Structural Biology of Macromolecular Complexes

大分子复合物的结构生物学

• 批准号: 7964886
• 负责人: ALASDAIR C. STEVEN
• 金额: $ 64.69万
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
• 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/7964886
• 关键词: 26S proteasome; ATP Hydrolysis; ATP phosphohydrolase; Apical; Auxilins; Binding; Biochemical; Biological Models; Brain; Caliber; Cattle; Cell Cycle Regulation; Cells; Classification; Clathrin; Cleaved cell; Coated vesicle; Code; Complex; DNA; DNA Sequence Rearrangement; Digestion; Dimensions; Distal; Electron Microscopy; Escherichia coli; Eukaryota; Face; Fullerenes; Generation of Antibody Diversity; Genes; Genetic Recombination; Image; Immune; Immunoglobulins; Individual; Intracellular Membranes; J segment gene; Left; Length; Macromolecular Complexes; Measures; Membrane; Membrane Protein Traffic; Modeling; Molecular; Molecular Chaperones; Movement; N Domain; N-terminal; Negative Staining; Neurotransmitters; Peptide Hydrolases; Peptide Signal Sequences; Peptides; Play; Population; Positioning Attribute; Process; Protein Binding; Protein Subunits; Proteins; Publications; Publishing; Quality Control; Reaction; Regulation; Relative (related person); Reporting; Role; Shapes; Signal Transduction; Site; Staging; Structure; Synapses; System; T-Cell Receptor; Technology; Tertiary Protein Structure; Tomogram; Ubiquitin; V(D)J Recombination; VDJ Recombinases; Vesicle; Virus Receptors; combinatorial; density; electron tomography; endopeptidase Clp; falls; genetic regulatory protein; macromolecule; multicatalytic endopeptidase complex; particle; programs; protease E; protein degradation; reconstruction; seal; structural biology; theories; trafficking; urinary gonadotropin fragment

(1) Role of Energy-dependent Proteases in Protein Quality Control and Cell Regulation. All cells must be capable of degrading aberrant and foreign proteins that would otherwise pollute them. Programmed degradation of regulatory factors also contributes to controlling the cell cycle and to generating peptides for immune presentation. These activities are all carried out by energy-dependent proteases, which generically consist of two parts - a peptidase and a chaperone-like ATPase. (i) For several years, we have studied the Clp proteases of E. coli, considered as a model system. We showed that the peptidase ClpP consists of two apposed heptameric rings and the cognate ATPase - either ClpA or ClpX - is a single hexameric ring. ClpA/X stack axially on one or both faces of ClpP in active complexes. We went on to show that substrate proteins bind to distal sites on the ATPase and are then unfolded and translocated axially into the digestion chamber of ClpP. In FY09, we completed a cryo-EM study of the ClpAP protease. Specifically, we reconstructed the ClpA hexamer as an integral part of the ClpAP complex. Two segments lining the axial channel were found to show anomalously low density, indicative of local mobility. By comparing a model for ClpA-ATPgS derived from cryo-EM with one built for ClpA_ADP, we infer that ATP hydrolysis is accompanied by substantial structural changes in the D2 but not the D1 tier of AAA. The entire N-domain is rendered invisible by large-scale fluctuations. When deletions of 10 and 15-residues were introduced into the linker, N-domain mobility was reduced but not eliminated and changes were observed in enzymatic activities. We also performed cryo-EM reconstructions to investigate the structure of ClpP with and without end-mounted ClpA hexamers. In the absence of ClpA, the apical region of ClpP is sealed; however, it opens up on ClpA binding, creating an access channel. This region is occupied by the N-terminal loops (residues 1 17) of ClpP. We were able to model the closed-to-open transition - which facilitates the translocation of substrates into the interior of ClpP - in terms of movements of these loops. These observations indicate that access to the ClpP degradation chamber is controlled by hinged movements of its N-terminal loops, which the binding of ClpA suffices to induce. (ii) The proteosome is responsible for ubiquitin-tagged protein degradation in eukaryotes. The 26S proteasome is composed by the 20s proteolytic chamber and the 19S regulatory complex. The 19S complex contains a hexameric ATPase ring (like ClpA - see above) and numerous regulatory proteins, of which the largest are Rpn1 and Rpn2 (both > 100 kDa). It has been predicted that each has a large domain of PC repeats with alpha-solenoid folds. We confirmed this prediction in an EM analysis of both proteins. The results support the alpha-toroid model in terms of molecular dimensions and shape, and indicate that the repeats are organized not as symmetrical circular toroids, but in less regular structures. This project, first reported in FY08, was completed and published in FY09. (2) Intracellular Trafficking: Interaction of Clathrin with Proteins that Regulate its Assembly. Clathrin plays a key role in intracellular trafficking, via its coating of membranous pits and vesicles (CCVs). Assembly of clathrin is promoted by accessory proteins such as auxilin and AP180, and disassembly is effected by the Hsc70 ATPase. In the 1980s, we studied the molecular composition of coated vesicles and the plasticity of the assembly unit, the clathrin triskelion. We returned to this system in FY05, equipped with cryo-EM technology, and compared the structures of coated vesicles with and without binding of the uncoating ATPase, Hsc70. From these observations, we developed a model for uncoating. In FY07, we extended studies initiated during the previous year in which cryo-electron tomography is used to study the structures of individual CCVs isolated from bovine brain. Their polyhedral coats surround cargoes of various shapes and sizes, including vesicles containing neurotransmitters or receptors and viruses. The coated particles reconstructed in the tomograms fall into two sub-populations: 20% contain vesicles and are true CCVs; the remainder lack internal membranes and are termed "clathrin baskets" (CBs). CCVs range from 80 to 134 nm in diameter, with vesicles of 30 to 68 nm. CBs range from 66 to 120 nm. While many small polyhedral forms of coat are possible in theory, many are not observed, suggesting that they are energetically disfavored. The common feature of "forbidden" polyhedra is that they have vertices with high curvature. In tomograms we see density between the clathrin N-termini and the membrane. The vesicle is always off-center relative to the coat. To further investigate the polyhedral coats, we generated a comprehensive set of fullerene polyhedra composed of 12 pentagons and 20 -60 hexagons. These were regularized and used to calculate a variety of energetic measures, such as inter-spar angles, polygon regularity and planarity, curvature and sphericity. These results are being prepared for publication. 3) The large repertoire of immunoglobulins and T cell receptors is generated by combinatorial rearrangement of an extensive array of variable (V), diversity (D), and joining (J) gene segments that are combined to encode the variable domains of the protein chains. The recombination signal sequences (RSS) that flank these gene segments are recognized, paired in a synaptic complex, and cleaved by the proteins RAG1 (110 kDa) and RAG2 (58 kDa). Most biochemical studies on the RAG proteins have been done with the core fragments, which suffice for the basic recombination reaction, because the full-length proteins are difficult to express and purify. The RAG1/2 complex catalyzes a double-strand break at the coding/signal junctions, leaving blunt cut signal ends and DNA hairpins on the ends of the flanking coding sequences. DNA cleavage is a two-step reaction.There are two types of RSS, differing in the length of the spacer (12 or 23bp) between a conserved DNA heptamer and a nonamer). The heptamer sequence borders the coding V, D, or J segment. Efficient V(D)J recombination requires one RSS of each type. To obtain structural information on the early stages of V(D)J recombination, we used negative staining electron microscopy with image classification and averaging to visualize a RAG1/2 complex with DNA containing a pair of cleaved RSS sequences. This analysis revealed an anchor-shaped particle with approximate two-fold symmetry. Consistent with a parallel arrangement of DNA and protein subunits, the N-termini of RAG1 and RAG2 are positioned at opposing ends of the complex, and the DNA chains beyond the RSS nonamer emerge from the same face of the complex, near to RAG1 N-termini. These images of SEC, the post-cleavage V(D)J recombinase, provide a framework for modeling RAG domains and their interactions with DNA.

(1) 能量依赖性蛋白酶在蛋白质质量控​​制和细胞调节中的作用。所有细胞都必须能够降解异常和外来蛋白质，否则会污染它们。调节因子的程序性降解也有助于控制细胞周期和产生用于免疫呈递的肽。这些活动都是由能量依赖性蛋白酶进行的，这些蛋白酶通常由两部分组成：肽酶和分子伴侣样 ATP 酶。 (i) 多年来，我们研究了大肠杆菌的 Clp 蛋白酶，将其视为模型系统。我们证明肽酶 ClpP 由两个并置的七聚环组成，而同源 ATP 酶（ClpA 或 ClpX）是单个六聚环。 ClpA/X 在活性复合物中轴向堆叠在 ClpP 的一个或两个面上。我们继续证明底物蛋白与 ATP 酶的远端位点结合，然后展开并轴向易位到 ClpP 的消化室中。 2009 财年，我们完成了 ClpAP 蛋白酶的冷冻电镜研究。具体来说，我们将 ClpA 六聚体重建为 ClpAP 复合物的组成部分。发现轴向通道内衬的两个部分显示出异常低的密度，表明局部流动性。通过将源自冷冻电镜的 ClpA-ATPgS 模型与为 ClpA_ADP 构建的模型进行比较，我们推断 ATP 水解伴随着 AAA 的 D2 层而非 D1 层的实质性结构变化。整个N域因大规模的波动而变得不可见。当连接子中引入 10 个和 15 个残基的缺失时，N 结构域的流动性降低但并未消除，并且观察到酶活性发生变化。 我们还进行了冷冻电镜重建，以研究带有和不带有末端安装 ClpA 六聚体的 ClpP 的结构。在没有 ClpA 的情况下，ClpP 的顶端区域被密封；然而，它在 ClpA 绑定上打开，创建一个访问通道。该区域被 ClpP 的 N 末端环（残基 1 17）占据。我们能够根据这些环的运动来模拟封闭到开放的转变——这有助于底物易位到 ClpP 的内部。这些观察结果表明，进入 ClpP 降解室是通过其 N 末端环的铰链运动控制的，ClpA 的结合足以诱导这种运动。 (ii) 蛋白酶体负责真核生物中泛素标记的蛋白质降解。 26S蛋白酶体由20S蛋白水解室和19S调节复合物组成。 19S 复合体包含一个六聚体 ATP 酶环（如 ClpA - 见上文）和许多调节蛋白，其中最大的是 Rpn1 和 Rpn2（均 > 100 kDa）。 据预测，每个都有一个大的 PC 重复区域，带有 α-螺线管折叠。我们通过对两种蛋白质的电镜分析证实了这一预测。结果在分子尺寸和形状方面支持α-环形模型，并表明重复序列不是以对称的圆形环形组织，而是以不太规则的结构组织。该项目于 2008 财年首次报告，于 2009 财年完成并发布。 (2) 细胞内运输：网格蛋白与调节其组装的蛋白质的相互作用。 网格蛋白通过其膜坑和膜泡 (CCV) 涂层在细胞内运输中发挥关键作用。网格蛋白的组装由辅助蛋白（例如辅助蛋白和 AP180）促进，而分解则由 Hsc70 ATP 酶实现。在 20 世纪 80 年代，我们研究了包被囊泡的分子组成和组装单元网格蛋白三链的可塑性。我们在 2005 财年重新使用了该系统，配备了冷冻电镜技术，并比较了有或没有结合未包被 ATP 酶 Hsc70 的包被囊泡的结构。根据这些观察，我们开发了一个脱涂层模型。 2007 财年，我们扩展了前一年启动的研究，其中使用冷冻电子断层扫描来研究从牛脑中分离出的个体 CCV 的结构。它们的多面体外壳包围着各种形状和大小的货物，包括含有神经递质或受体和病毒的囊泡。断层扫描中重建的涂层颗粒分为两个亚群：20% 含有囊泡，是真正的 CCV；20% 含有囊泡，是真正的 CCV；20% 含有囊泡，是真正的 CCV。其余的缺乏内膜，被称为“网格蛋白篮”(CB)。 CCV 直径范围为 80 至 134 nm，囊泡直径为 30 至 68 nm。 CB 范围为 66 至 120 nm。虽然许多小的多面体形式的皮毛在理论上是可能的，但许多都没有被观察到，这表明它们在能量上是不受欢迎的。 “禁止”多面体的共同特征是它们具有高曲率的顶点。在断层图中，我们可以看到网格蛋白 N 末端和膜之间的密度。囊泡相对于皮毛总是偏离中心。为了进一步研究多面体涂层，我们生成了一套全面的富勒烯多面体，由 12 个五边形和 20 -60 个六边形组成。这些被正则化并用于计算各种能量测量，例如翼梁间角度、多边形规则性和平面性、曲率和球形度。这些结果正在准备发表。 3) 免疫球蛋白和 T 细胞受体的大库是通过大量可变 (V)、多样性 (D) 和连接 (J) 基因片段的组合重排产生的，这些基因片段组合起来编码蛋白质链的可变域。这些基因片段侧翼的重组信号序列 (RSS) 被识别，在突触复合体中配对，并被 RAG1 (110 kDa) 和 RAG2 (58 kDa) 蛋白切割。大多数关于RAG蛋白的生化研究都是用核心片段进行的，这足以进行基本的重组反应，因为全长蛋白难以表达和纯化。 RAG1/2 复合物在编码/信号连接处催化双链断裂，在侧翼编码序列的末端留下平切信号末端和 DNA 发夹。 DNA 切割是一个两步反应。RSS 有两种类型，其不同之处在于保守的 DNA 七聚体和九聚体之间的间隔区长度（12 或 23bp）。七聚体序列与编码 V、D 或 J 片段接壤。有效的 V(D)J 重组需要每种类型一个 RSS。为了获得 V(D)J 重组早期阶段的结构信息，我们使用负染色电子显微镜进行图像分类和平均，以可视化 RAG1/2 复合物与含有一对切割 RSS 序列的 DNA。该分析揭示了具有近似二次对称性的锚形颗粒。与 DNA 和蛋白质亚基的平行排列一致，RAG1 和 RAG2 的 N 末端位于复合物的相对两端，并且 RSS 九聚体之外的 DNA 链从复合物的同一面出现，靠近 RAG1 N-终点站。 SEC（切割后 V(D)J 重组酶）的这些图像为 RAG 结构域及其与 DNA 相互作用的建模提供了框架。