Structural Biology of Macromolecular Complexes

大分子复合物的结构生物学

基本信息

批准号：
8559282
负责人：
ALASDAIR C. STEVEN
金额：
$ 62.12万
依托单位：
NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
依托单位国家：
美国
项目类别：
财政年份：
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/8559282
关键词：
ATP phosphohydrolase Affect Algae Amyloid Antibiotics Behavior Binding Biological Models Caliber Cell Cycle Regulation Cell surface Cells Charge Chlamydomonas Chlamydomonas reinhardtii Chloroplasts Complex Cryoelectron Microscopy Digestion Distal Electron Microscopy Electron Spin Resonance Spectroscopy Endocytosis Environment Escherichia coli Exhibits Exposure to Face Genetic Genitourinary system Goals Gonorrhea Gram-Negative Bacteria Gram-Positive Bacteria Green Algae Human Hybrids Immune In Situ In Vitro Infection Intracellular Membranes Invaded Iron Learning Lipid Bilayers Lipids Macromolecular Complexes Membrane Membrane Protein Traffic Membrane Proteins Meningitis Metabolism Micelles Modeling Molecular Chaperones Morphology Muramidase N Domain Nasopharynx Neck Negative Staining Neisseria Neurologic Outcome Parkinson Disease Peptide Hydrolases Peptides Peptidoglycan Peripheral Pesticin Physiological Plants Process Protein Binding Proteins Publications Quality Control Reaction Regulation Resistance Serum Shapes Siderophores Site Solutions Source Staging Structure Structure-Activity Relationship Synapses Synaptic Vesicles System Systemic infection Techniques Testing Therapeutic Tissues Toxin Transferrin Transferrin Receptor Transferrin-Binding Protein A Tube Vesicle Virulence Virulence Factors Virus Width Yersinia Yersinia pestis alpha synuclein amphiphysin combat density design dimer genetic regulatory protein hybrid protein killings lysin macromolecule particle pathogenic bacteria programs reconstruction relating to nervous system structural biology unfoldase

项目摘要

The goal of this project is to elucidate structure-function relationships in macromolecular machines. During FY12, we studied: chaperone-assisted proteases involved in protein quality control and cell regulation; membrane remodeling; and two components of pathogenic bacteria. (1) All cells must be capable of degrading aberrant and foreign proteins that would otherwise pollute them. Programmed degradation of regulatory factors also contributes to controlling the cell cycle and to generating peptides for immune presentation. These activities are all carried out by energy-dependent proteolytic machines, which generically consist of two subcomplexes - a protease and an ATPase/unfoldase. Since 1995, we have studied the Clp complexes of E. coli, considered as a model system. We showed that the protease 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 s. We went on to show that substrate proteins bind to distal sites on the ATPase and are then unfolded and translocated axially into the digestion chamber of ClpP. These studies all dealt with homomeric ring complexes. ClpP in plants depart from this paradigm in being heteromeric. Chloroplast ClpPs associate multiple different subunits. In FY12, we completed our structural analysis of ClpP from the green alga Chlamydomonas reinhardtii (1). As with land plants, this ClpP has both active subunits (3 ClpPs) and inactive subunits (5 ClpRs). It also has two ClpT subunits which, like land plant ClpTs, show Clp-N domains. ClpTs are believed to function in substrate binding and/or assembly of the two heptameric rings. Negative staining electron microscopy showed that the Chlamydomonas ClpP complex retains the barrel-like shape of homomeric ClpPs, with 4 additional peripheral masses which may represent either the additional IS1 domain of ClpP1 (a feature unique to algae), or ClpTs, or extensions of ClpR subunits. (2) Membrane Remodeling. Remodeling, a process in which lipid bilayer structures are reconfigured by interacting proteins, is central to the functioning and metabolism of cells. We are investigating this phenomenon by using cryo-electron microscopy to characterize the effects of remodeling proteins on large lipid vesicles in vitro. Endophilin A1 is a BAR (Bin/Amphiphysin /Rvs) protein abundant in neural synapses that senses and induces membrane curvature, contributing to neck formation in pre-synaptic vesicles. We found that, on exposure to endophilin, vesicles convert rapidly to coated tubules whose morphology reflects the local concentration of endophilin. Their diameters and curvature resemble those of synaptic vesicles in situ. 3D reconstructions of quasi-cylindrical tubes revealed arrays of BAR dimers, flanked by densities that we equate with amphipathic. We also observed the compression of bulbous coated tubes into 7nm-wide cylindrical micelles, which appear to mimic the penultimate (hemi-fission) stage of endocytosis. Starting in FY11, this project was extended to alpha-synuclein (aS). Natively unfolded in solution, aS accumulates as amyloid in neurological tissue in Parkinsons disease, and also interacts with membranes under both physiological and pathological conditions. We used cryo-EM in conjunction with electron paramagnetic resonance (EPR) and other techniques to characterize the effect of aS on lipid vesicles. The products obtained depend on the protein : lipid ratio. At a molar ratio of < 1: 40, POPG vesicles are converted into cylindrical micelles 5nm in diameter, together with bilayer tubes of various widths (15 - 50 nm ). Between 1 : 20 and 1 : 10, cylindrical micelles are produced exclusively. Other negatively charged lipids (DMPG, DLPG, DAPG) exhibit generally similar behavior. At higher protein: lipid ratios (> 1: 10), cylindrical micelles are replaced by discoid particles, 7 - 10 nm across. In FY12, these studies were completed and have been accepted for publication (5). 3) The resistance of pathogenic bacteria to conventional antibiotic drugs is a growing problem in combating human infections. An alternative therapeutic strategy uses virus-derived proteins, called lysins, to kill Gram-positive bacteria by damaging the peptidoglycan layer in their envelopes. However, a major obstacle to this approach is that lysins cannot cross the protective outer membrane. In this study, a chimeric lysin was produced and shown to kill Gram-negative bacteria. A crystal structure was obtained for a Yersinia pestis toxin called pesticin and used to guide the design of a hybrid lysin in which a domain from pesticin was fused withT4 lysozyme, an archetypal lysin. This hybrid protein was shown to cross the outer membrane and kill model E. coli cells as well as Yersinia strains. Cryo-EM of whole bacterial cells treated with the lysin showed extensive disruption of their membranes (4). Importantly, killing only affects cells that produce the membrane protein FyuA4, a virulence factor, and can therefore be used against essentially any pathogenic strain that expresses FyuA4. 4) Neisseria are Gram-negative bacteria of which two pathogenic species invade the human urogenital tract and nasopharynx, causing gonorrhea, meningitis and other systemic infections. Neisseria require iron for survival and virulence. While most Gram-negative bacteria secrete siderophores to scavenge iron from the environment, Neisseria extracts iron directly from human sources such as transferrin. The Neisserial transferrin transport system consists of two large surface proteins: transferrin binding protein A (TbpA), a 100 kDa integral outer membrane protein, and TbpB, an 80 kDa transferrin receptor. TbpA binds apo and holo transferrin, whereas TbpB associates only with the iron-bound form of transferrin. In order to learn how TbpA and TbpB interact to bind human transferrin and extract its tightly bound iron at physiological pH, a combined structural approach was used. A crystal structure determined for the TbpA-(apo)hTf dimer and a SAXS structure for the TbpB-(holo)hTf complex led to a model for the TbpA-TbpB-(holo)hTf triple complex. We tested this model - with a positive outcome - by negative staining electron microscopy supplemented by computational class-averaging (6). Our results suggest that Neisseria cannot utilize the whole serum transferrin iron supply and the primary function of TbpB is to select and concentrate on the cell surface only those forms of transferrin that can be taken up.

该项目的目标是阐明大分子机器中的结构-功能关系。 2012 财年，我们研究了：参与蛋白质质量控制和细胞调节的分子伴侣辅助蛋白酶；膜重塑；和致病菌的两种成分。 (1) 所有细胞必须能够降解异常的和外来的蛋白质，否则会污染它们。调节因子的程序性降解也有助于控制细胞周期和产生用于免疫呈递的肽。这些活动均由能量依赖性蛋白水解机进行，该蛋白水解机通常由两个子复合物组成 - 蛋白酶和 ATP 酶/解折叠酶。自 1995 年以来，我们研究了大肠杆菌的 Clp 复合物，将其视为模型系统。我们证明蛋白酶 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。我们继续证明底物蛋白与 ATP 酶的远端位点结合，然后展开并轴向易位到 ClpP 的消化室中。这些研究都涉及同聚环复合物。植物中的 ClpP 与此范例不同，它是异聚体。叶绿体 ClpP 关联多个不同的亚基。 2012 财年，我们完成了对绿藻莱茵衣藻 ClpP 的结构分析 (1)。与陆地植物一样，该 ClpP 具有活性亚基（3 个 ClpP）和非活性亚基（5 个 ClpR）。它还具有两个 ClpT 亚基，与陆地植物 ClpT 一样，显示 Clp-N 结构域。 ClpT被认为在底物结合和/或两个七聚环的组装中起作用。负染色电子显微镜显示，衣藻 ClpP 复合体保留了同聚 ClpP 的桶状形状，具有 4 个额外的外围质量，可能代表 ClpP1 的额外 IS1 结构域（藻类独有的特征）、ClpT 或 ClpR 的延伸亚单位。 (2)膜重塑。重塑是通过相互作用的蛋白质重新配置脂质双层结构的过程，是细胞功能和代谢的核心。我们正在通过使用冷冻电子显微镜来研究这种现象，以表征重塑蛋白对体外大脂质囊泡的影响。 Endophilin A1 是一种神经突触中丰富的 BAR (Bin/Amphiphysin /Rvs) 蛋白，可感知并诱导膜弯曲，有助于突触前小泡颈的形成。我们发现，暴露于内亲素后，囊泡迅速转化为包被的小管，其形态反映了内亲素的局部浓度。它们的直径和曲率类似于原位突触小泡的直径和曲率。准圆柱形管的 3D 重建揭示了 BAR 二聚体阵列，其两侧的密度相当于两亲性。我们还观察到球状涂层管被压缩成 7nm 宽的圆柱形胶束，这似乎模仿了内吞作用的倒数第二（半裂变）阶段。从 2011 财年开始，该项目扩展到 α-突触核蛋白 (aS)。 aS 在溶液中自然展开，在帕金森病的神经组织中以淀粉样蛋白的形式积累，并且在生理和病理条件下也与细胞膜相互作用。我们使用冷冻电镜结合电子顺磁共振 (EPR) 和其他技术来表征 aS 对脂质囊泡的影响。获得的产品取决于蛋白质：脂质的比例。在<1:40的摩尔比下，POPG囊泡转化为直径5nm的圆柱形胶束，以及各种宽度（15-50nm）的双层管。在 1:20 和 1:10 之间，专门生产圆柱形胶束。其他带负电荷的脂质（DMPG、DLPG、DAPG）通常表现出类似的行为。在较高的蛋白质：脂质比例 (> 1: 10) 下，圆柱形胶束被直径 7 - 10 nm 的盘状颗粒取代。 2012 财年，这些研究已完成并已接受发表 (5)。 3）病原菌对传统抗生素药物的耐药性是对抗人类感染的一个日益严重的问题。另一种治疗策略是使用病毒衍生的蛋白质（称为溶素）通过破坏革兰氏阳性细菌包膜中的肽聚糖层来杀死革兰氏阳性细菌。然而，这种方法的一个主要障碍是溶素不能穿过保护性外膜。在这项研究中，产生了一种嵌合溶素，并证明它可以杀死革兰氏阴性细菌。获得了鼠疫耶尔森氏菌毒素（称为 pesticin）的晶体结构，并用于指导混合溶素的设计，其中来自 pesticin 的结构域与 T4 溶菌酶（一种典型的溶素）融合。这种混合蛋白被证明可以穿过外膜并杀死模型大肠杆菌细胞以及耶尔森氏菌菌株。用溶素处理的整个细菌细胞的冷冻电镜显示其细胞膜发生广泛破坏 (4)。重要的是，杀伤作用仅影响产生膜蛋白 FyuA4（一种毒力因子）的细胞，因此可用于对抗基本上任何表达 FyuA4 的致病菌株。 4）奈瑟菌属革兰氏阴性菌，其中两种致病菌侵入人体泌尿生殖道和鼻咽部，引起淋病、脑膜炎等全身感染。奈瑟菌需要铁才能生存和发挥毒力。虽然大多数革兰氏阴性细菌分泌铁载体来清除环境中的铁，但奈瑟菌直接从人类来源（例如转铁蛋白）中提取铁。奈瑟氏球菌转铁蛋白转运系统由两种大型表面蛋白组成：转铁蛋白结合蛋白 A (TbpA)（一种 100 kDa 的完整外膜蛋白）和 TbpB（一种 80 kDa 转铁蛋白受体）。 TbpA 结合载脂蛋白和全转铁蛋白，而 TbpB 仅与铁结合形式的转铁蛋白结合。为了了解 TbpA 和 TbpB 如何相互作用以结合人转铁蛋白并在生理 pH 值下提取其紧密结合的铁，使用了组合结构方法。 TbpA-(apo)hTf 二聚体的晶体结构和 TbpB-(holo)hTf 复合物的 SAXS 结构确定，得出了 TbpA-TbpB-(holo)hTf 三重复合物的模型。我们通过负染色电子显微镜并辅以计算类平均（6）测试了该模型，并取得了积极的结果。我们的结果表明奈瑟氏球菌不能利用全部血清转铁蛋白铁供应，TbpB 的主要功能是仅选择可被摄取的转铁蛋白形式并将其集中在细胞表面。