Structural Analysis of Macromolecular Complexes by Electron Microscopy

我们对E1E2丙酮酸脱氢酶复合物结构的测定引出了一些关于这些酶复合物中活性位点偶联的分子机制的重要问题。例如，E1和E3酶在外壳中的排列方式是否会有所不同？链接区域是结构化的还是捆绑在一起的，因为它们从核心出现？复合物的大小是由蛋白与蛋白在外壳的相互作用决定的，还是由连接体的性质决定的？将丙酮酸脱羧与乙酰辅酶a合成结合的酶的摆动臂的轨迹是什么？这些和相关的问题构成了我们对这个复合体的持续研究。利用低温电子显微镜，我们用60个同源二聚体100 kDa二氢脂酰脱氢酶（E3）修饰的E2核心进行了三维重建。E2E3配合物在乙酰转移酶结构域的内二十面体组装和E3同型二聚体的外壳之间具有类似的约75的环状间隙。将E3坐标自动拟合到地图中表明，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良好的对应关系。就像E1E2复合体中的E1一样，E3同型二聚体的中心2折轴大致沿着壳的外围定向，使得酶的活性位点可以从E2核心和外壳之间的环形间隙进入。E1E2和E2E3配合物在结构上的相似性表明，在需要摆动的脂酰结构域穿过环隙运动的两个关键阶段，即乙酰辅酶a的合成和脂酰结构域二硫烷环的再生中，活性位点耦合的机制基本相似。在一组相关的研究中，我们解决了另一个重要的未解决的问题，即确定中央E2核心和外酶壳之间的间隙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亚基结合域的连接子区域的刚度维持的。结合圆二色性、分析性超离心和溶液核磁共振研究，我们已经获得证据，证明连接区域对应的肽具有延长的构象，持续长度为75-89，与观察到的间隙大小一致。低温电子断层扫描的单个复合物具有不同的占用酶在外壳明确地证实，即使在非常低的E1或E3占用核和外壳之间的环形保持。这些研究表明，PDH酶的内连接区域是关键的结构元件，用于维持外壳中丙酮酸脱羧与核心中乙酰辅酶a合成耦合所需的环状间隙。

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.