MOLECULAR COMPLEXES OF THE ACTIN CYTOSKELETON

肌动蛋白细胞骨架的分子复合物

• 批准号: 7598557
• 负责人: ROBERTO DOMINGUEZ
• 金额: $ 1.68万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7598557
• 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; Tyrosine Phosphorylation; 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 ¿-actinin and zyxin. ¿-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. ¿-Actinin was originally described as an actin-crosslinking protein, but it has now become evident that ¿-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 ¿-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 ¿-actinin with the focal-adhesion protein zyxin. To understand this interaction, which could serve as a paradigm for many of ¿-actinins interactions in focal adhesion complexes, we co-crystallized an ¿-actinin-zyxin complex. The complex consists of the spectrin repeat dimer of ¿-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 ¿-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 Missing-in-Metastasis. Structure 15, 145-155. 9. Kishi, Y., Clements, C., Mahadeo, D.C., Cotter, D.A., and Sameshima, M. (1998). High levels of actin tyrosine phosphorylation: correlation with the dormant state of Dictyostelium spores. J Cell Sci 111 (Pt 19), 2923-2932. 10. Liu, X., Shu, S., Hong, M.S., Levine, R.L., and Korn, E.D. (2006). Phosphorylation of actin Tyr-53 inhibits filament nucleation and elongation and destabilizes filaments. Proc Natl Acad Sci U S A 103, 13694-13699.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 真核细胞的肌动蛋白细胞骨架不断重塑，产生各种类型的细胞骨架结构，如丝状伪足、板状伪足、应力纤维和粘着斑等[1]。这些动态结构在许多细胞功能中发挥着重要作用，包括运动、胞质分裂、成纤维细胞迁移和内吞作用。肌动蛋白是一种 42 kDa ATP 酶，是真核细胞中最丰富的蛋白质，是细胞骨架的主要成分。肌动蛋白以两种形式存在，单体（G-肌动蛋白）形式和丝状（F-肌动蛋白）形式。 F-肌动蛋白最常被描述为头尾相互作用的肌动蛋白单体的双螺旋[2]。灯丝在结构和动力学上是不对称的。除了肌动蛋白的核苷酸水解之外，细胞骨架结构的时间、位置、关联率和特定类型由信号蛋白以及大量肌动蛋白结合蛋白（ABP）的作用决定。 我们研究的主要目标是了解蛋白质-蛋白质相互作用网络如何将细胞骨架支架、成核和延伸因子结合在一起以完成细胞功能。为了实现这一目标，我们结合使用结构和生物物理方法。该提案的目的是要求 X 射线数据收集时间以获得肌动蛋白细胞骨架大分子复合物的高分辨率结构。 需要时间收集以下项目的数据： 1. ¿-肌动蛋白和zyxin复合物的研究。 ¿-肌动蛋白是一种反平行同二聚体。每个亚基由一个 N 端肌动蛋白结合结构域 (ABD) 组成，该结构域由串联钙调蛋白同源 (CH) 结构域组成，后面是四个血影蛋白重复序列​​，以及一个 C 端钙调蛋白样 (CaM) 结构域。 ¿-肌动蛋白最初被描述为一种肌动蛋白交联蛋白，但现在已经证明，Ф-肌动蛋白拥有异常大量的分子伴侣，其中大多数是粘着斑和致密体相关蛋白[3-5]。 β-肌动蛋白与这些伙伴的相互作用通常由血影蛋白重复区域介导[6]。该区域（也称为杆结构域）的结构已被确定并显示形成具有保守酸性表面的扭曲反平行二聚体，这被认为在目标识别中发挥作用[7]。特别是，该区域被认为介导β-肌动蛋白与焦点粘附蛋白zyxin的相互作用。 为了理解这种相互作用，它可以作为粘着斑复合物中许多 β-肌动蛋白相互作用的范例，我们共结晶了 β-肌动蛋白-zyxin 复合物。该复合物由 β-辅肌动蛋白的血影蛋白重复二聚体和 zyxin 的 N 末端片段组成，其中包含一个基本序列，该序列被认为结合在血影蛋白重复序列​​的酸性片段中。复合物的总质量约为 120 kDa。我们获得了两种不同形式的晶体。晶体使用外部源将 X 射线衍射至低分辨率 (~4¿)。我们有许多这两种形式的晶体冷冻在甘油中作为冷冻保护剂。将晶体储存在液氮中。我们现在要求收集数据以确定该复合物的高分辨率结构。 2. ¿-actinin-4 肌动蛋白结合域的结构。 Alpha-actinin-4 广泛表达。我们与哈佛医学院的 Martin Pollak 团队合作，正在研究 ¿-actinin-4 的 ABD 点突变，该突变会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来确定它们的高分辨率结构。这些结构有望揭示突变体与肌动蛋白相互作用受损的分子基础。将 X 射线衍射至相对高分辨率的晶体是可用的，并且已被冷冻。 3. 膜磷脂转移缺失的 IMD 结构。 转移缺失 (MIM) 是一种多结构域衔接蛋白，它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 端 250 个氨基酸 IMD 和 C 端 WH2 组成。我们最近确定了该域的晶体结构[8]。该结构与BAR域的结构相关。与 BAR 结构域一样，IMD 与膜结合有关。然而，结构的比较表明，两个域的膜结合表面具有相反的曲率，这可以确定相互作用膜的曲率类型。 为了了解 IMD 如何感知膜磷脂并与之相互作用，我们使用表面等离子共振 (SPR) 检查了 IMD 对不同类型脂质的特异性和亲和力。我们还使 IMD 与其中一些脂质共结晶，并希望请求数据收集时间来确定结构。 4. 弓丝蛋白-肌动蛋白复合物的结构。 许多人类病原体利用宿主细胞的肌动蛋白细胞骨架进行感染，包括弓形虫，一种与疟疾病原体疟原虫相关的顶复门寄生虫。弓形虫表达最丰富的蛋白质之一是弓形虫蛋白，一种参与入侵的单体肌动蛋白结合蛋白。 我们正在使用生物物理和结构方法研究弓丝蛋白与肌动蛋白的相互作用。我们的研究表明，toxofilin 最多可以结合三个肌动蛋白单体。我们最近确定了 1:1 复合物中 toxofilin 与肌动蛋白的晶体结构（Lee 等人，正在准备中）。我们现在还结晶了 1:2 的 toxofilin:actin 复合物。我们希望请求数据收集时间来确定这个新结构。这些结构将提供关于弓丝蛋白-肌动蛋白相互作用的重要见解，这对于开发针对顶复门寄生虫的治疗方法可能具有医学意义。 5. 磷酸化后盘基网柄菌肌动蛋白的构象变化。 一旦营养物质被去除，细胞粘菌盘基网柄菌的变形虫就会分化成休眠孢子，在饥饿应激下存活。在此阶段，Tyr-53 上发生高水平的肌动蛋白磷酸化，并与肌动蛋白细胞骨架的重排和细胞形状的变化密切相关 [9, 10]。 Tyr-53 磷酸化显着抑制肌动蛋白丝尖端的成核和伸长，并减少有刺末端的伸长。为了了解磷酸化时的分子机制和肌动蛋白构象变化，必须确定盘基网柄菌肌动蛋白磷酸化和非磷酸化形式的高分辨率结构。我们与 NIH 的 Ed Korn 合作，将盘基网柄菌肌动蛋白的这两种磷酸化状态结晶化为与维生素 D 结合蛋白 (DBP) 和 profilin 的复合物。我们要求收集数据以确定结构。 参考 1. Pollard, T.D. 和 Borisy, G.G. （2003）。细胞运动由肌动蛋白丝的组装和分解驱动。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。自然 347, 44-49。 3. Otey, C.A. 和 Carpen, O. (2004)。阿尔法肌动蛋白重温：老玩家的新视角。细胞动力细胞骨架 58, 104-111。 4. Broderick, M.J. 和 Winder, S.J. （2005）。血影蛋白、α-肌动蛋白和肌营养不良蛋白。高级蛋白质化学 70, 203-246。 5. Zaidel-Bar, R.、Cohen, M.、Addadi, L. 和 Geiger, B. (2004)。细胞基质粘附复合物的分层组装。生物化学 Soc Trans 32, 416-420。 6. Djinovic-Carugo, K.、Gautel, M.、Ylanne, J. 和 Young, P. (2002)。血影蛋白重复序列​​：细胞骨架蛋白组装的结构平台。 FEBS Lett 513, 119-123。 7. Ylanne, J.、Scheffzek, K.、Young, P. 和 Saraste, M. (2001)。 α-辅肌动蛋白棒的晶体结构揭示了广泛的扭转。结构 9，597-604。 8. Lee, S.H.、Kerff, F.、Chereau, D.、Ferron, F.、Klug, A. 和 Dominguez, R. (2007)。转移缺失的肌动蛋白结合功能的结构基础。结构 15、145-155。 9. Kishi, Y.、Clements, C.、Mahadeo, D.C.、Cotter, D.A. 和 Sameshima, M. (1998)。高水平的肌动蛋白酪氨酸磷酸化：与盘基网柄菌孢子休眠状态的相关性。 《细胞科学杂志》111（第 19 部分），2923-2932。 10. Liu, X.、Shu, S.、Hong, M.S.、Levine, R.L. 和 Korn, E.D. （2006）。肌动蛋白 Tyr-53 的磷酸化会抑制丝的成核和伸长并使丝不稳定。美国国家科学院院报 103, 13694-13699。