SECONDARY STRUCTURE OF MYRISTOYLATED RECOVERIN DETERMINED BY 3-DIMENSIONAL HETERONUCLEAR NMR - IMPLICATIONS FOR THE CALCIUM MYRISTOYL SWITCH
SECONDARY STRUCTURE OF MYRISTOYLATED RECOVERIN DETERMINED BY 3-DIMENSIONAL HETERONUCLEAR NMR - IMPLICATIONS FOR THE CALCIUM MYRISTOYL SWITCH
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DOI：
10.1021/bi00201a023
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发表时间：
1994-09-06
期刊：
影响因子：
2.9
通讯作者：
IKURA, M
中科院分区：
文献类型：
--
作者：
AMES, JB;TANAKA, T;IKURA, M
Recovery蛋白是EF-hand超家族的新成员，在视觉中作为Ca 2+传感器。肉豆蔻酰基或相关的N-酰基共价连接在其N-末端，并通过一种新的钙-肉豆蔻酰基开关机制在钙依赖性膜靶向中起着重要作用。含有单一结合Ca 2+的未豆蔻酰化恢复素的结构最近已通过X射线晶体学解决[Flaherty，K. M.，Zozulya，S.，斯特雷尔湖，& McKay，D. B。（1993）Cell 75，709-716]。在这里，我们报告多维heterozygosity NMR研究Ca 2 +-免费的，豆蔻酰化的recoverin（201个残基，23 kDa）。完整的多肽骨架H-1，N-15和C-13共振分配和二级结构。我们发现了11个螺旋段和两对反平行的β-折叠，与晶体结构中的四个EF-手一致雅阁。本NMR研究还揭示了一些独特的结构特征的钙自由豆蔻酰化蛋白。EF-2的N-末端螺旋在肉豆蔻酰化的无钙蛋白中是灵活的，而在非肉豆蔻酰化的钙结合形式中具有明确的结构。这种差异表明，Ca 2+与EF-3的结合诱导EF-2采取有利于第二个Ca 2+与recoverin结合的构象。此外，N-末端螺旋（K5-E16）的豆蔻酰化的Ca 2+自由recoverin是显着长于在unmyristoylated的Ca 2+结合蛋白。我们建议，该螺旋是稳定的所附的肉豆蔻酰基，并可能发挥作用，螯合肉豆蔻酰基内的蛋白质中的钙离子的状态。
Recoverin, a new member of the EF-hand superfamily, serves as a Ca2+ sensor in vision. A myristoyl or related N-acyl group is covalently attached at its N-terminus and plays an essential role in Ca2+-dependent membrane targeting by a novel calcium-myristoyl switch mechanism. The structure of unmyristoylated recoverin containing a single bound Ca2+ has recently been solved by X-ray crystallography [Flaherty, K. M., Zozulya, S., Stryer, L., & McKay, D. B. (1993) Cell 75, 709-716]. We report here multidimensional heteronuclear NMR studies on Ca2+-free, myristoylated recoverin (201 residues, 23 kDa). Complete polypeptide backbone H-1, N-15, and C-13 resonance assignments and secondary structure are presented. We find 11 helical segments and two pairs of antiparallel beta-sheets, in accord with the four EF-hands seen in the crystal structure. The present NMR study also reveals some distinct structural features of the Ca2+-free myristoylated protein. The N-terminal helix of EF-2 is flexible in the myristoylated Ca2+-free protein, whereas it has a well-defined structure in the unmyristoylated Ca2+-bound form. This difference suggests that the binding of Ca2+ to EF-3 induces EF-2 to adopt a conformation favorable for the binding of a second Ca2+ to recoverin. Furthermore, the N-terminal helix (K5-E16) of myristoylated Ca2+-free recoverin is significantly longer than that seen in the unmyristoylated Ca2+-bound protein. We propose that this helix is stabilized by the attached myristoyl group and may play a role in sequestering the myristoyl group within the protein in the Ca2+-free state.