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).

该项目的目标是阐明大分子机器中的结构-功能关系。 2014 财年，我们的研究重点是：α-突触核蛋白的膜重塑；以及新型细菌纳米隔室的表征。 (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。这些活动是由能量依赖性蛋白水解机进行的，该蛋白水解机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。我们描述了两个子复合物的结构，并表征了它们之间以及与结合的底物蛋白之间的相互作用。 2014 财年，该项目的活动规模较小，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。我们正在通过应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）来研究这种现象。在 2014 财年，我们的主要工作是表征蛋白质 α-突触核蛋白 (aS) 对脂质囊泡的影响以及这种相互作用对 aS 寡聚/聚合状态的影响 (1)。 阿尔法突触核蛋白 (aS) 是一种膜结合蛋白，与载脂蛋白和其他脂质携带蛋白具有序列相似性，能够形成含脂质纳米颗粒，有时称为圆盘。迄今为止，尚不清楚 aS 是否也拥有此属性。使用冷冻电子显微镜和光散射，我们发现aS可以将磷脂酰甘油囊泡重塑为纳米颗粒，其形状（椭圆体）和尺寸（7-10 nm范围内）类似于载脂蛋白形成的纳米颗粒。它们的aS与脂质的摩尔比约为1:20并且aS是寡聚体（包括三聚体和四聚体）。当将 aS 添加到线粒体脂质囊泡中时，会形成类似的纳米颗粒。这一观察结果表明了先前报道的 aS 破坏线粒体膜的机制。 圆二色性和 4 脉冲 DEER 实验表明，在纳米颗粒中，aS 呈现破碎的螺旋构象，与 aS 与完整囊泡或膜管结合时采用的延伸螺旋构象不同。 我们还观察到在油酸存在下αS依赖性小管和纳米颗粒的形成，这意味着αS可以以类似的方式与脂肪酸和脂质相互作用。与 aS 相关的纳米颗粒可能在以前归因于该蛋白质的脂质和脂肪酸运输功能中发挥作用。 3）最近我们参与了两个项目，旨在确定参与遗传转座的蛋白质复合物的结构。 3a) 第一个涉及 MuB，一种由噬菌体 Mu 编码的酶 (2)。此前，人们对 MuB 功能的机制理解一直因其溶解度差而受到阻碍。我们发现 MuB 是一种 AAA+ ATP 酶，可形成螺旋丝来控制 DNA 转座的靶标选择。为此，我们结合了生物信息学、诱变学、生化学和电子显微镜学。我们证明 MuB 在有或没有 DNA 的情况下形成 ATP 依赖性细丝。我们还鉴定了其 ATP 酶、DNA 结合、蛋白质聚合和 MuA 相互作用活性的关键残基。通过 EM，我们发现 DNA 结合在 MuB 丝的轴孔中。这些发现，加上 MuB 丝大小对链转移效率的影响，得出了一个模型，其中 MuB 施加的对称性使 MuB 丝边界处的 DNA 短暂变形，并导致有利于转座的弯曲 DNA。 3b) Hermes 蛋白是 hAT 转座子超家族的成员，该超家族在许多真核物种中具有活跃的代表，包括 McClintock 的原型 Ac 移动遗传元件。我们的同事确定了 Hermes 转座酶-DNA 复合物的晶体结构，结果表明 Hermes 形成八聚体，并且每个单体都结合了一个切割的转座子末端。我们贡献了 EM 分析，定位了晶体中不可见的 BED 域，即排列不良的区域 (3)。总体情况是催化单元是二聚体：然而，只有八聚体在体内具有活性。这表明它们提供了关键的多个特异性 DNA 结合域，可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。这种不寻常的结构解释了 hAT 转座子末端二分 DNA 识别的基础，为转座子末端不对称提供了基本原理，并展示了八聚体如何提供多个相互作用位点，使转座酶能够在染色体 DNA 海洋中定位其转座子末端。 3）病毒衣壳状纳米室，可储存铁并保护细菌免受氧化应激。 活细胞划分材料和酶促反应以提高其代谢效率。 真核生物使用膜结合的细胞器，而细菌和古细菌主要依赖蛋白质结合的纳米区室。 封装蛋白构成了最近发现的一类纳米室，广泛存在于细菌和古细菌中。迄今为止，它们的功能尚不清楚。我们对黄色粘球菌的封装蛋白纳米室的结构进行了表征，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)。由于该项目涉及铁资源的细菌管理，因此该项目与我们去年报道的涉及奈瑟菌吸收铁的分子机器相吻合。 M. xanthus 的纳米区室由具有内部内容物的蛋白质壳组成。它具有一个外壳蛋白（EncA，32.5 kDa）和三个内部蛋白（EncB，17 kDa；EncC，13 kDa；EncD，11 kDa）。 使用冷冻电子显微镜，我们确定大肠杆菌中表达的 EncA 自组装成直径 32 nm（内径 26 nm）的二十面体壳，该壳由 180 个亚基组成，其折叠首先在噬菌体 HK97 衣壳中观察到。内部蛋白（其中 EncB 和 EncC 具有铁蛋白样结构域）附着在其内表面。 天然纳米室具有致密的富铁核心。从功能上讲，它们类似于铁蛋白（笼状铁储存蛋白），但容量大得多（铁蛋白为 30,000 个铁原子，铁原子为 3,000 个）。 生理数据显示，在营养生长过程中，很少有纳米区室被组装，但它们在饥饿时会增加五倍，通过铁螯合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) 临床受影响儿童中突变的 STING 残基的分子模型。在由 Raphaela Goldbach-Mansky 博士 (NIAMS) 领导的一个联盟进行的一项多方参与的多方面研究中，一组 6 名患者被确定患有早发性全身炎症、血管炎和肺部炎症。这些患者被发现在编码干扰素刺激基因（STING）的 TMEM173 中存在从头功能获得性突变。我们通过使用分子图形和建模将突变残基映射到 STING 二聚体的同源模型上来参与这项工作。有趣的是，突变往往映射在二聚体界面处或附近 (5)。