Structure, topology, and dynamics of myristoylated recoverin bound to phospholipid bilayers.
Structure, topology, and dynamics of myristoylated recoverin bound to phospholipid bilayers.
复制标题
与磷脂双层结合的肉豆蔻酰化恢复蛋白的结构、拓扑和动力学。
DOI：
10.1021/bi0206816
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发表时间：
2003
期刊：
影响因子：
2.9
通讯作者：
Ames,JamesB
中科院分区：
文献类型：
--
作者：
Valentine,KathleenG;Mesleh,MichaelF;Opella,StanleyJ;Ikura,Mitsuhiko;Ames,JamesB
Recoverin是EF-Hand蛋白超家族的成员之一，在视网膜视杆细胞中作为钙传感器发挥作用。恢复素的N-末端共价连接的肉豆蔻基通过一种称为钙-肉豆蔻基开关的机制促进其与视网膜盘膜的结合。根据固体核磁共振谱中观察到的15N和31P化学位移的分析，15N标记的钙结合的肉豆蔻酰化恢复素各向异性地结合到磷脂膜上。根据A2H核磁共振序参数的分析，恢复素含有一个完全的氚肉豆蔻基，N-末端的肉豆蔻基团似乎位于脂质双层内。~1H-−~(15)N-PISEMA的二维固体核磁共振(~1H-~(15)N-PISEMA)谱表明，钙离子结合蛋白位于膜表面，使其分子长轴相对于膜法线方向为∼45°。恢复素的N-末端区域指向膜表面，与碱性残基K5、K11、K22、K37、R43和K84形成紧密接触。膜结合蛋白的这种取向允许靠近膜表面的暴露的疏水缝隙作为目标蛋白视紫红质激酶的潜在结合部位。恢复素的实验和计算的固体核磁共振谱之间的密切一致表明，膜结合的恢复素保持了与其在溶液中相同的整体三维结构。这些结果表明，恢复素与膜的结合主要是通过在双层内插入肉豆蔻基实现的，而蛋白质结构的重排显然很少。
Recoverin, a member of the EF-hand protein superfamily, serves as a calcium sensor in retinal rod cells. A myristoyl group covalently attached to the N-terminus of recoverin facilitates its binding to retinal disk membranes by a mechanism known as the Ca2+-myristoyl switch. Samples of15N-labeled Ca2+-bound myristoylated recoverin bind anisotropically to phospholipid membranes as judged by analysis of15N and31P chemical shifts observed in solid-state NMR spectra. On the basis of a2H NMR order parameter analysis performed on recoverin containing a fully deuterated myristoyl group, the N-terminal myristoyl group appears to be located within the lipid bilayer. Two-dimensional solid-state NMR (1H−15N PISEMA) spectra of uniformly and selectively15N-labeled recoverin show that the Ca2+-bound protein is positioned on the membrane surface such that its long molecular axis is oriented ∼45° with respect to the membrane normal. The N-terminal region of recoverin points toward the membrane surface, with close contacts formed by basic residues K5, K11, K22, K37, R43, and K84. This orientation of the membrane-bound protein allows an exposed hydrophobic crevice, near the membrane surface, to serve as a potential binding site for the target protein, rhodopsin kinase. Close agreement between experimental and calculated solid-state NMR spectra of recoverin suggests that membrane-bound recoverin retains the same overall three-dimensional structure that it has in solution. These results demonstrate that membrane binding by recoverin is achieved primarily by insertion of the myristoyl group inside the bilayer with apparently little rearrangement of the protein structure.