Structural Analysis of Macromolecular Complexes by High

大分子复合物的结构分析

• 批准号: 7291784
• 负责人: JACQUELINE MILNE
• 金额: --
• 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/7291784
• 关键词: Structural; Analysis; Macromolecular; Complexes

Cells contain thousands of multimolecular complexes which work together much like miniature factory machines. A detailed understanding of the structure and function of these molecular devices is a problem of great interest in cell biology. Our research efforts focus on electron cryo-microscopic analysis of single particles, a powerful method to determine the three-dimensional architectures of complex cellular assemblies. We have defined and interpreted the structure of an icosahedral pyruvate dehydrogenase multienzyme complex, a prototypical example of a multi-step catalytic machine which couples the activity of three component enzymes (E1, E2, and E3) in the oxidative decarboxylation of pyruvate to generate acetyl CoA at the junction of glycolysis and the tricarboxylic acid cycle. The three-dimensional model for a 11 MDa, icosahedral PDH complex, composed of 60 E2 enzymes and 60 E1 enzymes, was obtained by combining a 28 structure derived from electron cryo-microscopy with previously determined atomic coordinates of the individual components of the complex (Milne et al. EMBO J. 21, 5587, 2002). Analysis of the model provides a number of novel insights into the design and function of this molecular machine. A key feature is that the E1 molecules are located on the periphery in an orientation that allows each of the 60 mobile lipoyl domains tethered to the inner E2 enzyme to access multiple E1 active sites from inside the icosahedral complex. This unanticipated architecture provides a highly efficient mechanism for active site coupling and catalytic rate enhancement, which we propose is achieved by the motion of the lipoyl domain in the restricted annular region between the inner and outer cores of the complex. We have just completed refinement of a second PDH complex comprised of 60 E2 enzymes and 60 E3 enzymes to determine the structural basis of the final regeneration phase of the reaction catalyzed by this enzyme complex. Our three-dimensional reconstruction this complex indicates that, similar to the E1E2 complex described above, an annular gap of 75 exists between the inner core and the outer shell of E3 homodimers. Image analysis of partial occupancy complexes, formed by decorating the E2 core with 10, 20, 40 or 60 E1 tetramers or with 10, 20, 40 or 60 E3 homodimers, also indicates a 75-95 separation of the E2 and E1 or E2 and E3 densities. Thus, interactions occurring between the enzymes of the outer protein shell are not responsible for maintenance of the size of the complex. Rather, the E2 inner linkers that connect the E2 catalytic domains to the E2 peripheral-subunit binding domains must be fairly rigid radial spokes that provide a scaffold for an organization of E1 and E3 molecules. E3 localizes slightly closer to the core, suggesting that the swinging lipoyl domain can effectively access the active sites of all three enzymes, without leaving the annular space. This architecture provides further evidence of the highly efficient mechanism for active site coupling during both the production of acetyl CoA and the subsequent enzyme regeneration step that is required to initiate a new cycle of acetyl CoA production. We are also working actively to identify conditions that lead to outstanding microscopic images, to develop methods to select and accurately align the best molecular images for three-dimensional reconstructions, to reliably interpret these structures, and to develop automated procedures to facilitate the process of obtaining high quality three dimensional models of macromolecular complexes. To this end, we have developed algorithms for automated data collection, automated fitting of X-ray structures into density maps derived by cro electron microscopy, and optimized methods for the computational analysis of molecular images.

细胞包含成千上万的多分子复合物，它们就像微型工厂机器一样一起工作。对这些分子器件的结构和功能的详细了解是细胞生物学中非常感兴趣的问题。我们的研究工作集中在单粒子的电子冷冻显微镜分析，这是一种确定复杂细胞组装体三维结构的有力方法。我们已经定义和解释的二十面体丙酮酸脱氢酶多酶复合物的结构，一个典型的例子，一个多步骤的催化机器，耦合的活性的三个组成部分的酶（E1，E2和E3）在丙酮酸的氧化脱羧生成乙酰辅酶A的交界处的糖酵解和三羧酸循环。通过将来自电子冷冻显微镜的28结构与先前确定的复合物的各个组分的原子坐标相结合，获得了由60种E2酶和60种E1酶组成的11 MDa、二十面体PDH复合物的三维模型（Milne等人，EMBO J.21，5587，2002）。对该模型的分析为这种分子机器的设计和功能提供了一些新的见解。一个关键特征是E1分子位于外围，其取向允许60个移动的硫辛酰基结构域中的每一个连接到内部E2酶，以从二十面体复合物内部访问多个E1活性位点。这种意想不到的架构提供了一个高效的机制，活性位点耦合和催化速率的增强，我们建议是通过运动的硫辛酰域之间的复杂的内部和外部核心的限制性环形区域。我们刚刚完成了由60种E2酶和60种E3酶组成的第二PDH复合物的精制，以确定由该酶复合物催化的反应的最终再生阶段的结构基础。我们的三维重建这个复杂的表明，类似于上述的E1 E2复合物，一个环形间隙75之间存在的内核和外壳的E3同源二聚体。通过用10、20、40或60个E1四聚体或用10、20、40或60个E3同源二聚体装饰E2核心形成的部分占据复合物的图像分析也表明E2和E1或E2和E3密度的75-95分离。因此，蛋白质外壳的酶之间发生的相互作用并不负责维持复合物的大小。相反，连接E2催化结构域与E2外周亚基结合结构域的E2内部连接体必须是相当刚性的放射状辐条，为E1和E3分子的组织提供支架。E3定位在稍微靠近核心的位置，这表明摆动的硫辛酰结构域可以有效地进入所有三种酶的活性位点，而不离开环形空间。这种结构提供了在乙酰辅酶A的生产和随后的酶再生步骤期间活性位点偶联的高效机制的进一步证据，所述酶再生步骤是启动乙酰辅酶A生产的新循环所需的。我们还积极致力于确定导致出色的显微图像的条件，开发选择和准确对齐三维重建的最佳分子图像的方法，可靠地解释这些结构，并开发自动化程序以促进获得高质量大分子复合物三维模型的过程。为此，我们已经开发了自动数据收集算法，自动拟合的X-射线结构的密度图来自cro电子显微镜，和优化的方法计算分析的分子图像。