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以协调方式作为大分子机器的工作部分发挥作用的大型复合物。复合物还主要在包括皮肤和肌肉在内的许多组织中扮演生物材料的结构角色。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，重点是这些观察的功能含义。它由四个子标记组成。 （1）蛋白质质量控​​制是由能量依赖性蛋白酶（由肽酶和具有伴侣样性质的ATPase组成的大复合物）进行的重要功能。这种蛋白酶的原型是蛋白酶体，除其他活性外，还产生用于抗体生产的抗原肽。关于蛋白酶体的详细机械研究受到其ATPase的复杂亚基组成的阻碍。我们的研究集中于大肠杆菌的CLP蛋白酶，其ATPases只是同源环，作为一个有吸引力的模型系统。在较早的工作中，我们证明了肽酶CLPP由两个符合的七聚环组成，同源ATPase -CLPA或CLPX由一个六聚环组成，该环在CLPP的一个或两个面上轴上堆叠。这些观察结果是当前范式的基础，即ATPase识别底物，将其展开并将其馈入蛋白酶内部的消化室。在01财年，我们试图表征这些蛋白酶与蛋白质底物的相互作用，从而区分了反应产物的识别，易位，消化和分散的步骤。我们描述了CLPXP通过CLPAP和LAMBDA-O的底物处理。两种底物都与ATPase的远端表面结合，然后将其转移到肽酶的消化室中。对于CLPAP，我们证明了易位途径是轴向的。我们继续研究CLPXP与ATPase环在肽酶的两个面上的合作性。发现ATPG不仅支持CLPXP的组装及其结合Lambda-O的结合，而且还支持展开和易位，尽管比ATP慢了100倍。我们按时间分辨的EM研究了易位，发现一次易位是一次从一端发生的，这意味着负面的合作。在CLPYQ中，CLPY ATPase在其ATPase域中插入一个“中间”域。我们证明该域突出至ATPase域的环。此后在几项晶体学研究中得到了证实。我们得出的结论是，CLPXP以及其他蛋白酶的底物处理在整个复合物的水平上是协调的。易位是限速步骤，一次从复合物的一侧进行。 （2）末端分化角质形成细胞的蜂蜜细胞信封（CE）是覆盖其表面的脂蛋白层。由于其蛋白质的共价交联，因此CE具有弹性，主要是Loricrin，它具有赋予身体弹性和不可渗透性的能力。我们通过多种EM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建模的组成推断，我们开发了CE的模型作为交联的Loricrin分子层。因此，我们将CE视为复合生物材料。这种情况允许根据不同上皮的要求来调节其生物力学特性，通过调整矩阵与交联的比率。在01财年，我们完成了三项在洛里克林合成中受影响的转基因和基因敲除小鼠中CE生物发生的研究。其中一种转基因模拟了vohwinkel综合征和进行性对称性红细胞抑制剂的人类患者遇到的左licrin的框架变速，遗传性皮肤疾病的遗传性皮肤疾病的症状包括鱼质病和数字的自动警不观。令人惊讶的是，敲除洛里克林小鼠本质上是正常的。尽管没有洛里克林，但其表皮的薄部分显示出具有正常CES的角膜细胞。分离的CE在厚度和质量单位区域的正常状态，但它们在细胞质表面的结构发生了变化，并且蛋白质组成改变了。可以通过一种备用系统来解释洛里克林敲除小鼠的正常表型，该系统提供了另一种用于组装正常表现的CES的蛋白质。我们还从表达Loricrin突变形式的表皮研究了表达的表皮，该突变型Loricrin形式类似于其异常的C- terminus，c-terminus in VoHinkellys syndrome syndrome syndrome syndrome syndrome syndrome。我们检测到突变洛里克林在颗粒层细胞的细胞质和核中的异常沉积。在Vohwinkel转基因小鼠中，突变的洛里克林不进入CE，而是将其转运到粒细胞的核中，在这些核细胞的核中似乎会导致对核功能导致疾病症状的一般性干扰：即后者不能归因于由突变洛里卡林掺入CE的变化。 （3）酵母具有已被鉴定为王子的非孟德尔遗传元素。它们的细胞向细胞传播方式是通过具有异常构象（淀粉样蛋白）的蛋白质形式的细胞质传播，类似于哺乳动物的神经病（例如海绵状脑病）的哺乳动物prions。然而，酵母菌prion表型表现为缺乏代谢功能，而不是细胞病变作用。在01财年，我们试图在体外形成淀粉样细丝与受感染酵母细胞中存在的蛋白质之间建立相关性。我们还测量了在正常和过表达[URE3]和[URE-0]单元格中URE2P的拷贝数。先前的光学显微镜研究表明，在[URE3]（含有prion的）细胞中，URE2P聚集。我们发现，过表达URE2P的[URE3]细胞在其细胞质中包含独特的网络，并通过免疫标记证明了它们含有URE2P。在[URE3]细胞提取物中，ure2p位于聚集体中，仅通过在SDS和尿素中煮沸而被部分溶解。在这些骨料中，N末端prion域（长80个残基长，异常富含谷氨酰胺）是抗体无法接近的，而C端氮调节结构域则可访问。我们的数据支持prion域堆叠形成细丝骨干的概念，该主链被C末端域包围。正常表达细胞中URE2P的量很小，每个细胞分子约为3000个，解释了为什么我们没有检测到其中的细丝：即，细丝很少，以至于不太可能在随机的薄部分中进行采样。 （4）造成百日咳的病原体Bordetella budtussis通过其外表面上显示的粘附素分子粘附在呼吸道上。丝状血凝素（FHA）是其最突出的粘附蛋白，具有多种功能，是细胞疫苗的组成部分。我们旨在建立结构性基础，以理解其粘合剂和免疫学特性。在较早的工作中，我们设计了FHA的两个分子模型，该模型是50 nm的单体杆：（1）两个反平行β片的发夹； （2）单个β-螺旋由三个平行的beta表组成。在FY00中，我们基于序列分析和计算模型构建，开发了有利于Beta-helix的参数。在01财年，我们获得了通过截短变体的EM来支持该模型的实验证据，并且观察到了具有相同宽度的杆，大约是天然FHA的长度的一半。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碎片，预计它们可能比完整的FHA更容易结晶。