Structural Biology Of Macromolecular Complexes

许多重要的细胞功能是由大分子复合体执行的，它们的运作就像大分子机器一样。复合体还主要作为生物材料在许多组织中发挥结构作用，包括皮肤和肌肉。旨在阐明这两类配合物的结构、组装性质和相互作用，并密切关注其功能内涵。在过去的一年里，我们开展了三个主要项目。 (1)依赖能量的蛋白水解酶。蛋白质质量控制对于消除异常蛋白质至关重要，否则这些蛋白质会污染细胞，例如通过淀粉样蛋白的形成。这种活性在很大程度上是由能量依赖的蛋白水解酶完成的，它一般由两个亚复合体组成--一个多肽酶和一个类似伴侣的ATPase。我们的研究主要集中在大肠杆菌的CLP蛋白水解酶，这为我们提供了一个易于处理的模型系统。早些时候，我们证明了肽酶ClpP由两个相对的七聚体环组成，而同源的ATPase-ClpA或ClpX-是一个单一的六聚体环。ATPase轴向堆积在ClpP的一侧或两侧形成活性络合物。我们继续研究了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上的远端结合，然后沿着轴向路径转移到ClpP内的消化室。结果。(A)在我们以1.2 nm分辨率对ClpA六角体的冷冻EM重建中，看到了与ATPase结构域(D1，D2)相对应的两个六聚体环，其间有一个空腔。然而，尽管N-末端结构域相当大，每个17 kDa，但几乎没有迹象。显然，N-结构域具有很高的流动性。然而，我们设法通过侧视图的方差映射和N结构域缺失突变体的平均侧视图的差异映射来可视化它们。我们还通过分子建模测量了它们的迁移率，表明每个N-结构域的迁移率高达3.5 nm。(B)ClpP可以与ATPase中的任何一个合作？ClpA还是ClpX？每个ClpP寡聚体可以结合两个ATPase。它能绑定每个ATPase的一个拷贝吗？如果是这样的话，杂化络合物是否具有底物结合和内化的功能？我们通过对启动ClpA特异性底物和ClpX特异性底物移位后记录的显微照片的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)酵母蛋白Ure2p形成淀粉样丝。淀粉样蛋白是一种纤维状的蛋白质聚集体(S)，具有抗蛋白酶、富含β-折叠的非天然构象。淀粉样蛋白在包括类风湿性关节炎在内的多种疾病中积聚。Prion(感染蛋白)是一种可传播的淀粉样蛋白，与某些神经疾病有关，包括海绵状脑病。为了研究淀粉样蛋白的结构和形成机制，我们研究了酵母蛋白。与哺乳动物不同，它们的表型表现为缺乏新陈代谢功能，而不是细胞病变效应。这大大简化和加速了他们的学习。我们关注的是Ure2p，这是一种通常参与氮代谢的蛋白质。其Prion表型表现为不能在贫乏的氮源上生长。在早期的工作中，我们证明了Ure2p在体外形成细丝，并在感染Pron的细胞中存在细丝。结果：我们专注于证实我们1999年建立的淀粉样蛋白脊椎模型.Ure2p有一个N-末端的Prion结构域和一个C-末端的结构域，这是形成纤维所必需的，C-末端的结构域起到氮调节作用。根据该模型，在细丝中，Pron结构域形成被C-末端结构域包围的淀粉样蛋白骨架，而在可溶性Ure2p中，Prion结构域是展开的。这个模型成功地预测了Pron结构域与外源蛋白的融合也应该形成细丝。通过生化实验和EM实验对Ure2p纤维和融合蛋白纤维进行了表征。用酶消化直径为25 nm的Ure2p丝，将其切割成4 nm的丝，质谱分析表明它由Pron结构域片段组成。蛋白水解法将直径为14~25 nm的融合蛋白纤维同样还原为4 nm的纤维。在每种情况下，Pron结构域在细丝形成时从最敏感的部分转变为最不敏感的部分，这意味着一个很大的构象变化。冷冻-EM或钒酸盐染色后的细丝在STEM上显示一个中心的4 nm核心和球状附属物。对未染色的细丝进行茎质量单位长度测量，每0.45 nm产生一种单体。这些观察结果都支持淀粉样蛋白主干模型。 (3)角化细胞膜(CES)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。CES被认为对这些组织具有物理弹性和不可渗透性。我们研究了它们的生物发生，并应用了各种EM方法，包括分离的CES和原位。包括基于氨基酸组成数学模型的组成推断，我们开发了一个CES模型，作为蛋白质的单分子层，Clicrin，直接和通过少量CE蛋白质交联。我们设想行政长官是一个复合体？含有基质物质(氯化物)和交联剂(次要蛋白质)的生物材料。结果。通过冰冻切片的免疫金标-EM，我们发现新生小鼠皮肤中的角化包膜标记为LEPS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氯氰菊酯基因敲除小鼠。与野生型相比，主要的区别是LEP似乎同时从外部和内部标记在LKO动物中发现的替代(无氯)CES。

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.