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: 7601513
• 负责人: Bradley J Nolen
• 金额: $ 0.03万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7601513
• 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.

该副本是利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，这不一定是调查员的机构。 肌动蛋白是一种高度保守的真核蛋白，将聚合分成细丝，形成细胞骨架的主要成分。肌动蛋白折叠由四个子域（总MW约40kD）组成，并由许多其他与肌动蛋白相关的蛋白（ARP）共享，它们具有同一个序列，并且可能与肌动蛋白相同。肌动蛋白和最类似肌动蛋白的ARP结合并水解ATP。在肌动蛋白的情况下，认为磷酸γ的解离会产生构象变化，从而在肌动蛋白的生化特性中产生临界差异，这取决于腺嘌呤核苷酸的结合。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-和ATP-肌动蛋白丝对某些丝状结合蛋白具有不同的亲和力。这些差异被细胞利用以控制肌动蛋白动力学。 ARP3是一种与肌动蛋白相关的蛋白质，是ARP2/3复合物的一部分，ARP2/3复合物是一种蛋白质组件，可响应细胞外信号的肌动蛋白丝核。 In Vito FRET实验表明，ATP结合会导致ARP3的构象变化，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，磷酸解离后核苷酸结合裂缝发生了细微的变化，并且这些变化将传递到DNase结合环中，该环路经历了A alpha Helix Transition的A经历。相反，电子显微镜表明，在细丝的背景下，肌动蛋白在ADP结合时采用开放构象，并且在ATP结合时闭合构型。 ARP2/3复合物的最新晶体结构支持了这一想法。 ATP与ARP3结合导致裂缝关闭，而ADP绑定到开放式裂口。我们试图使用ATP-肌动蛋白（封闭），ADP-肌动蛋白（封闭），Apo-arp3（open），ADP-arp3（open）和ATP-arp3的分子动力学（MD）模拟的ATP-肌动蛋白（封闭），ADP-ARP3（apo-arp3（open），apo-arp3（open），apo-arp3（开放），apo-arp3（apo-arp3（open）（apo-arp3（open）），核苷酸结合和构象之间的关系。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相同，并寻找构象变化。我们的第二轮模拟将涉及在给定结构中改变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或关闭的僵化身体运动，并在裂缝中进行更多的局部变化，并将其与可用的晶体学快照进行比较。通过阐明核苷酸结合与构象之间的关系，我们希望了解肌动蛋白的聚合和解聚的机理，以及通过ARP2/3复合物对肌动蛋白丝的成核。