Three-dimensional structure and subunit topology of the V(1) ATPase from Manduca sexta midgut.
Three-dimensional structure and subunit topology of the V(1) ATPase from Manduca sexta midgut.
复制标题
烟草天蛾中肠 V(1) ATP 酶的三维结构和亚基拓扑。
作者：
G. Grüber;M. Radermacher;T. Ruiz;J. Godovac;B. Cañas;D. Kleine;M. Huss;W. Harvey;H. Wieczorek
从阴性染色标本的电子显微镜下，以3.2 nm分辨率确定了Manduca sexta中肠V(1) atp酶的三维结构。V(1)配合物呈桶状结构，高11nm，直径13.5 nm。在俯视图中，它是六边形的，而在侧面视图中，六个大亚基A和B在其大部分长度（9 nm）上是交叉的。通过蛋白酶消化、对混乱剂的抗性、MALDI-TOF质谱和CuCl(2)诱导的二硫化物形成，研究了V(1)复合物的拓扑结构和单个亚基的重要性。用胰蛋白酶或朝向碘化物处理V(1)导致D亚基从酶中快速裂解或释放，表明该亚基暴露在复合物中。胰蛋白酶切割V(1)使atp酶活性降低，其时间过程与切割B、C、G和f亚基的时间过程一致。在CaADP存在的情况下，将CuCl(2)加入V(1)，生成交联产物a - e - f和B- h。在CaATP预孵育后加入CuCl(2)的实验中，形成交联产物E-F和E-G。这些亚基E与邻近亚基交联的变化支持了E亚基的核苷酸依赖性构象变化的假设。
The three-dimensional structure of the Manduca sexta midgut V(1) ATPase has been determined at 3.2 nm resolution from electron micrographs of negatively stained specimens. The V(1) complex has a barrel-like structure 11 nm in height and 13.5 nm in diameter. It is hexagonal in the top view, whereas in the side view, the six large subunits A and B are interdigitated for most of their length (9 nm). The topology and importance of the individual subunits of the V(1) complex have been explored by protease digestion, resistance to chaotropic agents, MALDI-TOF mass spectrometry, and CuCl(2)-induced disulfide formation. Treatment of V(1) with trypsin or chaotropic iodide resulted in a rapid cleavage or release of subunit D from the enzyme, indicating that this subunit is exposed in the complex. Trypsin cleavage of V(1) decreased the ATPase activity with a time course that was in line with the cleavage of subunits B, C, G, and F. When CuCl(2) was added to V(1) in the presence of CaADP, the cross-linked products A-E-F and B-H were generated. In experiments where CuCl(2) was added after preincubation of CaATP, the cross-linked products E-F and E-G were formed. These changes in cross-linking of subunit E to near-neighbor subunits support the hypothesis that these are nucleotide-dependent conformational changes of the E subunit.