INVESTIGATION OF THE RELATIONSHIP BETWEEN NUCLEOTIDE BINDING STATE AND CONFORMA

核苷酸结合态与构形关系的研究

• 批准号: 7723250
• 负责人: Bradley J Nolen
• 金额: $ 0.05万
• 依托单位: CARNEGIE-MELLON 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 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7723250
• 关键词: ADP-G-actin; ATP Hydrolysis; ATP-G-actin; Actins; Adenine Nucleotides; Adopted; Affect; Affinity; Binding; Binding Proteins; Biochemical; Cells; Cleaved cell; Complex; Computer Retrieval of Information on Scientific Projects Database; Cytoskeleton; Deoxyribonuclease I; Deoxyribonucleases; Dissociation; Electron Microscopy; Filament; Fluorescence Resonance Energy Transfer; Funding; Grant; Institution; Investigation; Microfilaments; Molecular Conformation; Motion; Nucleotides; Numbers; Property; Protein Binding; Proteins; Research; Research Personnel; Resources; Running; Signal Transduction; Source; Structure; Thinking; United States National Institutes of Health; alpha helix; depolymerization; extracellular; inorganic phosphate; molecular dynamics; monomer; polymerization; research study; response; simulation

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. Actin is a highly conserved eukaryotic protein which polymerizes into filaments that form a major component of the cytoskeleton. The actin fold consists of four subdomains (a total MW of about 40kD) and is shared by a number of other actin-related proteins (Arps), which have homolgous sequences and presumably the same fold as actin. Actin and the most actin-like Arps bind and hydrolyze ATP. In the case of actin, the dissociation of the gamma phosphate is thought to produce a conformational change which creates critical differences in the biochemical properties of actin, depending on which adenine nucleotide is bound. ADP-actin and ATP-actin monomers, for instance, show markedly different polymerization properties, and ADP- and ATP-actin filaments have different affinities for certain filament binding proteins; these differences are exploited by the cell in order to control actin dynamics. Arp3 is an actin-related protein which is part of the Arp2/3 complex, a protein assembly that nucleates actin filaments in response to extracellular signals. In vito FRET experiments have shown that ATP-binding causes a conformational change in Arp3 that is necessary for the function of the complex. Crystal structures of actin with either ADP or ATP bound have shown that subtle changes occur in the nucleotide binding cleft after phosphate dissociation, and that these changes are relayed to the DNAse binding loop, which undergoes a unfolded to alpha helix transition. In contrast, electron microscopy showed that in the context of the filament, actin adopts an open conformation when ADP is bound and a closed conformation when ATP is bound. Recent crystal structures of Arp2/3 complex supported this idea; ATP binding to Arp3 caused the cleft to close, while ADP bound to an open cleft. We seek to understand the relationship between nucleotide-binding and conformation using the available crystal structures of ATP-actin (closed), ADP-actin (closed), apo-Arp3 (open), ADP-arp3 (open), and ATP-arp3 in molecular dynamics (MD) simulations. In our first set of simulations we will keep the nucleotide-binding state the same as in the crystal structure and look for conformational changes. Our second round of simulations will involve changing the nucleotide binding state in a given structure and running a 5ns simulation to determine how changing the nucleotide affects conformation. We will look for rigid body motions that result in cleft opening or closing and for more local changes in the cleft and compare these to the available crystallographic snapshots. By clarifying the relationship between nucleotide-binding and conformation, we hope to understand both the mechanism of polymerization and depolymerization of actin and nucleation of actin filaments by Arp2/3 complex.

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。 肌动蛋白是一种高度保守的真核蛋白质，它聚合成细丝，形成细胞骨架的主要组成部分。肌动蛋白折叠由四个子结构域组成（总分子量约为 40kD），由许多其他肌动蛋白相关蛋白 (Arps) 共享，这些蛋白具有同源序列，并且可能与肌动蛋白具有相同的折叠。肌动蛋白和最像肌动蛋白的 Arps 结合并水解 ATP。就肌动蛋白而言，γ磷酸的解离被认为会产生构象变化，从而根据所结合的腺嘌呤核苷酸，在肌动蛋白的生化特性上产生关键差异。例如，ADP-肌动蛋白和ATP-肌动蛋白单体表现出明显不同的聚合特性，并且ADP-肌动蛋白和ATP-肌动蛋白丝对某些丝结合蛋白具有不同的亲和力；细胞利用这些差异来控制肌动蛋白动力学。 Arp3 是一种肌动蛋白相关蛋白，是 Arp2/3 复合体的一部分，Arp2/3 复合体是一种响应细胞外信号而使肌动蛋白丝成核的蛋白组装体。体外 FRET 实验表明，ATP 结合会导致 Arp3 发生构象变化，这对于复合物的功能是必需的。与 ADP 或 ATP 结合的肌动蛋白的晶体结构表明，磷酸盐解离后，核苷酸结合裂隙中会发生微妙的变化，并且这些变化会传递到 DNAse 结合环，而 DNAse 结合环会经历展开到 α 螺旋的转变。相比之下，电子显微镜显示，在丝状体中，肌动蛋白在 ADP 结合时采用开放构象，在 ATP 结合时采用闭合构象。最近的 Arp2/3 复合物晶体结构支持了这一想法； ATP 与 Arp3 结合导致裂口闭合，而 ADP 与开放裂口结合。我们试图利用 ATP-actin（闭合）、ADP-actin（闭合）、apo-Arp3（开放）、ADP-arp3（开放）和 ATP-arp3 的可用晶体结构来了解核苷酸结合和构象之间的关系分子动力学 (MD) 模拟。在我们的第一组模拟中，我们将保持核苷酸结合状态与晶体结构中相同，并寻找构象变化。我们的第二轮模拟将涉及改变给定结构中的核苷酸结合状态并运行 5ns 模拟以确定改变核苷酸如何影响构象。我们将寻找导致裂缝打开或关闭的刚体运动，以及裂缝中更多的局部变化，并将这些与可用的晶体快照进行比较。通过阐明核苷酸结合和构象之间的关系，我们希望了解肌动蛋白的聚合和解聚机制以及Arp2/3复合物对肌动蛋白丝的成核机制。