Structural Analysis of Macromolecular Complexes by Electron Microscopy

我们对E1E2丙酮酸脱氢酶复合体的结构的测定导致了关于这些酶复合体中活性部位偶联的分子机制的一些重要问题。例如，E1和E3酶在外壳中的排列方式是否会有所不同？当链接区从核心出现时，它们是结构化的还是捆绑在一起的？复合体的大小是由外壳的蛋白质-蛋白质相互作用决定的，还是由连接物的性质决定的？连接丙酮酸脱羧基和乙酰辅酶A合成的酶的摆动臂的轨迹是什么？这些问题和相关问题构成了我们对这个综合体正在进行的研究。利用冷冻电子显微镜，我们对装饰有60个拷贝的同源二聚体100 kDa二氢硫酰脱氢酶(E3)的E2核心进行了三维重建。E2E3复合体在乙酰基转移酶结构域的内部二十面体组装和E3同源二聚体的外壳之间有类似的约75的环状间隙。自动将E3坐标拟合到地图中表明，外壳地图的密度与E3的两个最佳拟合方向的位置之间具有很好的一致性。与E1E2复合体中的E1一样，E3同源二聚体的中心2倍轴大致沿壳的外围定向，使酶的活性部位可以从E2核心和外壳之间的环隙中获得。E1E2和E2E3在结构上的相似性表明，活性部位偶联的机制基本相似，这两个阶段涉及需要摆动的硫辛基结构域跨越环隙的两个关键阶段，即乙酰辅酶A的合成和硫辛基结构域二硫杂环戊环的再生。在一系列相关的研究中，我们解决了另一个重要的悬而未决的问题，即确定中央E2核心和外部酶外壳之间的差距是通过外壳中的蛋白质-蛋白质相互作用维持的，还是通过连接核心与外围亚单位结合域的连接区的硬度来维持的。利用圆二色谱、分析超速离心法和溶液核磁共振研究相结合的方法，我们得到了与连接区相对应的多肽具有延长的构象的证据，其持续长度为75-89，与观察到的缺口大小一致。冷冻电子断层扫描证实，即使在很低的E_1或E_3的情况下，核心和外壳之间的环状结构仍然保持不同的酶在外壳中所占的比例。这些研究表明，PDH酶的内部连接区是关键的结构元件，用于维持将外壳中丙酮酸的脱羧基与核心中乙酰辅酶A的合成偶联所需的环隙。

Our determination of the structure of the E1E2 pyruvate dehydrogenase complex led to a number of important questions about the molecular mechanisms of active site coupling in these enzyme complexes. For example, would there be differences in the way E1 and E3 enzymes would be arranged in the outer shell? Are the linker regions structured or bundled together as they emerge from the core? Is the size of complex determined by protein-protein interactions at the outer shell, or by properties of the linker? What is the trajectory of the swinging arm of the enzyme that couples pyruvate decarboxylation to acetyl CoA synthesis? These and related questions constitute our ongoing studies of this complex. Using cryo-electron microscopy, we carried out a three-dimensional reconstruction of the E2 core decorated with 60 copies of the homodimeric 100 kDa dihydrolipoyl dehydrogenase (E3). The E2E3 complex has a similar annular gap of about 75 between the inner icosahedral assembly of acetyltransferase domains and the outer shell of E3 homodimers. Automated fitting of the E3 co-ordinates into the map suggests excellent correspondence between the density of the outer shell map and the positions of the two best fitting orientations of E3. As in the case of E1 in the E1E2 complex, the central 2-fold axis of the E3 homodimer is roughly oriented along the periphery of the shell, making the active sites of the enzyme accessible from the annular gap between the E2 core and the outer shell. The similarities in architecture of the E1E2 and E2E3 complexes indicate fundamental similarities in the mechanism of active site coupling involved in the two key stages requiring motion of the swinging lipoyl domain across the annular gap, namely the synthesis of acetyl CoA and regeneration of the dithiolane ring of the lipoyl domain. In a related set of studies we have addressed another important unresolved question, which is to determine whether the gap between the central E2 core and the outer enzyme shell is maintained by virtue of protein-protein interactions in the outer shell or by stiffness of the linker region connecting the core to the peripheral subunit binding domain. Using a combination of circular dichroism, analytical ultracentrifugation and solution NMR studies we have obtained evidence that the peptide corresponding to the linker region has an extended conformation with a persistence length of 75-89 , consistent with the observed size of the gap. Cryo electron tomography of individual complexes with varying occupancies of enzymes in the outer shell confirmed unequivocally that the annular between the core and the outer shell was maintained even at very low E1 or E3 occupancies. These studies demonstrate that the inner linker region of PDH enzymes are critical structural elements, serving to maintain the annular gap required for coupling the decarboxylation of pyruvate in the outer shell to the synthesis of acetyl CoA in the core.