Structural Analysis of Macromolecular Complexes by Electron Microscopy

电子显微镜对大分子复合物的结构分析

• 批准号: 7733026
• 负责人: JACQUELINE MILNE
• 金额: $ 24.89万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7733026
• 关键词: Acetyl Coenzyme A; Acetyltransferase; Active Sites; Address; Architecture; Binding; Biological; Biological Models; Cells; Circular Dichroism; Complex; Couples; Coupling; Cryoelectron Microscopy; Decarboxylation; Electron Microscopy; Elements; Energy Metabolism; Enzymes; Individual; Length; Macromolecular Complexes; Maps; Molecular; Molecular Conformation; Molecular Machines; Motion; Multienzyme Complexes; Natural regeneration; Numbers; Peptides; Peripheral; Property; Pyruvate; Pyruvate Dehydrogenase Complex; Pyruvates; Relative (related person); Roentgen Rays; Role playing therapy; Solutions; Staging; Structure; Techniques; Upper arm; Work; analytical ultracentrifugation; dihydrolipoamide dehydrogenase; electron tomography; fascinate; insight; interest; protein protein interaction; pyruvate dehydrogenase; size

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.

使用低温电子显微镜，我们已经扩展了我们以前的确定， 详细分析了E1和E2复合物的结构，并用60个拷贝修饰了E2核心 同源二聚体100 kDa二氢硫辛酰脱氢酶（E3）。E2 E3复合体具有类似的 在乙酰转移酶的内部二十面体组装之间的约75 A的环形间隙 结构域和外壳的E3同源二聚体。与E1 E2复合体中的E1一样， E3同二聚体的中心2重轴大致沿着壳的外围取向， 使酶的活性位点可从E2核心之间的环形间隙进入， 外壳。E1 E2和E2 E3复合物结构的相似性表明， 两个关键的活性位点偶联机制的基本相似性 需要摆动硫辛酰基结构域穿过环形间隙运动的阶段，即 乙酰辅酶A的合成和硫辛酰结构域的二硫戊环的再生。中 在一系列相关研究中，我们讨论了另一个重要的悬而未决的问题，即 确定中心E2核和外部酶壳之间的差距是否保持 通过外壳中的蛋白质-蛋白质相互作用或通过接头的刚性 连接核心与外周亚基结合结构域的区域。结合使用 圆二色性、分析超离心和溶液核磁共振研究 对应于接头区的肽具有延伸构象的证据， 持续长度为75-89，与观测到的差距大小一致。低温电子 在外壳中具有不同酶占据率的单个复合物的断层摄影术 明确地证实，核心和外壳之间的环形保持 即使在非常低的E1或E3占用率下。这些研究清楚地表明， 接头，而不是外壳酶之间的相互作用，这是负责 将子单元保持在核心上方。我们的结论是PDH酶的内部连接区 是关键的结构元件，用于保持耦合所需的环形间隙 外壳中丙酮酸的脱羧作用转化为核心中乙酰辅酶A的合成。 因此，我们的工作揭示了对建筑和内部运作的独特见解， 这是一个迷人的细胞机器，它是多酶复合物的一个范例， 使用移动的摆动臂来耦合远距离分离的活性位点。更广泛地说，我们 在单个多酶复合物中绘制单个酶的演示将可能 被证明是一种获得结构信息的宝贵方法， 平均化，在不适合的大的和结构异质的生物组件上， 通过NMR或X射线晶体学技术进行分析。