Structural Analysis of Macromolecular Complexes by Electron Microscopy

使用冷冻电子显微镜，我们扩展了之前对E1E2复合体结构的确定，详细分析了装饰有60个拷贝的同源二聚体100 kDa二氢硫辛基脱氢酶(E3)的E2核心。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75A的环状间隙。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。这些研究明确地证明，负责将亚基保持在核心之上的是连接子，而不是外壳酶之间的相互作用。我们的结论是，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。因此，我们的工作揭示了对一种迷人的细胞机器的体系结构和内部工作的独特见解，这种机器是多酶复合体的典范，它的功能是使用移动摆动的手臂来耦合遥远的活性部位。更广泛地说，我们在单个多酶复合体中绘制单个酶的图谱的演示可能会被证明是一种宝贵的方法，可以在没有分子平均的情况下获得关于大型和结构不同的生物组合体的结构信息，这些生物组合体不适合用核磁共振或X射线结晶学技术进行分析。

Using cryo-electron microscopy, we have extended our previous determination of the structure of the E1E2 complex with a detailed analysis 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 A between the inner icosahedral assembly of acetyltransferase domains and the outer shell of E3 homodimers. 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 unambiguously that it is the linker, rather than interactions between the outer shell enzymes, that are responsible for holding the subunits above the core. We conclude 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. Our work thus reveals unique insights into the architecture and inner workings of a fascinating cellular machine that is a paradigm among multi-enzyme complexes that function using mobile swinging arms to couple distantly separated active sites. More broadly, our demonstration of mapping individual enzymes within single multi-enzyme complexes will likely prove to be an invaluable approach to obtain structural information, without molecular averaging, on large and structurally heterogeneous biological assemblies that are not amenable to analysis by NMR or X-ray crystallographic techniques.