Structural Biology Of Macromolecular Complexes

许多重要的细胞功能是由大型复合物完成的，它们像大分子机器一样运作。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。我们的目的是阐明这两种类型的配合物的结构，组装性能，和相互作用，密切关注的功能内涵。在过去一年中，我们开展了三个主要项目。 (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是必不可少的，否则这些蛋白质会污染细胞，例如通过淀粉样蛋白的形成。这种活性主要由能量依赖性蛋白酶进行，所述能量依赖性蛋白酶一般由两个亚复合物-肽酶和伴侣样ATP酶组成。本研究主要对大肠杆菌Clp蛋白酶进行了研究。大肠杆菌，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由两个并列的七聚体环组成，而同源ATP酶--ClpA或ClpX --是一个六聚体环。ATP酶轴向堆叠在ClpP的一面或两面上以形成活性复合物。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，底物蛋白最初结合到ATP酶上的远端位点，然后沿沿着轴向途径移位到ClpP内的消化室中。结果(a)在我们以1.2 nm分辨率对ClpA六聚体进行的冷冻-EM重建中，可以看到对应于ATP酶结构域（D1，D2）的两个六聚体环，其间有一个空腔。然而，几乎没有N-末端结构域的迹象，尽管它们相当大，每个17 kDa。显然，N域是高度移动的。然而，我们设法可视化他们的方差映射的侧视图和差异映射与平均侧视图的N-结构域缺失突变体。我们还通过分子建模测量了它们的迁移率，表明每个N结构域的移动高达3.5 nm。(b)ClpP可以与ATP酶结合吗？ClpA还是ClpX？并且每个ClpP寡聚体可以结合两个ATP酶。它能结合每个ATP酶的一个拷贝吗？如果是这样的话，杂交复合物在底物结合和内化中是否起作用？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Ure 2 p形成淀粉样蛋白丝。淀粉样蛋白是蛋白质的纤维聚集体，具有蛋白酶抗性、富含β折叠、非天然构象。淀粉样蛋白在包括类风湿性关节炎在内的许多疾病中积累。朊病毒（感染性蛋白质）是一种可传播的淀粉样蛋白，与某些神经病变有关，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。这大大简化和加速了他们的学习。我们专注于Ure 2 p，通常参与氮代谢的蛋白质。其朊病毒表型表现为不能在贫氮源上生长。在早期的工作中，我们证明了丝的形成Ure 2 p在体外和存在的细丝朊病毒感染的细胞。结果：我们专注于充实我们的？淀粉样蛋白骨架模型，1999年制定。Ure 2 p有一个N-末端朊病毒结构域，这是必要的细丝形成和C-末端结构域，在氮调节执行。根据该模型，在细丝中，朊病毒结构域形成被C-末端结构域包围的淀粉样骨架，而在可溶性Ure 2 p中，朊病毒结构域是未折叠的。该模型成功地预测了朊病毒结构域与外源蛋白的融合也应该形成细丝。我们通过生化和EM实验表征了Ure 2 p丝和融合蛋白丝。蛋白酶消化25 nm直径的Ure 2 p细丝将其修剪成4 nm细丝，质谱显示其由朊病毒结构域片段组成。直径为14至25 nm的融合蛋白丝通过蛋白水解类似地减少到4 nm丝。在每种情况下，朊病毒结构域转换从最不蛋白酶敏感的部分后，细丝的形成，这意味着一个大的构象变化。冷电镜或钒酸盐染色后，STEM成像的灯丝显示一个中心4纳米的核心与球状附属物。在每种情况下，未染色细丝的每单位长度STEM质量测量产生1个单体/0.45nm。这些观察结果都支持淀粉样蛋白骨架模型。 (3)Corpus Cell Envelopes（CE）的结构和组装。CE是一种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起源，并已应用各种EM方法，无论是孤立的CE和原位。包括基于氨基酸组成的数学建模的组成推断，我们开发了一个模型的CE作为单层的分子的蛋白质，兜甲蛋白，直接和通过次要的CE蛋白交联。我们设想行政长官是一个？合成的？具有基质物质（兜甲蛋白）和交联剂（次要蛋白质）的生物材料。结果免疫胶体金电镜观察发现，新生小鼠皮肤皮质被膜和颗粒层中的颗粒呈LEPs阳性。我们最近将这些观察结果扩展到兜甲蛋白敲除小鼠。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（无兜甲菌素）CE的外部和内部标记。

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.