Nalefski E A, Slazas M M, Falke J J
Department of Chemistry and Biochemistry, University of Colorado at Boulder 80309-0215, USA.
Biochemistry. 1997 Oct 7;36(40):12011-8. doi: 10.1021/bi9717340.
The C2 domain is a Ca2+-dependent, membrane-targeting motif originally discovered in protein kinase C and recently identified in numerous eukaryotic signal-transducing proteins, including cytosolic phospholipase A2 (cPLA2) of the vertebrate inflammation pathway. Intracellular Ca2+ signals recruit the C2 domain of cPLA2 to cellular membranes where the enzymatic domain hydrolyzes specific lipids to release arachidonic acid, thereby initiating the inflammatory response. Equilibrium binding and stopped-flow kinetic experiments reveal that the C2 domain of human cPLA2 binds two Ca2+ ions with positive cooperativity, yielding a conformational change and membrane docking. When Ca2+ is removed, the two Ca2+ ions dissociate rapidly and virtually simultaneously from the isolated domain in solution. In contrast, the Ca2+-binding sites become occluded in the membrane-bound complex such that Ca2+ binding and dissociation are slowed. Dissociation of the two Ca2+ ions from the membrane-bound domain is an ordered sequential process, and release of the domain from the membrane is simultaneous with dissociation of the second ion. Thus, the Ca2+-signaling cycle of the C2 domain passes through an active, membrane-bound state possessing two occluded Ca2+ ions, one of which is essential for maintenance of the protein-membrane complex.
C2结构域是一种依赖钙离子的膜靶向基序，最初在蛋白激酶C中发现，最近在许多真核信号转导蛋白中也被鉴定出来，包括脊椎动物炎症途径中的胞质磷脂酶A2（cPLA2）。细胞内钙离子信号将cPLA2的C2结构域招募到细胞膜上，在那里酶结构域水解特定脂质以释放花生四烯酸，从而引发炎症反应。平衡结合和停流动力学实验表明，人cPLA2的C2结构域以正协同性结合两个钙离子，导致构象变化和膜对接。当去除钙离子时，两个钙离子在溶液中从分离的结构域迅速且几乎同时解离。相反，在膜结合复合物中钙离子结合位点被封闭，使得钙离子的结合和解离变慢。两个钙离子从膜结合结构域的解离是一个有序的顺序过程，并且结构域从膜上的释放与第二个离子的解离同时发生。因此，C2结构域的钙离子信号循环经过一个具有两个封闭钙离子的活性膜结合状态，其中一个钙离子对于维持蛋白质 - 膜复合物至关重要。
