Computational studies of Arp2/3 activation and deactivation

 描述(申请人提供)：真核细胞依赖肌动蛋白纤维执行大量不同的功能，包括细胞分裂、黏附和运动。它们的形成受到细胞的仔细调控，细胞使用各种机制来控制具有空间和时间特异性的聚合。这样的系统是由一个复杂的蛋白质相互作用网络控制的。该系统在细胞功能中的重要性意味着它在生物功能障碍中具有相当大的重要性。任何成分的突变都可能导致遗传病。病原体也可以滥用这些系统，通过几种途径帮助感染健康细胞。癌细胞转移发生在细胞从肿瘤体内移开时，这是肌动蛋白网络机制上调的结果。更全面地了解肌动蛋白的组装和分解对于人类疾病的治疗至关重要。 该系统的核心成分之一是肌动蛋白相关蛋白复合体Arp2/3，它由两个蛋白Arp2和Arp3以及5个辅因子蛋白ArpC1-5组成。Arp2/3与先前存在的肌动蛋白细丝结合，形成子丝的起点，子丝以70度角分叉。有证据表明，Arp2/3复合体默认处于不活跃状态，即在没有与其他细胞成分相互作用的情况下，它不会启动分支。相反，实验表明了使复合体失活的机制，促进了去分支。由于这种蛋白质组装的大小和复杂性，到目前为止，研究Arp2/3系统的计算研究相对较少。我建议用计算方法研究Arp2/3的激活/失活的三个方面，以促进我们对这些蛋白质最近的实验结果的理解。 首先，我将研究Arp2/3与肌动蛋白界面的离子结合效应。最近的WOR表明，在Arp2/3与肌动蛋白结合的界面上可能存在特定的位置，从而开始一个新的阳离子可以结合的细丝。我们将检验这样的假设，即在这些位置上有离子可以稳定界面并促进聚合。其次，我将研究被称为成核促进因子(NPF)的特殊辅助因子如何与Arp2/3合作，通过结合Arp2/3和肌动蛋白将它们结合在一起来促进分支，并导致Arp2/3的构象变化，使其能够与新的肌动蛋白单体形成良好的界面。第三，我将研究Arp2/3构象对其将ATP转化为ADP的能力的影响，ADP是促进去分支的调控之一。 这些研究将在原子水平上加深我们对细胞用来控制肌动蛋白原纤维网络的关键机制的理解。我的工作将带来方法论上的改进，我得到的数据将有助于解释以前的实验以及新实验的设计。

 DESCRIPTION (provided by applicant): Eukaryotic cells depend on actin fibrils to perform a large number of diverse functions, including cell division, adhesion, and movement. Their formation is carefully regulated by the cells, which employ a variety of mechanisms to control polymerization with spatial and temporal specificity. Such a system is governed by a complex network of protein interactions. The importance of this system in cellular function means it has a commensurately large importance in biological malfunction. Mutations in any component can result in genetic disease. Pathogens can also abuse these systems to aid in infection of healthy cells through several pathways. Cancer cell metastasis occurs when cells move themselves away from a tumorous body, and this results from up-regulation of the actin network machinery. A fuller understanding of actin assembly and disassembly is crucial for the treatment of human disease. One of the central components of this system is the actin-related protein complex Arp2/3, which consists of two proteins, Arp2 and Arp3, as well as 5 cofactor proteins ArpC1-5. Arp2/3 binds to a preexisting actin filament and forms the beginnings of a daughter filament, which branches off at a 70º angle. There is evidence that the Arp2/3 complex is by default in an inactive state, i.e.it does not initiate branching without interaction with other cellular components. Conversely, experiments suggest mechanisms that deactivate the complex, promoting debranching. Due to the size and complexity of this protein assembly, relatively few computational studies to date have investigated the Arp2/3 system. I propose to study three aspects of the activation/deactivation of Arp2/3 using computational methodologies to advance our understanding of recent experimental results on these proteins. First, I will study the effect of ion binding at the interface between Arp2/3 and actin. Recent wor suggests that there may be specific sites at the interface where Arp2/3 binds actin to begin a new filament where cations can bind. We will test the hypothesis that having ions in these sites stabilizes the interface and promotes polymerization. Second, I will study the way in which special cofactors called nucleation promoting factors (NPFs) work with Arp2/3 to promote branching by binding Arp2/3 and actin to bring them together and to cause a conformational change in Arp2/3 that allows it to form a good interface with new actin monomers. Third, I will study the effect on Arp2/3 conformation on its ability to transform ATP to ADP, which is one of the controls that promotes debranching. These studies will enhance our understanding at the atomic level of key mechanisms used by cells to control networks of actin fibrils. My work will produce methodological improvements, and my resulting data will aid in the interpretation of previous experiments as well as the design of new ones.