Structural Biology of Macromolecular Complexes

大分子复合物的结构生物学

• 批准号: 8939411
• 负责人: ALASDAIR C. STEVEN
• 金额: $ 54.31万
• 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/8939411
• 关键词: ATP phosphohydrolase; Adopted; Affect; Apolipoproteins; Archaea; Bacteria; Bacteriophage mu; Bacteriophages; Binding; Binding Proteins; Biochemical; Biochemical Reaction; Bioinformatics; Biological Models; Caliber; Capsid; Cells; Child; Circular Dichroism; Cleaved cell; Complex; Cryoelectron Microscopy; DNA; DNA Binding; DNA Binding Domain; DNA-Binding Proteins; Data; Dimensions; Electron Microscopy; Enzymes; Escherichia coli; Eukaryota; Fatty Acids; Ferritin; Filament; Genes; Genetic; Goals; Growth; Homeostasis; Homology Modeling; Inflammation; Interferons; Intracellular Membranes; Investigation; Iron; Life; Lipid Bilayers; Lipids; Macromolecular Complexes; Maps; Membrane; Membrane Protein Traffic; Metabolic; Metabolism; Minor; Mitochondria; Mobile Genetic Elements; Modeling; Molecular; Molecular Conformation; Molecular Machines; Molecular Models; Mutate; Mutation; Myxococcus xanthus; National Institute of Arthritis and Musculoskeletal and Skin Diseases; Neisseria; Oleic Acids; Organelles; Oxidative Stress; Participant; Patients; Peptide Hydrolases; Phosphatidylglycerols; Physiologic pulse; Physiological; Play; Pneumonia; Process; Property; Protein Binding; Proteins; Reaction; Reporting; Resources; Role; Sea; Shapes; Site; Solubility; Starvation; Structure; Structure-Activity Relationship; Surface; System; Transposase; Vasculitis; Vesicle; Virus; alpha synuclein; base; cohort; dimer; early onset; electron tomography; fatty acid transport; gain of function mutation; genetic regulatory protein; in vivo; light scattering; macromolecule; member; mitochondrial membrane; molecular modeling; monomer; nano; nanoparticle; novel; polymerization; protein complex; research study; structural biology; unfoldase; uptake

The goal of this project is to elucidate structure-function relationships in macromolecular machines. During FY14, our studies focussed on: membrane remodeling by alfa-synuclein; and characterization of a novel bacterial nano compartment. (1) All cells must be capable of degrading aberrant and foreign proteins that would otherwise pollute them. These activities are carried out by energy-dependent proteolytic machines, which 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 described the structures of the two sub complexes and characterized the interactions between them and with bound substrate proteins. In FY14, activity on this project was minor but it is still in our portfolio. (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 cryo-electron microscopy (EM) and cryo-electron tomography (ET) applied to several systems. In FY14, our main effort was directed towards characterizing the effects of the protein alpha-synuclein (aS) on lipid vesicles and the influence of this interaction on the oligomeric/polyeric state of aS (1). Alfa-Synuclein (aS) is a membrane-binding protein with sequence similarity to apolipoproteins and other lipid-carrying proteins, which are capable of forming lipid-containing nanoparticles, sometimes referred to as discs. Hitherto it has been unclear whether aS also possesses this property. Using cryo-electron microscopy and light scattering, we found that aS can remodel phosphatidyl glycerol vesicles into nanoparticles whose shape (ellipsoidal) and dimensions (in the 7-10 nm range) resemble those formed by apolipoproteins. Their molar ratio of aS to lipid is approximately 1 : 20 and aS is oligomeric (including trimers and tetramers). Similar nanoparticles form when aS is added to vesicles of mitochondrial lipids. This observation suggests a mechanism for the previously reported disruption of mitochondrial membranes by aS. Circular dichroism and 4-pulse DEER experiments reveal that in nanoparticles aS assumes a broken helical conformation distinct from the extended helical conformation adopted when aS is bound to intact vesicles or membrane tubules. We also observed aS-dependent tubule and nanoparticle formation in the presence of oleic acid, implying that αS can interact with fatty acids and lipids in similar manners. aS-related nanoparticles might play a role in lipid and fatty acid transport functions previously attributed to this protein. 3) Recently we have participated in two projects aimed at determining the struures of protein complexes involved in genetic transposition. 3a) The first concerns MuB, an enzyme encoded by bacteriophage Mu (2). Mechanistic understanding of MuB function had previously been hindered by its poor solubility. We found that MuB is an AAA+ ATPase that forms helical filaments to control target selection for DNA transposition. To do so, we combined bioinformatic, mutagenic, biochemical and electron microscopy. We demonstrated that MuB forms ATP-dependent filaments with or without DNA. We also identified critical residues for its ATPase, DNA binding, protein polymerization and MuA interaction activities. By EM, we showed that DNA binds in the axial hole of the MuB filament. These findings, together with the influence of MuB-filament size on strand-transfer efficiency, led to a model in which MuB-imposed symmetry transiently deforms the DNA at the boundary of the MuB filament and results in a bent DNA favored for transposition. 3b) The Hermes protein is a member of the hAT transposon superfamily which has active representatives, including McClintock's archetypal Ac mobile genetic element, in many eukaryotic species. Our colleagues determined the crystal structure of the Hermes transposase-DNA complex which revealed that Hermes forms an octamer, and that each monomer has bound a cleaved transposon end. We contributed EM analyses that localized the BED domains what were invisible, i.e. poorly ordered, in the crystal (3). The overall picture is that the catalytic unit is a dimer: however, only octamers are active in vivo. This suggests that they provide crucial multiple specific DNA binding domains that recognize repeated subterminal sequences and non-specific DNA binding surfaces for target capture. The unusual structure explains the basis of bipartite DNA recognition at hAT transposon ends, provides a rationale for transposon end asymmetry, and demonstrates how an octamer could provide multiple sites of interaction to allow the transposase to locate its transposon ends amidst a sea of chromosomal DNA. 3) A virus capsid-like nanocompartment that stores iron and protects bacteria from oxidative stress. Living cells compartmentalize materials and enzymatic reactions to increase their metabolic efficiency. While eukaryotes use membrane-bound organelles, bacteria and archaea rely primarily on protein-bound nanocompartments. Encapsulins constitute a recently discovered class of nanocompartments that are widespread in bacteria and archaea. Hitherto, their functions have been unclear. We have characterized the structure of the encapsulin nanocompartment from Myxococcus xanthus and shown that its role is to sequester cytosolic iron, thereby to protect the cells from oxidative stress (4). Inasmuch as this project relates to bacterial management of iron resources, this project dovetails with the one reporting molecular machine involved in the uptake of iron by Neisseria that we reported last year. The nanocompartment of M. xanthus consists of a protein shell with internal contents. It has a shell protein (EncA, 32.5 kDa) and three internal proteins (EncB, 17 kDa; EncC, 13 kDa; EncD, 11 kDa). Using cryo-electron microscopy, we determined that EncA expressed in E. coli self-assembles into an icosahedral shell 32 nm in diameter (26 nm internal diameter), built from 180 subunits with the fold first observed in bacteriophage HK97 capsid. The internal proteins, of which EncB and EncC have ferritin-like domains, attach to its inner surface. Native nanocompartments have dense iron-rich cores. Functionally, they resemble ferritins, cage-like iron storage proteins, but with a massively greater capacity (30,000 Fe atoms vs. 3,000 in ferritin). Physiological data reveal that few nanocompartments are assembled during vegetative growth, but they increase five-fold upon starvation, protecting cells from oxidative stress through iron sequestration. Further investigation of this system is ongoing. 4) Molecular modeling of mutated STING residues in clinically affected children. In a multi-participant multi-faceted study by a consortium led by Dr Raphaela Goldbach-Mansky (NIAMS), a cohort of 6 patients was identified with early onset systemic inflammation, vasculitis, and pulmonary inflammation. These patients were found to have de novo gain-of-function mutations in TMEM173, which encodes STimulator of Interferon Genes (STING). We participated in this effort by using molecular graphics and modeling to map the mutated residues on a homology model of a STING dimer. Intriguingly, the mutations tend to map at or near the dimer interface (5).

该项目的目的是阐明大分子机器中的结构 - 功能关系。在2014财年期间，我们的研究集中在：阿尔法 - 核蛋白的膜重塑；和新型细菌纳米室的表征。 （1）所有细胞都必须能够降解异常和异物蛋白质，否则会污染它们。这些活性是由能量依赖性蛋白水解机进行的，该机器由两个亚复合物组成 - 一种蛋白酶和ATPase/fromoldase。自1995年以来，我们研究了大肠杆菌的CLP复合物，被认为是模型系统。我们描述了两个亚复合物的结构，并表征了它们与结合底物蛋白之间的相互作用。在2014财年，该项目的活动很小，但仍在我们的投资组合中。 （2）膜重塑。重塑是通过相互作用蛋白重新配置脂质双层结构的过程，是细胞功能和代谢的核心。我们正在研究用于多个系统的冷冻电子显微镜（EM）和冷冻电子断层扫描（ET）的这种现象。在2014财年，我们的主要努力是为了表征蛋白质α-核蛋白（AS）对脂质囊泡的影响，以及这种相互作用对AS（1）寡聚/聚体状态的影响。 阿尔法 - 核蛋白（AS）是一种膜结合蛋白，与载脂蛋白和其他脂肪蛋白具有序列相似，能够形成含脂质的纳米颗粒，有时称为椎间盘。迄今为止，尚不清楚是否也拥有该属性。使用冷冻电子显微镜和光散射，我们发现，重塑磷脂酰甘油囊泡的形状（椭圆形）和尺寸（在7-10 nm范围内）类似于载脂蛋白形成的磷脂酰甘油囊泡。它们的脂质与脂质的摩尔比大约为1：20，并且是寡聚的（包括三聚体和四聚体）。当添加到线粒体脂质囊泡中时，类似的纳米颗粒形成。该观察结果提出了一种先前报道的通过AS破坏线粒体膜的机制。 圆二色性和4脉冲鹿实验表明，在纳米颗粒中，假设与完整的囊泡或膜小管所结合时采用的伸长螺旋构象与扩展的螺旋构象不同。 我们还观察到在存在油酸的情况下AS依赖性小管和纳米颗粒的形成，这意味着αs可以以相似的方式与脂肪酸和脂质相互作用。与此相关的纳米颗粒可能在先前归因于该蛋白质的脂质和脂肪酸转运功能中起作用。 3）最近，我们参加了两个项目，旨在确定与遗传转座有关的蛋白质复合物的束缚。 3a）第一个问题是MUB，一种由噬菌体MU编码的酶（2）。以前，对MUB功能的机械理解因其溶解度差而受到阻碍。我们发现MUB是一种AAA+ ATPase，形成螺旋丝，以控制DNA转座的靶标选择。为此，我们结合了生物信息学，诱变，生化和电子显微镜。我们证明了MUB形成具有或不具有DNA的ATP依赖性细丝。我们还确定了其ATPase，DNA结合，蛋白质聚合和MUA相互作用活性的关键残基。通过EM，我们表明DNA在MUB细丝的轴向孔中结合。这些发现，加上Mub丝尺寸对链转移效率的影响，导致了一个模型，其中MUB施加的对称性瞬时在MUB细丝的边界处变形DNA，并导致弯曲的DNA偏向于转置。 3b）爱马仕蛋白是许多真核物种中有活跃代表的HAT Transposon超家族的成员，其中包括McClintock的原型AC移动遗传元素。我们的同事们确定了爱马仕（Hermes）转座酶-DNA复合物的晶体结构，该复合体揭示了爱马仕（Hermes）形成八聚体，并且每个单体都结合了裂解的转座子端。我们贡献了EM分析，该分析将床域定位于晶体中（3）中的床域，即有序不良的分析。总体情况是催化单元是一个二聚体：但是，只有八聚体在体内活跃。这表明它们提供了至关重要的多个特定DNA结合结构域，这些结构域识别重复的亚末端序列和非特异性DNA结合表面以捕获目标捕获。不寻常的结构解释了在帽子转座子末端识别两分的DNA识别的基础，为转座端端不对称提供了理由，并演示了八聚体如何提供多个相互作用的位点，以允许转座酶定位其转座子的转座子末端，并在染色体DNA的海中定位。 3）一种类似衣壳的纳米室，可保护铁并保护细菌免受氧化应激。 活细胞将材料和酶促反应分隔以提高其代谢效率。 虽然真核生物使用膜结合的细胞器，但细菌和古细菌主要依赖于蛋白质结合的纳米室。 封装构成了最近发现的一类纳米室，在细菌和古细菌中广泛。迄今为止，他们的功能尚不清楚。我们已经表征了叶th虫球菌的结构结构，并表明其作用是隔离胞质铁，从而保护细胞免受氧化应激（4）。由于该项目与铁资源的细菌管理有关，因此该项目与我们去年报道的Neisseria摄入的一台报道分子机相吻合。 黄叶菌的纳米室由具有内部内容物的蛋白质壳组成。它具有壳蛋白（ENCA，32.5 kDa）和三个内部蛋白质（ENCB，17 kDa; encc，13 kDa; encd; encd，11 kDa）。 使用冷冻电子显微镜，我们确定在大肠杆菌自组装中表达的ENCA直径为32 nm的二十面体壳（26 nm内径），该壳是由180个亚基建造的，该亚基首先在phinephiophage hk97 capsid中首先观察到倍数。内部蛋白质（ENCB和ENCC）具有类似铁蛋白的结构域，附着在其内表面。 天然纳米室具有密集的铁核。从功能上讲，它们类似于铁蛋白，类似笼子的铁储存蛋白，但容量较大（30,000 Fe原子与铁蛋白中的3,000）。 生理数据表明，在营养生长过程中，很少有纳米室被组装，但在饥饿时增加了五倍，可以通过铁隔离来保护细胞免受氧化应激。对该系统的进一步调查正在进行中。 4）突变刺激残基在临床受影响儿童中的分子建模。在由Raphaela Goldbach-Mansky（NIAMS）领导的一项财团的多方多面研究中，发现了6例患者的队列，患有早期发作的全身性炎症，血管炎，血管炎和肺部炎症。发现这些患者在TMEM173中具有从头获得功能的突变，该突变编码了干扰素基因的刺激剂（Sting）。我们通过使用分子图形和建模来绘制Sting Dimer的同源性模型的突变残基来参与这项工作。有趣的是，突变倾向于在二聚体界面或附近映射（5）。