Structural Biology of Macromolecular Complexes

大分子复合物的结构生物学

• 批准号: 7137971
• 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/7137971
• 关键词: Escherichia coli; adenosinetriphosphatase; amylin; amyloid proteins; bacterial proteins; chemical models; clathrin; cryoelectron microscopy; endopeptidases; fungal proteins; macromolecule; molecular assembly /self assembly; nuclear magnetic resonance spectroscopy; prions; protein degradation; protein protein interaction; proteolysis; structural biology; three dimensional imaging /topography

Most important biological and biomedical processes are carried out not by isolated macromolecules but by complexes of interacting macromolecules. We are studying the structures of several such assemblies and the interactions among their components, using electron microscopy in combination with other approaches. The complexes currently under study are important for: intracellular proteolyis of defective and regulatory proteins; amyloidosis, with particular reference to the connection between amyloid formation and prion biology; clathrin-coated vesicles and their disassembly by the Hsc70 ATPase. (1) Role of Energy-dependent Proteases in Protein Quality Control and Cell Regulation. All cells must be capable of degrading aberrant and foreign proteins that would otherwise pollute the cell. Programmed degradation of regulatory factors is a major factor in controlling the cell cycle. Both activities are carried out by energy-dependent proteases, which consist of two parts - a peptidase and a chaperone-like ATPase. The archetypal complex of this kind is the proteasome. For several years, our studies focussed on the Clp proteases of E. coli, a model system with a similar repertoire. We showed that the peptidase ClpP consists of two apposed heptameric rings and the cognate ATPase - either ClpA or ClpX - is a single hexameric ring. ClpA/X stack axially on one or both faces of ClpP to form active complexes. We went on to show that substrate proteins bind to distal sites on the ATPase and are then translocated axially into the digestion chamber inside ClpP. In FY05, we continued working to reconcile our cryo-EM reconstruction of the ClpA hexamer (ATPgS state) at 1.1 nm resolution with the published crystal structure of the ClpA monomer (ADP state). Good agreement is obtained in the hexameric ring of D1 ATPase domains, which is the most static part of the structure. The D2 ring, which has higher ATPase activity, shows discrepancies in the interior, suggesting local mobility. We posit that this mobility is exploited in the processing of substrate proteins. (2) We studied the interaction of the 20S proteasome, its peptidase, with PA200, a 200-kDa nuclear protein that stimulates proteasomal hydrolysis of peptides. Monomers of PA200 bind to one or both ends of the 20S core. At 2.3 nm resolution, PA200 is seen to have an asymmetric dome-like structure with major and minor lobes. Its structure is likely to be an irregular folding of an alpha-helical solenoid composed of HEAT-like repeats. PA200 binding induces an opening of the axial channel through the alpha-ring of 20S. Thus PA200 activates via allosteric effects on the 20S core particle, perhaps facilitating release of digestion products or the entrance of substrates. (2) Amyloid Filament Formation by the Yeast Prions, Ure2p and Sup35p, and Other Amyloidogenic Proteins. Amyloid is fibrous aggregates of protein(s) in protease-resistant, beta-sheet-rich, non-native conformations that accumulate in disease situations, including rheumatoid arthritis. Prions (infectious proteins) are transmissible amyloids that have been implicated in neuropathies, including the spongiform encephalopathies. To investigate amyloids and the mechanisms that underlie their formation, we started studying the structures of yeast prions in 1998. We focused initially on Ure2p, a protein that normally functions as a negative regulator of nitrogen catabolism. Our previous work has shown that the N-terminal "prion domain" of Ure2p is responsible for filament formation, and the C-terminal domain which performs its regulatory function remains folded in the filamentous state but is inactivated by a steric mechanism. In our "amyloid backbone" model of the filament, the prion domains form a backbone surrounded by the C-terminal domains. In FY05, we performed experiments to test this model and to discriminate between it and an alternative model whereby filament formation is envisaged instead to be driven by interactions between N-domains and C-domains. Electron diffraction and X-ray diffraction were used to demonstrate that the backbone does indeed consist of polymerized N-domains in cross-beta conformation. NMR spectroscopy was used to confirm that in soluble Ure2p, the N-domain is highly mobile, indicative of complete disordering: this mobility disappears in the filamentous (amyloid) state. To further demonstrate that intracellular filaments are structurally indistinguishable from filaments assembled in vitro, we refined the electron tomograms of thin sections of infected yeast cells reported last year. In 2004, we formulated the "beta super-pleated structure" model for the amyloid species found in the backbone of Ure2p filaments. It envisages an array of parallel beta-sheets generated by stacking monomers, each of which adopts a planar "beta-serpentine" fold. The model accounts for all current data on Ure2p filaments. We also adapted this model to apply to filaments of the human protein, amylin, whose fibrillation is implicated in type 2 diabetes. This model has only three beta-strands per subunit serpentine as compared to eight or more in Ure2p, and explains why rat amylin does not fibrillize on account of its having amino acid substitutions in a few key positions compared to human amylin. We also started electron microscopic work on filaments of the fungal prion protein, Het-S which differs from Ure2p in that its prion domain does not have high concentrations of asparagine and is at the C-terminal end, not the N-terminal end, of the molecule. (3) Interaction of clathrin with proteins that regulate its assembly. The protein clathrin plays a key role in intracellular trafficking, via its polymerization into the coats of coated pits and vesicles. Assembly of clathrin is promoted by accessory proteins such as auxilin and AP180, and disassembly is effected by the Hsc70 ATPase. In the 1980s, we studied the molecular composition of coated vesicles and the plasticity of the assembly subunit, the clathrin triskelion. We have now returned to this system, equipped with cryo-EM technology, and are investigating the interaction of regulatory proteins - in particular, Hsc70 - with clathrin lattices. In FY05, we completed the current phase of our investigation into the binding of Hsc70 to clathrin baskets. The chimera C58J is a minimal construct capable of supporting both reactions, i.e assembly and disassembly. It consists of the C58 moiety of AP180, which facilitates clathrin assembly, fused with the J-domain of auxilin, which recruits Hsc70 to baskets. We studied the first steps in disassembly by using cryo-electron microscopy to identify the binding site of Hsc70 on clathrin-C58J baskets at pH 6: under these conditions, disassembly does not proceed further. Hsc70 interactions involve two sites: (i) its major interaction is with the sides of spars of the clathrin lattice, close to the triskelion hubs; and (ii) a site at the N-terminal hooks of the clathrin heavy chains, presumably via the J-domain of C58J. We have proposed that individual triskelions may be extricated from the clathrin lattice by the concerted action of up to six Hsc70 molecules, which intercalate between clathrin leg segments, prying them apart. Three Hsc70s remain bound to the dissociated triskelion, close to its trimerization hub.

最重要的生物和生物医学过程不是由孤立的大分子进行的，而是由相互作用的大分子的复合物进行的。我们正在使用电子显微镜结合其他方法来研究几个此类组件的结构及其组件之间的相互作用。目前正在研究的复合物对于以下方面很重要：缺陷蛋白和调节蛋白的细胞内蛋白水解；淀粉样变性，特别是淀粉样蛋白形成与朊病毒生物学之间的联系；网格蛋白包被的囊泡及其被 Hsc70 ATP 酶分解。 (1) 能量依赖性蛋白酶在蛋白质质量控​​制和细胞调节中的作用。所有细胞都必须能够降解异常和外来蛋白质，否则会污染细胞。调节因子的程序性降解是控制细胞周期的主要因素。这两种活动均由能量依赖性蛋白酶进行，该蛋白酶由两部分组成：肽酶和分子伴侣样 ATP 酶。这种复合体的原型是蛋白酶体。多年来，我们的研究重点是大肠杆菌的 Clp 蛋白酶，这是一个具有相似功能的模型系统。我们证明肽酶 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。 ClpA/X 轴向堆叠在 ClpP 的一个或两个面上，形成活性复合物。我们继续证明底物蛋白与 ATP 酶的远端位点结合，然后轴向转移到 ClpP 内的消化室中。在 2005 财年，我们继续致力于以 1.1 nm 分辨率对 ClpA 六聚体（ATPgS 态）进行的冷冻电镜重建与已发布的 ClpA 单体（ADP 态）晶体结构进行协调。在 D1 ATP 酶结构域的六聚环中获得了良好的一致性，这是结构中最静态的部分。 D2 环具有较高的 ATP 酶活性，其内部显示出差异，表明局部流动性。我们假设这种流动性在底物蛋白质的加工中被利用。 (2) 我们研究了 20S 蛋白酶体（其肽酶）与 PA200（一种刺激肽蛋白酶体水解的 200 kDa 核蛋白）的相互作用。 PA200 的单体结合到 20S 核心的一端或两端。在 2.3 nm 分辨率下，PA200 具有不对称的圆顶状结构，带有主瓣和小瓣。它的结构很可能是由类似 HEAT 的重复序列组成的 α 螺旋螺线管的不规则折叠。 PA200 结合诱导穿过 20S α 环的轴向通道打开。因此，PA200 通过 20S 核心颗粒的变构效应激活，可能促进消化产物的释放或底物的进入。 (2) 酵母朊病毒、Ure2p 和 Sup35p 以及其他淀粉样蛋白形成淀粉样蛋白丝。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的纤维聚集体，在疾病情况下积累，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，与神经病有关，包括海绵状脑病。为了研究淀粉样蛋白及其形成机制，我们于 1998 年开始研究酵母朊病毒的结构。我们最初关注的是 Ure2p，一种通常充当氮分解代谢负调节因子的蛋白质。我们之前的工作表明，Ure2p的N端“朊病毒结构域”负责丝状形成，而执行其调节功能的C端结构域保持折叠在丝状状态，但通过空间机制失活。在我们的丝状体“淀粉样蛋白主链”模型中，朊病毒结构域形成被 C 端结构域包围的主链。在 2005 财年，我们进行了实验来测试该模型，并将其与另一种模型区分开来，在另一种模型中，丝状形成被设想为由 N 域和 C 域之间的相互作用驱动。电子衍射和 X 射线衍射被用来证明主链确实由交叉 β 构象的聚合 N 结构域组成。使用核磁共振波谱证实，在可溶性 Ure2p 中，N 结构域具有高度可移动性，表明完全无序：这种移动性在丝状（淀粉样蛋白）状态下消失。为了进一步证明细胞内丝在结构上与体外组装的丝没有区别，我们改进了去年报告的受感染酵母细胞薄片的电子断层图。 2004年，我们为Ure2p丝主链中发现的淀粉样蛋白种类制定了“β超折叠结构”模型。它设想通过堆叠单体产生一系列平行的β-折叠，每个折叠都采用平面“β-蛇形”折叠。该模型考虑了 Ure2p 丝的所有当前数据。我们还对该模型进行了改造，以应用于人类蛋白质胰岛淀粉样蛋白的细丝，其纤维颤动与 2 型糖尿病有关。该模型每个蛇纹石亚基只有 3 个 β 链，而 Ure2p 中的每个亚基蛇纹石只有 3 个 β 链，这解释了为什么大鼠胰岛淀粉样多肽不会纤维化，因为与人胰岛淀粉样多肽相比，其在几个关键位置具有氨基酸取代。我们还开始对真菌朊病毒蛋白 Het-S 的细丝进行电子显微镜研究，Het-S 与 Ure2p 的不同之处在于，其朊病毒结构域没有高浓度的天冬酰胺，并且位于分子的 C 末端，而不是 N 末端。 (3)网格蛋白与调节其组装的蛋白质的相互作用。蛋白质网格蛋白通过聚合形成包被的凹坑和囊泡的外壳，在细胞内运输中发挥关键作用。网格蛋白的组装由辅助蛋白（例如辅助蛋白和 AP180）促进，而分解则由 Hsc70 ATP 酶实现。在 20 世纪 80 年代，我们研究了包被囊泡的分子组成和组装亚基网格蛋白 triskelion 的可塑性。我们现在回到了这个配备了冷冻电镜技术的系统，并正在研究调节蛋白（特别是 Hsc70）与网格蛋白晶格的相互作用。 2005 财年，我们完成了当前阶段的 Hsc70 与网格蛋白篮结合的研究。嵌合体 C58J 是一种能够支持组装和拆卸两种反应的最小结构。它由 AP180 的 C58 部分组成，促进网格蛋白组装，与辅助蛋白的 J 结构域融合，将 Hsc70 招募到篮子中。我们通过使用冷冻电子显微镜研究了拆卸的第一步，以确定 pH 6 下网格蛋白-C58J 篮上 Hsc70 的结合位点：在这些条件下，拆卸不会进一步进行。 Hsc70 相互作用涉及两个位点：（i）其主要相互作用是与网格蛋白晶格的翼梁侧面，靠近 triskelion 中心； (ii) 网格蛋白重链 N 端钩的位点，可能通过 C58J 的 J 结构域。我们提出，通过多达六个 Hsc70 分子的协同作用，可以将单个三链体从网格蛋白晶格中解脱出来，这些分子插入网格蛋白腿段之间，将它们撬开。三个 Hsc70 仍与分离的 triskelion 结合，靠近其三聚化中心。