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来执行的。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。我们的目标是阐明两种复合物的结构、组装特性和相互作用，并密切关注其功能内涵。去年我们实施了三个主要项目。 (1) 能量依赖性蛋白酶。蛋白质质量控​​制对于消除异常蛋白质至关重要，否则会污染细胞，例如通过淀粉样蛋白形成。这种活性主要由能量依赖性蛋白酶进行，这些蛋白酶通常由两个子复合物组成 - 肽酶和分子伴侣样 ATP 酶。我们的研究重点是大肠杆菌的 Clp 蛋白酶，它提供了一个易于处理的模型系统。早些时候，我们表明肽酶 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。 ATP 酶在 ClpP 的一个或两个面上轴向堆积，形成活性复合物。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 ATP 酶的远端位点结合，然后沿着轴向路径转移到 ClpP 内的消化室中。结果。 (a) 在我们以 1.2 nm 分辨率对 ClpA 六聚体进行冷冻电镜重建时，可以看到对应于 ATPase 结构域（D1、D2）的两个六聚环，其间有一个空腔。然而，尽管 N 端结构域相当大（每个 17 kDa），但几乎没有迹象表明它们存在。显然，N 域具有高度的移动性。尽管如此，我们还是设法通过侧视图的方差映射和 N 域删除突变体的平均侧视图的差异映射来可视化它们。我们还通过分子模型测量了它们的移动性规模，表明每个 N 域涉及高达 3.5 nm 的移动。 (b) ClpP 可能与 ATP 酶结合？ ClpA 还是 ClpX ？每个 ClpP 寡聚体可以结合两个 ATP 酶。它可以结合每种 ATP 酶的一个副本吗？如果是这样，杂合复合物是否在底物结合和内化方面发挥作用？我们通过对 ClpA 特异性和 ClpX 特异性底物开始易位后记录的显微照片进行统计分析来解决这些问题。这两个问题的答案都是肯定的。 (2) 酵母朊病毒蛋白 Ure2p 形成淀粉样蛋白丝。淀粉样蛋白是具有蛋白酶抗性、富含β折叠、非天然构象的蛋白质纤维状聚集体。淀粉样蛋白在许多疾病中积累，包括类风湿性关节炎。朊病毒（传染性蛋白质）是一种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及其形成机制，我们研究了酵母朊病毒。与哺乳动物朊病毒不同，它们的表型表现为缺乏代谢功能而不是细胞病变效应。这极大地简化并加速了他们的学习。我们关注 Ure2p，一种通常参与氮代谢的蛋白质。其朊病毒表型表现为无法在贫乏的氮源上生长。在早期的工作中，我们在体外证明了 Ure2p 的细丝形成以及朊病毒感染的细胞中细丝的存在。结果：我们专注于证实 1999 年制定的淀粉样蛋白骨架模型。Ure2p 具有丝形成所必需的 N 端朊病毒结构域和执行氮调节的 C 端结构域。根据该模型，在丝状结构中，朊病毒结构域形成被 C 端结构域包围的淀粉样蛋白主链，而在可溶性 Ure2p 中，朊病毒结构域是展开的。该模型成功预测朊病毒结构域与外源蛋白的融合也应形成丝状结构。我们通过生化和电镜实验对 Ure2p 丝和融合蛋白丝进行了表征。蛋白酶消化 25 纳米直径的 Ure2p 细丝，将其修剪成 4 纳米细丝，质谱分析显示该细丝由朊病毒结构域片段组成。直径为 14 至 25 nm 的融合蛋白丝同样通过蛋白水解减少为 4 nm 丝。在每种情况下，朊病毒结构域在丝形成时从对蛋白酶最敏感的部分转变为最不敏感的部分，这意味着较大的构象变化。通过冷冻电镜成像或通过 STEM 钒酸盐染色后的细丝显示出具有球形附属物的中央 4 纳米核心。对未染色的细丝进行 STEM 单位长度质量测量，每种情况下每 0.45 nm 产生 1 个单体。这些观察结果都支持淀粉样蛋白骨架模型。 (3) 角质化细胞膜 (CE) 的结构和组装。 CE 是共价交联的蛋白质层，排列在终末分化的角质形成细胞的细胞质表面。 CE 被认为有助于这些组织的物理弹性和不可穿透性。我们研究它们的生物发生，并应用了各种电磁方法，包括分离的 CE 和原位。包括基于氨基酸组成数学模型的组成推断，我们开发了一种 CE 模型，作为蛋白质、兜甲蛋白分子的单层，直接交联或通过次要 CE 蛋白交联。我们将 CE 设想为“复合体”具有基质物质（兜甲素）和交联剂（次要蛋白质）的生物材料。结果。通过冷冻切片的免疫金电镜，我们发现新生小鼠皮肤中的角质化包膜被标记为 LEP 阳性，颗粒层中的颗粒也是如此。我们最近将这些观察扩展到了兜甲素基因敲除小鼠。与野生型相比的主要区别在于，LEP 似乎从 LKO 动物中发现的替代（无甲甲蛋白）CE 的外部和内部进行标记。