Structural Biology Of Macromolecular Complexes

大分子复合物的结构生物学

• 批准号: 6823052
• 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/6823052
• 关键词: adenosinetriphosphatase; adhesin; amyloid proteins; bacteria infection mechanism; bacterial proteins; bioenergetics; cell membrane; cell wall; chemical models; conformation; crosslink; cryoelectron microscopy; endopeptidases; fibrous protein; gene targeting; genetically modified animals; keratinization; keratinocyte; laboratory mouse; mathematical model; molecular assembly /self assembly; prions; protein folding; protein protein interaction; protein structure function; structural biology; tissue /cell culture

Many important cellular functions are performed by large complexes which operate like macromolecular machines. Complexes also play primarily structural roles as biomaterials in many tissues, including skin and muscle. Twe aim to elucidate the structures, assembly properties, and interactions of complexes of both kinds, with close attention to the functional connotations. We pursued three main projects over the past year. (1) Energy-dependent Proteases. Protein quality control is essential for eliminating aberrant proteins that would otherwise pollute the cell, for example by amyloid formation. This activity is largely carried out by energy-dependent proteases which generically consist of two subcomplexes - a peptidase and a chaperone-like ATPase. Our studies focus on the Clp proteases of E. coli which offer a tractable model system. Earlier we showed that peptidase ClpP consists of two apposed heptameric rings and the cognate ATPase - either ClpA or ClpX - is a single hexameric ring. The ATPases stack axially on one or both faces of ClpP to form active complexes. We went on to study the interaction of ClpAP and ClpXP with model substrates. In both cases, substrate proteins initially bind to distal sites on the ATPase and are then translocated along an axial pathway into the digestion chamber inside ClpP. Results. (a) In our cryo-EM reconstruction of the ClpA hexamer at 1.2 nm resolution, two hexameric rings corresponding to the ATPase domains (D1, D2) are seen with a cavity between. However, there is little sign of the N-terminal domains although they are quite large, 17 kDa each. Evidently, the N-domains, are highly mobile. Nevertheless, we managed to visualized them by variance mapping of sideviews and difference mapping with averaged sideviews of an N-domain-deleted mutant. We also measured the scale of their mobility by molecular modeling, showing it to involve movements of up to 3.5 nm for each N-domain. (b) ClpP may partner either ATPase ? ClpA or ClpX ? and each ClpP oligomer may bind two ATPases. Can it bind one copy of each ATPase? and if so, are the hybrid complexes functional in substrate binding and internalization? We addressed these questions by statistical analysis of micrographs recorded after initiating translocation of ClpA-spcific and ClpX-specific substrates. The answers to both questions are in the affirmative. (2) Amyloid filament formation by the yeast prion protein, Ure2p. Amyloid is fibrous aggregates of protein(s) in protease-resistant, beta-sheet-rich, non-native conformations. Amyloid accumulates in a number of disease situations including rheumatoid arthritis. Prions (infectious proteins) are transmissible amyloids that have been implicated in certain neuropathies, including the spongiform encephalopathies. To investigate the structure of amyloids and the mechanisms that underlie their formation, we study yeast prions. Unlike mammalian prions, their phenotypes are expressed as lack of metabolic functions rather than cytopathic effects. This greatly simplifies and accelerates their study. We focus on Ure2p, a protein normally involved in nitrogen metabolism. Its prion phenotype presents as an inability to grow on poor nitrogen sources. In earlier work, we demonstrated filament formation by Ure2p in vitro and the presence of filaments in prion-infected cells. Results: We focused on substantiating our ?amyloid backbone model, formulated in 1999. Ure2p has an N-terminal prion domain that is necessary for filament formation and a C-terminal domain that performs in nitrogen regulation. According to the model, in filaments, prion domains form an amyloid backbone that is surrounded by the C-terminal domains, whereas in soluble Ure2p, the prion domain is unfolded. This model successfully predicted that fusions of the prion domain with exogenous proteins should also form filaments. We characterized Ure2p filaments and fusion protein filaments by biochemical and EM experiments. Protease digestion of 25-nm diameter Ure2p filaments trimmed them to 4-nm filaments which mass spectrometry showed to be composed of prion domain fragments. Fusion protein filaments with diameters of 14 to 25 nm were similarly reduced to 4-nm filaments by proteolysis. In each case, the prion domain transforms from the most to the least protease-sensitive part upon filament formation, implying a large conformational change. Filaments imaged by cryo-EM or after vanadate staining by STEM revealed a central 4-nm core with globular appendages. STEM mass-per-unit-length measurements of unstained filaments yielded 1 monomer per 0.45nm in each case. These observations all support the amyloid backbone model. (3) Structure and Assembly of Cornified Cell Envelopes (CEs). The CE is a covalently cross-linked layer of protein that lines the cytoplasmic surface of terminally differentiated keratinocytes. CEs are thought to contribute physical resilience and impenetrability to these tissues. We study their biogenesis, and have applied a variety of EM approaches, both to isolated CEs and in situ. Including compositional inferences based on mathematical modeling of amino acid compositions, we developed a model of CEs as monolayers of molecules of the protein, loricrin, cross-linked both directly and via minor CE proteins. We envisage the CE as a ?composite? biomaterial with a matrix substance (loricrin) and cross-linkers (the minor proteins). Results. By immunogold-EM of cryosections, we found that the cornified envelopes in newborn mouse skin labeled positive for LEPs, as did granules in the stratum granulosum. We have recently extended these observations to loricrin knockout mice. The main difference compared to wildtype is that LEP appears to label from both the outside and the inside of the surrogate (loricrin-less) CEs found in LKO animals.

许多重要的细胞功能是由像大分子机器一样运行的大型复合物进行的。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。 TWE的目的是阐明两种复合物的结构，组装特性和相互作用，并密切关注功能含义。在过去的一年中，我们从事了三个主要项目。 （1）能量依赖性蛋白酶。蛋白质质量控​​制对于消除否则会污染细胞的异常蛋白质，例如通过淀粉样蛋白形成至关重要。该活性在很大程度上是由能量依赖性蛋白酶进行的，这些蛋白酶通常由两个亚复合物组成 - 肽酶和一个类似伴侣的ATPase。我们的研究集中于大肠杆菌的CLP蛋白酶，该蛋白酶提供了可拖动的模型系统。早些时候，我们表明肽酶CLPP由两个含有的七聚环组成，而cognate atpase -clpa或clpx是一个单个六聚体环。 ATPases轴向堆叠在CLPP的一个或两个面上以形成活跃的复合物。我们继续研究CLPAP和CLPXP与模型底物的相互作用。在这两种情况下，底物蛋白最初与ATPase上的远端位点结合，然后沿轴向途径易位到CLPP内的消化室。结果。 （a）在我们以1.2 nm分辨率的CLPA六聚体重建中，可以看到两个与ATPase域（D1，D2）相对应的六聚体环（D1，D2）。但是，尽管它们很大，但几乎没有迹象，每个末端域都有17 kDa。显然，n域高度流动性。然而，我们设法通过侧视图的差异图和差映射的差异来可视化它们，并使用n域删除的突变体的平均侧视图进行了映射。我们还通过分子建模测量了它们的迁移率的规模，表明它涉及每个N域的运动最高3.5 nm。 （b）CLPP可以搭配ATPase？ CLPA还是CLPX？并且每个CLPP低聚物可能结合两个ATPases。它可以绑定每个ATPase的一个副本吗？如果是这样，混合复合物在底物结合和内在化中是否功能性？我们通过对CLPA-SPCCIFIFIC和CLPX特异性底物的易位后记录的显微照片进行统计分析来解决这些问题。两个问题的答案都是肯定的。 （2）酵母菌prion蛋白Ure2p形成淀粉样细丝。淀粉样蛋白是抗蛋白质蛋白质蛋白质的纤维聚集体，抗蛋白酶富含β-折叠的非母构象。淀粉样蛋白在包括类风湿关节炎在内的多种疾病情况下积累。 prions（感染性蛋白质）是可传播的淀粉样蛋白，与某些神经病有关，包括海绵状脑病。为了研究淀粉样蛋白的结构以及其形成的基础的机制，我们研究了酵母菌王。与哺乳动物的王室不同，它们的表型被表示为缺乏代谢功能，而不是细胞病变作用。这极大地简化了他们的研究。我们专注于URE2P，这是一种通常参与氮代谢的蛋白质。它的prion表型表现为无法在贫困较差的氮源上生长。在较早的工作中，我们在体外表现出了URE2P的细丝形成，并且在受prion感染的细胞中存在细丝。结果：我们的重点是证实1999年制定的淀粉样蛋白主链模型。URE2P具有一个N末端prion域，这是细丝形成所必需的，并且在氮调节中执行的C末端结构域是必要的。根据该模型，在细丝中，prion域形成一个淀粉样蛋白的骨干，被C末端结构域围绕，而在可溶性URE2P中，prion域则展开。该模型成功地预测了与外源蛋白质的prion结构域的融合也应形成细丝。我们通过生化和EM实验表征了URE2P丝和融合蛋白丝。直径25 nm Ure2p丝的蛋白酶消化将它们修剪成4 nm丝，质谱显示由prion域片段组成。通过蛋白水解将直径为14至25 nm的融合蛋白丝类似地降低至4 nm丝。在每种情况下，prion域在细丝形成后从最小蛋白酶敏感的部分转变为最少的部分，这意味着大构象变化。由冷冻EM或STEM质染色后成像的细丝显示出具有球状附属物的中央4 nm核心。在每种情况下，未染色细丝的茎质量单位长度测量值每0.45nm产生1个单体。这些观察结果都支持淀粉样蛋白骨干模型。 （3）晶状体细胞信封（CES）的结构和组装。 CE是一个共价交联的蛋白质层，该蛋白质在末端分化的角质形成细胞的细胞质表面上。 CE被认为对这些组织有助于物理弹性和不可穿透性。我们研究了它们的生物发生，并应用了各种EM方法，包括孤立的CE和原位。包括基于氨基酸组成的数学模型的组成推断，我们开发了CES的模型，作为蛋白质分子Loricrin的分子单层，直接并通过次要CE蛋白交联。我们将CE视为“综合”？具有基质物质（Loricrin）和交联（小蛋白）的生物材料。结果。通过冷冻切除术的免疫原状EM，我们发现新生小鼠皮肤中的糖化信封标记为LEP呈阳性，而颗粒中的颗粒也是颗粒。我们最近将这些观察结果扩展到洛里克林敲除小鼠。与WildType相比，主要区别在于，LEP似乎在LKO动物中发现的替代物（loricrin-nime）CES的外部和内部标记。