revealed that the protein's two core domains exhibit a great degree of flexibility and are able to undergo a relative conformational change that could potentially initiate contacts between membranes [Avila-Sakar, A. J., et al. (2000)J. Struct. Biol.130, 54−62]. To assess the possibility of a regulatory role of phosphorylation in this behavior, the crystal structure of a phosphorylation-mimicking mutant (T356D in the flexible connector region of human annexin VI) was determined to 2.65 Å resolution. When the mutant is compared to the wild-type annexin VI, subtle differences are seen at the site of the mutation, while larger changes are evident in one of the calcium-binding loops and in the presence of five calcium ions. Furthermore, biochemical studies provide evidence for additional conformational differences between the T356D and wild-type solution structures. Fluorescence emission and acrylamide quenching suggest a higher level of solvent exposure of Trp-343 in the connector region of T356D in the presence of calcium. Comparisons of retardation coefficients in native gel electrophoresis reveal that T356D has a more extended shape, while proteolytic studies show a greater accessibility of a trypsin cleavage site inside the linker region, indicating a conformation more open than the wild-type form. These data provide insights into a possible regulatory mechanism leading to a higher degree of flexibility and possibly a higher calcium binding affinity of annexin VI upon phosphorylation.