INVESTIGATION OF THE RELATIONSHIP BETWEEN NUCLEOTIDE BINDING STATE AND CONFORMA

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 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亚域组成(总分子量约40kD)，并与许多其他肌动蛋白相关蛋白(ARP)共享，这些ARP具有同源序列，推测与肌动蛋白具有相同的折叠。肌动蛋白和最类似肌动蛋白的Arp结合并水解三磷酸腺苷。在肌动蛋白的情况下，伽马磷酸的解离被认为会产生构象变化，从而导致肌动蛋白的生化性质产生关键的差异，这取决于结合的腺嘌呤核苷酸。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性能，并且ADP-和ATP-肌动蛋白细丝对某些细丝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的动态。Arp3是一种肌动蛋白相关蛋白，是Arp2/3复合体的一部分，Arp2/3复合体是一种对细胞外信号做出反应而使肌动蛋白细丝有核的蛋白质组合。在体外FRET实验表明，ATP结合导致Arp3的构象变化，这是复合体功能所必需的。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解发生了细微的变化，这些变化被传递到DNA酶结合环，经历了一个未折叠到α螺旋的转变。相反，电子显微镜显示，在微丝的背景下，当ADP结合时，肌动蛋白采用开放构象，当ATP结合时，肌动蛋白采用闭合构象。最近Arp2/3复合体的晶体结构支持这一观点；ATP与Arp3结合导致裂隙闭合，而ADP与开放裂隙结合。我们试图通过分子动力学(MD)模拟中的ATP-肌动蛋白(闭合)、ADP-肌动蛋白(闭合)、apo-Arp3(开放)、ADP-arp3(开放)和ATP-arp3的晶体结构来理解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中的相同，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5 ns模拟来确定改变核苷酸对构象的影响。我们将寻找导致裂隙打开或闭合的刚体运动，以及裂隙中更多的局部变化，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与构象的关系，我们希望了解肌动蛋白的聚合和解聚机制，以及Arp2/3复合体对肌动蛋白细丝的成核作用。

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.