MOLECULAR COMPLEXES OF THE ACTIN CYTOSKELETON

肌动蛋白细胞骨架的分子复合物

• 批准号: 7721301
• 负责人: ROBERTO DOMINGUEZ
• 金额: $ 1.45万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7721301
• 关键词: ATP phosphohydrolase; Actin-Binding Protein; Actinin; Actins; Adaptor Signaling Protein; Affinity; Amoeba genus; Binding; C-terminal; Calmodulin; Cell Shape; Cell physiology; Cell-Matrix Junction; Cells; Collaborations; Complex; Computer Retrieval of Information on Scientific Projects Database; Cytokinesis; Cytoskeletal Proteins; Cytoskeleton; DNA Sequence Rearrangement; Data Collection; Dictyosteliida; Dictyostelium; Dictyostelium discoideum; Dystrophin; Elongation Factor; Endocytosis; Eukaryotic Cell; Excision; F-Actin; Fibroblasts; Filament; Filopodia; Focal Adhesions; Freezing; Funding; G Actin; Glycerol; Goals; Grant; Head; Helix (Snails); Home environment; Human; Hydrolysis; Infection; Institution; Kidney Failure; Link; Lipids; Liquid substance; Location; Macromolecular Complexes; Malaria; Mediating; Medical; Membrane; Methods; Microfilaments; Minus End of the Actin Filament; Modeling; Molecular; Molecular Structure; N-terminal; Nature; Neoplasm Metastasis; Nitrogen; Nucleotides; Numbers; Nutrient; Parasites; Phospholipids; Phosphorylation; Plasmodium; Play; Point Mutation; Preparation; Proteins; Rate; Reproduction spores; Research; Research Personnel; Resolution; Resources; Roentgen Rays; Role; Signal Transduction; Signaling Protein; Source; Specificity; Spectrin; Staging; Starvation; Stress; Stress Fibers; Structure; Surface; Surface Plasmon Resonance; Tail; Thinking; Time; Toxoplasma gondii; United States National Institutes of Health; Vitamin D-Binding Protein; ZYX gene; alpha Actinin; base; calponin; cell motility; dimer; ear helix; extracellular; insight; medical schools; migration; monomer; mutant; pathogen; profilin; protein crosslink; protein protein interaction; retinal rods; scaffold; toxofilin; ATP phosphohydrolase; Actin-Binding Protein; Actinin; Actins; Adaptor Signaling Protein; Affinity; Amoeba genus; Binding; C-terminal; Calmodulin; Cell Shape; Cell physiology; Cell-Matrix Junction; Cells; Collaborations; Complex; Computer Retrieval of Information on Scientific Projects Database; Cytokinesis; Cytoskeletal Proteins; Cytoskeleton; DNA Sequence Rearrangement; Data Collection; Dictyosteliida; Dictyostelium; Dictyostelium discoideum; Dystrophin; Elongation Factor; Endocytosis; Eukaryotic Cell; Excision; F-Actin; Fibroblasts; Filament; Filopodia; Focal Adhesions; Freezing; Funding; G Actin; Glycerol; Goals; Grant; Head; Helix (Snails); Home environment; Human; Hydrolysis; Infection; Institution; Kidney Failure; Link; Lipids; Liquid substance; Location; Macromolecular Complexes; Malaria; Mediating; Medical; Membrane; Methods; Microfilaments; Minus End of the Actin Filament; Modeling; Molecular; Molecular Structure; N-terminal; Nature; Neoplasm Metastasis; Nitrogen; Nucleotides; Numbers; Nutrient; Parasites; Phospholipids; Phosphorylation; Plasmodium; Play; Point Mutation; Preparation; Proteins; Rate; Reproduction spores; Research; Research Personnel; Resolution; Resources; Roentgen Rays; Role; Signal Transduction; Signaling Protein; Source; Specificity; Spectrin; Staging; Starvation; Stress; Stress Fibers; Structure; Surface; Surface Plasmon Resonance; Tail; Thinking; Time; Toxoplasma gondii; United States National Institutes of Health; Vitamin D-Binding Protein; ZYX gene; alpha Actinin; base; calponin; cell motility; dimer; ear helix; extracellular; insight; medical schools; migration; monomer; mutant; pathogen; profilin; protein crosslink; protein protein interaction; retinal rods; scaffold; toxofilin

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The actin cytoskeleton of eukaryotic cells undergoes constant remodeling, resulting in various types of cytoskeletal structures, such as filopodia, lamellipodia, stress fibers and focal adhesions [1]. These dynamic structures play essential roles in many cellular functions, including motility, cytokinesis, fibroblast migration and endocytosis. Actin, a 42-kDa ATPase and the most abundant protein in eukaryotic cells, is the primary component of the cytoskeleton. Actin exists is two forms, a monomeric (G-actin) form and a filamentous (F-actin) form. F-actin is most commonly described as a double helix of head-to-tail interacting actin monomers [2]. The filament is structurally and kinetically asymmetric. In addition to nucleotide hydrolysis by actin, the time, location, association rate and specific type of cytoskeletal structure are determined by signaling proteins as well as by the actions of a vast number of actin-binding proteins (ABPs). The main goal of our research is to understand how protein-protein interaction networks bring together cytoskeleton scaffolding, nucleation and elongation factors to accomplish cellular functions. To achieve this goal we use a combination of structural and biophysical methods. The aim of this proposal is to request X-ray data collection time to obtain high-resolution structures of macromolecular complexes of the actin cytoskeleton. Time is requested for data collection on the following projects: 1. Study of a complex of a-actinin and zyxin. a-Actinin is as an antiparallel homodimer. Each subunit consists of an N-terminal actin-binding domain (ABD), composed of tandem calponin-homology (CH) domains, followed by four spectrin repeats, and a C-terminal calmodulin-like (CaM) domain. a-Actinin was originally described as an actin-crosslinking protein, but it has now become evident that a-actinin possesses an exceptionally large number of molecular partners, most of which are focal adhesion and dense bodies-associated proteins [3-5]. The interactions of a-actinin with these partners are typically mediated by the spectrin repeat region [6]. The structure of this region (also known as the "rod" domain) has been determined and shown to form a twisted antiparallel dimer with a conserved acidic surface, which is thought to play a role in target recognition [7]. In particular, this region is believed to mediate the interaction of a-actinin with the focal-adhesion protein zyxin. To understand this interaction, which could serve as a paradigm for many of a-actinins interactions in focal adhesion complexes, we co-crystallized an a-actinin-zyxin complex. The complex consists of the spectrin repeat dimer of a-actinin and an N-terminal fragment of zyxin, containing a basic sequence, which is thought to bind in the acidic patch of the spectrin repeats. The total mass of the complex is ~120 kDa. We have obtained crystals in two different forms. The crystals diffract the X-rays to low resolution (~4¿ ) using out home source. We have many crystals of the two forms frozen in glycerol as cryo-protectant. The crystals were stored in liquid nitrogen. We now request data collection time to determine the high-resolution structure of this complex. 2. Structure of the actin-binding domain of ¿¿-actinin-4. Alpha-actinin-4 is ubiquitously expressed. In collaboration with the group of Martin Pollak at the Harvard Medical School, we are studying point mutations in ABD of a-actinin-4 that cause autosomal-dominant kidney failure. We have crystallized these mutants and would like to request data collection time to determine their high-resolution structures. The structures are expected to reveal the molecular basis for the compromised interactions of the mutants with actin. Crystals, diffracting the X-rays to relatively high resolution are available, and have been frozen. 3. Structure of the IMD of missing-in-metastasis with membrane phospholipids. Missing-in-metastasis (MIM) is a multi-domain adaptor protein that links extracellular signals to actin cytoskeleton remodeling. MIM contains independent F- and G-actin-binding domains, consisting respectively of an N-terminal 250-aa IMD and a C-terminal WH2. We have recently determined the crystal structure of this domain [8]. The structure is related to that of the BAR domain. Like the BAR domain, the IMD has been implicated in membrane binding. Yet, comparison of the structures reveals that the membrane-binding surfaces of the two domains have opposite curvatures, which may determine the type of curvature of the interacting membrane. To understand how the IMD senses and interacts with membrane phospholipids, we have examined the specificity and affinities of the IMD for different types of lipids using surface plasmon resonance (SPR). We have also co-crystallized the IMD with some of these lipids and would like to request data collection time to determine the structures. 4. Structure of the toxofilin-actin complex. Many human pathogens exploit the actin cytoskeleton of host cells for infection, including Toxoplasma gondii, an apicomplexan parasite related to Plasmodium the agent of malaria. One of the most abundantly expressed proteins of T. gondii is toxofilin, a monomeric actin-binding protein involved in invasion. We are studying the interaction of toxofilin with actin using biophysical and structural approaches. Our studies indicate that toxofilin may bind up to three actin monomers. We have recently determined the crystal structure of toxofilin with actin in a 1:1 complex (Lee et al., in preparation). We have now also crystallized a 1:2 toxofilin:actin complex. We would like to request data collection time to determine this new structure. The structures will provide important insights about the toxofilin-actin interaction, which may be of medical significance to develop treatments against apicomplexan parasites. 5. Conformational changes in Dictyostelium actin upon phosphorylation. Upon removal of nutrients, the amoebae of the cellular slime mold Dictyostelium discoideum differentiate into dormant spores, which survive under starvation stress. At this stage, high levels of actin phosphorylation on Tyr-53 occur and correlate closely with rearrangements of the actin cytoskeleton and changes in cell shape [9, 10]. Tyr-53 phosphorylation substantially inhibits nucleation and elongation from the pointed ends of actin filaments and reduces the elongation from the barbed ends. To understand the molecular mechanism and actin conformational change upon phosphorylation, high-resolution structures of both the phosphorylated and unphosphorylated forms of Dictyostelium actin must be determined. In collaboration with Ed Korn at the NIH, we have crystallized these two phosphorylation states of Dictyostelium actin in complexes with both vitamin-D binding protein (DBP) and profilin. We request data collection time to determine the structures. References 1. Pollard, T.D., and Borisy, G.G. (2003). Cellular motility driven by assembly and disassembly of actin filaments. Cell 112, 453-465. 2. Holmes, K.C., Popp, D., Gebhard, W., and Kabsch, W. (1990). Atomic model of the actin filament. Nature 347, 44-49. 3. Otey, C.A., and Carpen, O. (2004). Alpha-actinin revisited: a fresh look at an old player. Cell Motil Cytoskeleton 58, 104-111. 4. Broderick, M.J., and Winder, S.J. (2005). Spectrin, alpha-actinin, and dystrophin. Adv Protein Chem 70, 203-246. 5. Zaidel-Bar, R., Cohen, M., Addadi, L., and Geiger, B. (2004). Hierarchical assembly of cell-matrix adhesion complexes. Biochem Soc Trans 32, 416-420. 6. Djinovic-Carugo, K., Gautel, M., Ylanne, J., and Young, P. (2002). The spectrin repeat: a structural platform for cytoskeletal protein assemblies. FEBS Lett 513, 119-123. 7. Ylanne, J., Scheffzek, K., Young, P., and Saraste, M. (2001). Crystal structure of the alpha-actinin rod reveals an extensive torsional twist. Structure 9, 597-604. 8. Lee, S.H., Kerff, F., Chereau, D., Ferron, F., Klug, A., and Dominguez, R. (2007). Structural Basis for the Actin-Binding Function of Mi

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 真核细胞的肌动蛋白细胞骨架会经历恒定的重塑，从而导致各种类型的细胞骨架结构，例如丝状，层状脂蛋白，层状脂蛋白，应激纤维和局灶性粘合剂[1]。这些动态结构在许多细胞功能中起着至关重要的作用，包括运动，细胞因子，成纤维细胞迁移和内吞作用。肌动蛋白是42 kDa ATPase，是真核细胞中最丰富的蛋白质，是细胞骨架的主要成分。肌动蛋白的存在是两种形式，一种单体（G-肌动蛋白）和一种丝状（F-肌动蛋白）形式。 F-肌动蛋白最常被描述为头到尾相互作用肌动蛋白单体的双螺旋[2]。细丝在结构和动力学上是不对称的。除肌动蛋白通过核苷酸水解外，时间，位置，关联速率和特定类型的细胞骨架结构还通过信号蛋白以及大量肌动蛋白结合蛋白（ABP）的作用来确定。 我们研究的主要目的是了解蛋白质 - 蛋白质相互作用网络如何将细胞骨架脚手架，成核和伸长因子汇总在一起以实现细胞功能。为了实现这一目标，我们结合了结构和生物物理方法。该建议的目的是要求X射线数据收集时间以获得肌动蛋白细胞骨架的大分子复合物的高分辨率结构。 要求在以下项目上收集数据的时间： 1。研究A-肌动蛋白和Zyxin的复合物。 A-肌动蛋白是反平行同型二聚体。每个亚基由由串联calponin-sology（CH）结构域组成的N末端肌动蛋白结合结构域（ABD）组成，其次是四个光谱蛋白重复序列​​和C端钙调蛋白样（CAM）类结构域。 A-肌动蛋白最初被描述为肌动蛋白 - 跨链接蛋白，但现在已成为A-肌动蛋白具有异常数量的分子伴侣的证据，其中大多数是局灶性粘合剂和密集的身体相关蛋白[3-5]。 A-肌动蛋白与这些伴侣的相互作用通常由Spectrin重复区域介导[6]。该区域的结构（也称为“杆”结构域）已被确定，并显示出具有保守的酸性表面的扭曲的反平行二聚体，该二聚体被认为在目标识别中起作用[7]。特别是，该区域被认为可以介导a-肌动蛋白与局灶性粘附蛋白Zyxin的相互作用。 为了理解这种相互作用，它可以用作焦点粘合剂复合物中许多A-肌动蛋白相互作用的范式，我们共结晶了A-肌动蛋白 - Zyxin复合物。该复合物由a-肌动蛋白的光谱重复二聚体和二末端片段的Zyxin片段，其中包含基本序列，该序列被认为在光谱重复的酸性贴片中结合。该复合物的总质量约为120 kDa。我们以两种不同形式获得了晶体。晶体使用外源来源衍射X射线至低分辨率（〜4？）。我们有许多在甘油中冷冻的两种形式的晶体作为冷冻保护剂。晶体存储在液氮中。现在，我们请求数据收集时间以确定该复合物的高分辨率结构。 2。-actinin-4的肌动蛋白结合结构域的结构。 α-Actinin-4无处不在。在哈佛医学院与马丁·波拉克（Martin Pollak）的小组合作，我们正在研究A-Actinin-4的ABD中的点突变，该突变导致常染色体显性肾衰竭。我们已经结晶了这些突变体，并希望请求数据收集时间以确定其高分辨率结构。这些结构有望揭示突变体与肌动蛋白的相互作用的分子基础。晶体，将X射线衍射到相对较高的分辨率可用，并且已被冷冻。 3。与膜磷脂失踪中的IMD结构。缺失 - 中心（MIM）是一种多域衔接蛋白，将细胞外信号与肌动蛋白细胞骨架重塑联系起来。 MIM包含独立的F-和G-肌动蛋白结合域，分别由N末端250-AA IMD和C端WH2组成。我们最近确定了该结构域的晶体结构[8]。该结构与条形域的结构有关。像条形域一样，IMD已在膜结合中隐含。然而，对结构的比较表明，这两个结构域的膜结合表面具有相反的曲率，这可能决定了相互作用的膜的曲率类型。 为了了解IMD的感觉和与膜磷脂的相互作用，我们使用表面等离子体共振（SPR）检查了IMD对不同类型脂质的特异性和亲和力。我们还与其中一些脂质共结合了IMD，并希望请求数据收集时间以确定结构。 4。毒素 - 肌动蛋白复合物的结构。许多人类病原体探索宿主细胞的肌动蛋白细胞骨架感染，包括弓形虫弓形虫，弓形虫是与疟疾疟原虫相关的疟原虫寄生虫。 T. gondii的最丰富表达的蛋白质之一是毒素，这是一种参与侵袭的单体肌动蛋白结合蛋白。 我们正在研究使用生物物理和结构方法研究毒素与肌动蛋白的相互作用。我们的研究表明，毒素可能结合到三个肌动蛋白单体。我们最近在1：1复合物中用肌动蛋白确定了毒素蛋白的晶体结构（Lee等人，制备中）。现在，我们还结晶了1：2毒素：肌动蛋白复合物。我们想请求数据收集时间以确定这种新结构。这些结构将提供有关毒素 - 肌动蛋白相互作用的重要见解，这对于开发针对Apicomplexan寄生虫的治疗可能具有医学意义。 5.磷酸化时dictyostelium肌动蛋白的概念变化。去除营养成分后，细胞粘液霉菌的变形虫分化为休眠孢子，在饥饿应激下存活。在此阶段，Tyr-53上的高水平肌动蛋白磷酸化发生，并与肌动蛋白细胞骨架的重排和细胞形状变化密切相关[9，10]。 Tyr-53磷酸化基本上抑制了肌动蛋白丝的尖端末端的成核和伸长，并减少了带刺的末端的伸长。为了了解磷酸化时的分子机制和肌动蛋白会议变化，必须确定磷酸化和未磷酸化形式的肌动蛋白的高分辨率结构。在NIH与Ed Korn合作，我们已经在具有维生素-D结合蛋白（DBP）和叶酸蛋白的复合物中结晶了dictyostelium肌动蛋白的这两个磷酸化状态。我们要求数据收集时间以确定结构。 参考 1。Pollard，T。D.和Borisy，G。G.（2003）。由肌动蛋白丝的组装和拆卸驱动的细胞运动。单元112，453-465。 2。Holmes，K。C.，Popp，D.，Gebhard，W。和Kabsch，W。（1990）。肌动蛋白丝的原子模型。大自然347，44-49。 3。Otey，C.A。和Carpen，O。（2004）。重新审视α-肌动蛋白：对一位旧玩家的崭新观察。细胞Motil细胞骨架58，104-111。 4。Broderick，M.J。和S.J. Winder （2005）。光谱，α-肌动蛋白和肌营养不良蛋白。 ADV蛋白质化学70，203-246。 5。Zaidel-Bar，R.，Cohen，M.，Addadi，L。和Geiger，B。（2004年）。细胞矩阵粘合剂复合物的分层组件。 Biochem Soc Trans 32，416-420。 6。Djinovic-Carugo，K.，Gautel，M.，Ylanne，J。和Young，P。（2002）。光谱重复：细胞骨架蛋白质组件的结构平台。 FEB Lett 513，119-123。 7。Ylanne，J.，Scheffzek，K.，Young，P。和Saraste，M。（2001）。 α-肌动蛋白棒的晶体结构显示出广泛的扭转扭曲。结构9，597-604。 8。Lee，S.H.，Kerff，F.，Chereau，D.，Ferron，F.，Klug，A。和Dominguez，R。（2007）。 MI的肌动蛋白结合功能的结构基础