Structural Biology Of Virus Assembly

病毒组装的结构生物学

• 批准号: 6501315
• 负责人: ALASDAIR C. STEVEN
• 金额: --
• 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/6501315
• 关键词: capsid; coliphages; conformation; cryoelectron microscopy; hepatitis B virus group; herpes simplex virus 1; human immunodeficiency virus; microtubules; poliovirus; protamines; protein biosynthesis; protein engineering; protein folding; protein structure function; structural biology; virus DNA; virus RNA; virus assembly; virus protein; virus receptors

Many important cellular functions are performed by large complexes whose constituents function in a coordinated manner as working parts of macromolecular machines. Complexes also play primarily structural roles as biomaterials in many tissues, including skin and muscle. The goals of this project are to elucidate the structures, assembly properties, and interactions of complexes of both kinds, with emphasis on the functional connotations of these observations. It consists of four subprojects. (1)Protein quality control is a vital function carried out by energy-dependent proteases - large complexes consisting of a peptidase and an ATPase with chaperone-like properties. The archetypal such protease is the proteasome which, among other activities, generates antigenic peptides for antibody production. Detailed mechanistic studies on the proteasome are hampered by the complex subunit composition of its ATPase. Our studies focus on the Clp proteases of E. coli whose ATPases are simply homomeric rings, as an attractive model system. In earlier work, we showed that the peptidase ClpP consists of two apposed heptameric rings and the cognate ATPase - ClpA or ClpX - consists of a hexameric ring that stacks axially on one or both faces of ClpP . These observations underpin the current paradigm, whereby the ATPase recognizes substrates, unfolds them, and feeds them into a digestion chamber inside the protease. In FY01, we sought to characterize the interactions of these proteases with protein substrates, distinguishing the steps of recognition, translocation, digestion, and dispersal of reaction products. We described the processing of substrates, RepA by ClpAP, and lambda-O by ClpXP. Both substrates bind to the distal surface of the ATPase and are then translocated into the digestion chamber of the peptidase. For ClpAP, we demonstrated that the translocation pathway is axial. We went on to study cooperativity in ClpXP with ATPase rings on both faces of the peptidase. ATPgS was found to support not only the assembly of ClpXP and its binding of lambda-O but also, unfolding and translocation, albeit ~ 100-fold more slowly than with ATP. We studied translocation by time-resolved EM, finding that translocation takes place from one end at a time, implying negative cooperativity. In ClpYQ, the ClpY ATPase has an "intermediate" domain inserted into its ATPase domain. We demonstrated that this domain protrudes distal to the ring of ATPase domains. This orientation has since been confirmed in several crystallographic studies. We conclude that substrate processing by ClpXP and probably also other proteases is coordinated at the level of the complex as a whole. Translocation is the rate-limiting step, and proceeds from one side of the complex at a time. (2) The cornified cell envelopes (CEs) of terminally differentiated keratinocytes are lipoprotein layers covering their surfaces. CEs are resilient on account of covalent crosslinking of their proteins, principally loricrin, which confers physical resilience and impenetrability. We have investigated their biogenesis via a variety of EM approaches. Including compositional inferences based on mathematical modeling of their amino acid compositions, we developed a model of the CE as a layer of cross-linked loricrin molecules. Thus we envisage the CE as a composite biomaterial. This scenario allows for modulation of its biomechanical properties according to the requirements of different epithelia by adjusting the ratio of matrix to crosslinkers. In FY01, we finished off three studies of CE biogenesis in transgenic and knockout mice affected in loricrin synthesis. One of the transgenics emulates a frame-shift in loricrin encountered in human patients with Vohwinkel syndrome and Progressive Symmetric Erythrokeratoderma, genetic skin diseases whose symptoms include ichthyosis and autoamputation of digits. Surprisingly, knockout loricrin mice turned out to be essentially normal. Thin sections of their epidermis revealed corneocytes with normal-looking CEs, despite the absence of loricrin. Isolated CEs were normal in thickness and mass-per-unit-area, but they have an altered structure on their cytoplasmic surface, and altered protein compositions. The normal phenotype of loricrin knockout mice can be explained by a back-up system which supplies another protein(s) that is used to assemble normal-appearing CEs.We also studied epidermis from transgenic mice expressing a mutant form of loricrin that resembles, in its abnormal C-terminus, the protein produced in Vohwinkel?s syndrome. We detected unusual deposits of the mutant loricrin in the cytoplasm and nucleus of granular layer cells. In Vohwinkel transgenic mice, the mutant loricrin does not enter the CE but instead is transported into the nuclei of granulocytes where it appears to cause some generalized interference with nuclear function that results in the disease symptoms: i.e. the latter cannot be attributed to an alteration of the CE by incorporation by the mutant loricrin. (3) Yeast has non-Mendelian genetic elements that have been identified as prions. Their mode of cell-to-cell transmission is by cytoplasmic transmission of a polymeric form of the protein with an aberrant conformation (amyloid) resembling that of the mammalian prions implicated in neuropathies such as the spongiform encephalopathies. However, yeast prion phenotypes are manifested as lack of metabolic function rather than as cytopathic effects. In FY01, we sought to to establish a correlation between amyloid filament formation in vitro, and the protein present in infected yeast cells. We also measured the copy numbers of Ure2p in normal and overexpressing [URE3] and [ure-0] cells. Previous light microscopy studies showed that Ure2p is aggregated in [URE3] (prion-containing) cells. We found that [URE3] cells overexpressing Ure2p contain distinctive networks offilaments in their cytoplasm, and demonstrated by immunolabelling that they contain Ure2p. In [URE3] cell extracts, Ure2p is in aggregates that are only partially solubilized by boiling in SDS and urea. In these aggregates, the N-terminal prion domain (~ 80 residues long, and unusually rich in glutamine) is inaccessible to antibodies while the C-terminal nitrogen regulation domain is accessible. Our data support the concept that the prion domains stack to form the filament backbone, which is surrounded by C-terminal domains. The amount of Ure2p in normally-expressing cells is small, ~ 3000 molecules per cell, explaining why we have not detected filaments in them: i.e., the filaments are so few that they are unlikely to be sampled in random thin sections. (4) Bordetella pertussis, the pathogen responsible for whooping cough, adheres to the respiratory tract via adhesin molecules displayed on its outer surface. Filamentous hemagglutinin (FHA) is its most prominent adhesin, with multiple functionalities, and is a component of acellular vaccines. We aim to establish a structural basis for understanding its adhesive and immunological properties. In earlier work, we devised two molecular models for FHA, which is a 50-nm monomeric rod: (1) a hairpin of two antiparallel beta-sheets; (2) a single beta-helix composed of three parallel beta-sheets. In FY00, we developed arguments in favor of the beta-helix based on sequence analysis and computational model-building. In FY01, we obtained experimental evidence in support of this model by EM of a truncated variant, and observed rods of the same width and about half the length of native FHA. These observations concur with the prediction of the beta-helical model. We have also expressing fragments in E. coli, anticipating that they may crystallize more readily than intact FHA.

许多重要的细胞功能是由大型复合物执行的，这些复合物的组成部分作为大分子机器的工作部件以协调的方式发挥作用。复合物还在许多组织（包括皮肤和肌肉）中作为生物材料发挥主要的结构作用。该项目的目标是阐明两种复合物的结构、组装特性和相互作用，重点是这些观察结果的功能内涵。它由四个子项目组成。 (1) 蛋白质质量控​​制是由能量依赖性蛋白酶（由肽酶和具有分子伴侣特性的 ATP 酶组成的大型复合物）执行的一项重要功能。这种蛋白酶的原型是蛋白酶体，其除其他活性外，还产生用于抗体生产的抗原肽。蛋白酶体的详细机制研究因其 ATP 酶的复杂亚基组成而受到阻碍。我们的研究重点是大肠杆菌的 Clp 蛋白酶，其 ATP 酶只是同聚环，作为一个有吸引力的模型系统。在早期的工作中，我们表明肽酶 ClpP 由两个并置的七聚环组成，同源 ATP 酶 - ClpA 或 ClpX - 由轴向堆叠在 ClpP 的一个或两个面上的六聚环组成。这些观察结果支撑了当前的范例，即 ATP 酶识别底物，展开它们，并将它们送入蛋白酶内的消化室。在 2001 财年，我们试图表征这些蛋白酶与蛋白质底物的相互作用，区分反应产物的识别、易位、消化和分散步骤。我们描述了底物、ClpAP 的 RepA 和 ClpXP 的 lambda-O 的处理。两种底物均与 ATP 酶的远端表面结合，然后转移到肽酶的消化室中。对于 ClpAP，我们证明易位途径是轴向的。我们继续研究 ClpXP 与肽酶两面 ATPase 环的协同作用。人们发现 ATPgS 不仅支持 ClpXP 的组装及其与 lambda-O 的结合，而且还支持解折叠和易位，尽管比 ATP 慢约 100 倍。我们通过时间分辨电磁场研究了易位，发现易位一次从一端发生，这意味着负协同性。在 ClpYQ 中，ClpY ATPase 在其 ATPase 结构域中插入了一个“中间”结构域。我们证明该结构域突出于 ATP 酶结构域环的远端。此后，该取向已在多项晶体学研究中得到证实。我们得出的结论是，ClpXP 以及可能还有其他蛋白酶的底物处理在整个复合物水平上进行协调。易位是限速步骤，一次从复合体的一侧进行。 (2)终末分化的角质形成细胞的角质化细胞被膜(CE)是覆盖其表面的脂蛋白层。 CE 因其蛋白质（主要是兜甲素）的共价交联而具有弹性，这赋予了物理弹性和不可渗透性。我们通过各种 EM 方法研究了它们的生物发生。包括基于氨基酸组成数学模型的组成推断，我们开发了一个 CE 模型作为交联的兜甲素分子层。因此，我们将 CE 设想为一种复合生物材料。这种情况允许通过调整基质与交联剂的比例，根据不同上皮细胞的要求来调节其生物力学特性。在 2001 财年，我们完成了三项关于影响兜甲素合成的转基因和基因敲除小鼠中 CE 生物发生的研究。其中一种转基因技术模拟了患有沃温克尔综合征和进行性对称性红斑角化症的人类患者中所遇到的兜甲素的移码，这是一种遗传性皮肤病，其症状包括鱼鳞病和自动截肢。令人惊讶的是，敲除兜甲素的小鼠结果基本上是正常的。他们的表皮薄切片显示角质细胞具有正常的CE，尽管不存在兜甲蛋白。分离的 CE 的厚度和单位面积质量均正常，但其细胞质表面结构发生改变，蛋白质组成也发生改变。 Loricrin 敲除小鼠的正常表型可以通过备用系统来解释，该系统提供另一种蛋白质，用于组装外观正常的 CE。我们还研究了转基因小鼠的表皮，这些小鼠表达了 Loricrin 的突变形式，其异常的 C 末端类似于 Vohwinkel 综合征中产生的蛋白质。我们在颗粒层细胞的细胞质和细胞核中检测到突变体兜甲蛋白的异常沉积。在Vohwinkel转基因小鼠中，突变体兜甲蛋白不会进入CE，而是被转运到粒细胞的细胞核中，在那里它似乎对核功能造成一些普遍干扰，从而导致疾病症状：即后者不能归因于突变体兜甲蛋白掺入导致的CE改变。 (3) 酵母具有非孟德尔遗传元件，已被鉴定为朊病毒。它们的细胞间传播方式是通过具有异常构象（淀粉样蛋白）的聚合形式蛋白质的细胞质传播，类似于与海绵状脑病等神经病有关的哺乳动物朊病毒。然而，酵母朊病毒表型表现为缺乏代谢功能而不是细胞病变效应。在 2001 财年，我们试图建立体外淀粉样蛋白丝形成与受感染酵母细胞中存在的蛋白质之间的相关性。我们还测量了正常和过度表达的 [URE3] 和 [ure-0] 细胞中 Ure2p 的拷贝数。先前的光学显微镜研究表明，Ure2p 聚集在 [URE3]（含有朊病毒）细胞中。我们发现过表达 Ure2p 的 [URE3] 细胞的细胞质中含有独特的网络丝，并通过免疫标记证明它们含有 Ure2p。在 [URE3] 细胞提取物中，Ure2p 处于聚集体状态，在 SDS 和尿素中煮沸只能部分溶解。在这些聚集体中，抗体无法接近 N 端朊病毒结构域（约 80 个残基长，并且异常富含谷氨酰胺），而 C 端氮调节结构域则可接近。我们的数据支持这样的概念：朊病毒结构域堆叠形成细丝主链，该主链被 C 端结构域包围。正常表达的细胞中 Ure2p 的量很小，每个细胞约 3000 个分子，这解释了为什么我们没有在其中检测到细丝：即，细丝太少，以至于不可能在随机薄片中取样。 (4)百日咳博德特氏菌是引起百日咳的病原体，通过其外表面的粘附素分子粘附在呼吸道上。丝状血凝素 (FHA) 是其最重要的粘附素，具有多种功能，是非细胞疫苗的组成部分。我们的目标是建立理解其粘附和免疫特性的结构基础。在早期工作中，我们为 FHA（50 nm 单体杆）设计了两种分子模型：（1）两个反平行 β 折叠的发夹结构； (2)由三个平行的β-折叠组成的单个β-螺旋。在 2000 财年，我们基于序列分析和计算模型构建提出了支持 β 螺旋的论据。在 2001 财年，我们通过截断变体的 EM 获得了支持该模型的实验证据，并观察到与天然 FHA 具有相同宽度和大约一半长度的杆。这些观察结果与 β 螺旋模型的预测一致。我们还在大肠杆菌中表达了片段，预计它们可能比完整的 FHA 更容易结晶。