Manipulating Signaling Proteins for Target Binding and Recognition

描述(申请人提供)：这项建议的长期目标是了解钙调蛋白(CaM)如何在体外从数百个潜在结合靶点(CaMBT)中进行选择的机制，以便开发新的策略来指导体内特定结合伙伴的选择。鉴于CaM在调节细胞周期和细胞凋亡中的作用，这些策略很可能导致调节信号通路的能力，从而抑制细胞的不受控制的生长或诱导细胞凋亡。本研究的目的是利用计算、实验和生物信息学相结合的方法，表征CaM的不同结构状态，并确定细胞内因素(例如，钙离子结合和大分子拥挤)如何影响其靶标结合和识别。我们的假设是，在CaM中存在一种序列-结构-功能-环境关系，影响与其相互作用的特定类型的靶。这项研究的基本原理是，一旦我们表征了CaM在类似细胞的环境中结合和识别的自由能格局及其随各种因素的反应动力学，我们就可以设计能够控制选定CaMBT的形成的策略，以及扩展基于网络的CaM/CaMBT复合体的数据库，该数据库另外用这些新数据和细胞定位来注释。我们将通过追求以下三个具体目标来验证我们的中心假设：(1)在与细胞内发现的溶剂条件相关的溶剂条件下，通过计算机模拟表征目标结合和识别CaM的自由能图谱。(2)在与细胞内发现的构象相关的溶剂条件下，通过实验确定CaM填充的构象状态，并研究状态的时空分布变化如何影响钙离子和靶的结合。(3)用生物信息学方法诠释CaM/CaMBT复合体在不同细胞条件下的结构构象。这项研究具有创新性，因为它结合了多尺度分子动力学模拟、光谱学和生物信息学的方法，表征了结合和识别的CaM结构，并诠释了CaM在细胞中的生物学相关性。这一贡献意义重大，因为这是一系列研究的第一步，预计将揭示有关CaM结合的选择和操作的新策略，其中可能会指导人们在细胞分裂和凋亡之间的选择。这项研究具有重要意义，因为关于细胞内CaM特性控制的知识有望扩大对其他依赖构象灵活性发挥功能的信号蛋白的基本理解。这种机械的洞察力很可能导致细胞周期控制的创新方法，或许还会导致癌症治疗的治疗策略。

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to understand the mechanism of how calmodulin (CaM) selects among hundreds of its potential binding targets (CaMBTs) in vitro, so that novel strategies can be developed to direct the selection of specific binding partners in vivo. Given CaM's role in the regulation of the cell cycle and apoptosis, such strategies may well lead to the ability to regulate signaling pathways that allow suppression of uncontrolled cellular growth or to induce apoptosis. The objective of the present proposal is to characterize different structural states of CaM and to determine how intracellular factors (e.g., Ca2+ binding and macromolecular crowding) influence its target-binding and recognition using a combined computational, experimental and bioinformatics approach. Our hypothesis is that there is a sequence-structure-function- environment relationship in CaM that influences the specific types of targets with which it interacts. The rationale for the proposed research is that once we characterize the free energy landscape of binding and recognition and their kinetics for CaM in response to various factors in a cell-like environment, we can design strategies that are able to control the formation of selected CaMBTs as well as to expand a web-based database of CaM/CaMBT complexes additionally annotated with this new data and by cellular localization. We will test our central hypothesis by pursuing the following three specific aims: (1) Characterize the free energy landscape of target binding and recognition of CaM by computer simulations in solvent conditions relevant to those found inside cells. (2) Experimentally determine the conformational states populated by CaM in solvent conditions relevant to those found inside cells and examine how the altered spatiotemporal distribution of states influences Ca2+- and target-binding. (3) Annotate the structural conformation of CaM/CaMBT complexes in different cellular conditions with bioinformatics approaches. The research proposed is innovative because it combines the approaches of multiscale molecular dynamics simulation, spectroscopy, and bioinformatics in the characterization of CaM structures for binding and recognition and the annotation of CaM's biological relevance in a cell. The contribution is significant because it is the first step in a continuum of research that is expected to reveal new strategies regarding the selection and manipulation of CaM binding where one could potentially guide choices between, for example, cell division and apoptosis. The proposed research is of significance because the knowledge gained regarding the control of the properties of CaM in a cell is expected to expand the fundamental understanding of other signaling proteins that depend on conformational flexibility for their function. Such mechanistic insights could well lead to innovative approaches in cell cycle control and perhaps therapeutic strategies for cancer treatment.