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体完成的，这些复合体的成分作为大分子机器的工作部件以协调的方式起作用。复合物作为生物材料在许多组织中也起着主要的结构作用，包括皮肤和肌肉。该项目的目标是阐明这两种复合物的结构、组装性质和相互作用，重点是这些观察的功能内涵。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atp酶组成的大型复合物，具有伴侣样性质）执行的重要功能。这种蛋白酶的原型是蛋白酶体，它在其他活动中产生抗原肽以生产抗体。对蛋白酶体的详细机制研究受到其atp酶复杂亚基组成的阻碍。我们的研究集中在大肠杆菌的Clp蛋白酶，其atp酶是简单的同质环，作为一个有吸引力的模型系统。在早期的工作中，我们发现肽酶ClpP由两个相对的七聚体环组成，同源的atp酶ClpA或ClpX由一个六聚体环组成，该六聚体环轴向堆叠在ClpP的一面或两面。这些观察结果支持了目前的范式，即atp酶识别底物，展开它们，并将它们送入蛋白酶内部的消化室。在2001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、消化和分散的步骤。我们描述了底物的加工，用ClpAP处理RepA，用ClpXP处理lambda-O。这两种底物结合到atp酶的远端表面，然后被转运到肽酶的消化室。对于ClpAP，我们证明了易位途径是轴向的。我们继续研究ClpXP与肽酶两侧atp酶环的协同性。研究发现，atpg不仅支持ClpXP的组装及其与lambda-O的结合，还支持ClpXP的展开和易位，尽管其速度比ATP慢约100倍。我们通过时间分辨的电子显微镜研究了易位，发现易位一次只发生在一端，这意味着负合作。在ClpYQ中，ClpY ATPase在其ATPase域中插入了一个“中间”域。我们证明了这个结构域在atp酶结构域环的远端突出。这种取向已在几项晶体学研究中得到证实。我们得出结论，ClpXP和其他蛋白酶的底物加工在整个复合物水平上是协调的。易位是限速步骤，每次从复合体的一侧进行。(2)终末分化角质形成细胞的角化细胞包膜（CEs）是覆盖其表面的脂蛋白层。由于其蛋白质（主要是氯丙氨酸）的共价交联，ce具有弹性，从而赋予其物理弹性和不可穿透性。我们通过多种电子显微镜方法研究了它们的生物发生。包括基于其氨基酸组成的数学模型的成分推断，我们开发了一个模型的CE作为一个交联氯丙胺分子层。因此，我们设想CE是一种复合生物材料。这种情况允许根据不同上皮的要求，通过调节基质与交联剂的比例来调节其生物力学特性。在FY01，我们完成了三项在氯丙胺合成受转基因和敲除小鼠中CE生物发生的研究。其中一种转基因模拟了人类Vohwinkel综合征和进行性对称红角化皮病（遗传性皮肤病，其症状包括鱼鳞病和手指自体截肢）患者氯丙林的框架转移。令人惊讶的是，敲除loricrin的小鼠基本上是正常的。表皮的薄切片显示角质细胞具有正常外观的ce，尽管缺乏氯丙林。分离的ce在厚度和单位面积质量上是正常的，但它们的细胞质表面结构发生了变化，蛋白质组成也发生了变化。loricrin敲除小鼠的正常表型可以通过一个备用系统来解释，该系统提供另一种蛋白质，用于组装正常外观的ce。我们还研究了表达loricrin突变形式的转基因小鼠的表皮，其异常的c端类似于Vohwinkel？年代综合症。我们在颗粒层细胞的细胞质和细胞核中发现了不寻常的突变氯克林沉积。在Vohwinkel转基因小鼠中，突变的loricrin没有进入CE，而是被转运到粒细胞的细胞核中，在那里它似乎对细胞核功能造成一些普遍的干扰，从而导致疾病症状：即后者不能归因于突变的loricrin掺入CE而改变CE。(3)酵母具有非孟德尔遗传成分，已被鉴定为朊病毒。它们的细胞间传播模式是通过具有异常构象（淀粉样蛋白）的蛋白质的聚合形式的细胞质传播，类似于与海绵状脑病等神经性疾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年，我们试图在体外建立淀粉样蛋白细丝形成与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表达[URE3]和[ure0]细胞中Ure2p的拷贝数。先前的光镜研究表明，Ure2p聚集在[URE3]（含朊病毒）细胞中。我们发现过表达Ure2p的[URE3]细胞在其细胞质中含有独特的网络纤维，并通过免疫标记证明它们含有Ure2p。在[URE3]细胞提取物中，Ure2p以聚集体形式存在，在SDS和尿素中煮沸只能部分溶解。在这些聚集体中，抗体无法进入n端朊病毒结构域（约80个残基长，且异常富含谷氨酰胺），而c端氮调控结构域则可以进入。我们的数据支持这一概念，即朊病毒结构域堆叠形成细丝骨架，被c端结构域包围。在正常表达的细胞中，Ure2p的数量很少，每个细胞约3000个分子，这解释了为什么我们没有在它们中检测到细丝：即，细丝如此之少，以至于它们不太可能在随机的薄切片中取样。(4)百日咳的病原体百日咳杆菌通过其外表面的粘附分子附着在呼吸道上。丝状血凝素（FHA）是其最突出的黏附素，具有多种功能，是脱细胞疫苗的一个组成部分。我们的目的是建立一个结构基础，了解其粘接和免疫特性。在早期的工作中，我们设计了两种FHA分子模型，它是一个50纳米的单体棒：(1)两个反平行β片的发夹；(2)由三个平行的β片组成的单个β螺旋。在2008年，我们基于序列分析和计算模型构建提出了支持β -螺旋的论点。在FY01中，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相同宽度和大约一半长度的杆。这些观测结果与β -螺旋模型的预测一致。我们也在大肠杆菌中表达片段，预计它们可能比完整的FHA更容易结晶。