Analysis and Design of Coiled Coil Partnering

描述（由申请人提供）：拟议工作的广泛，长期目标是了解蛋白质如何在单个结构域水平上建立相互作用特异性，并在可用于计算预测和设计的模型中捕获这一点。这个问题是人类健康问题的核心。突变蛋白或失调蛋白之间不适当的相互作用可导致疾病，对其发生机制的深入了解非常重要。此外，设计新的蛋白质或肽来特异性抑制天然蛋白质复合物将为未来的治疗提供一条途径。相互作用特异性问题可以通过关注一个普遍存在但结构简单的蛋白质-蛋白质相互作用基序来简化：α -螺旋线圈。盘绕线圈存在于所有物种的蛋白质组中，并与多种功能相关。它们的序列和结构特性使它们易于进行计算分析，并且也便于使用生物物理方法进行实验研究。所提出的研究包括收集系统的实验相互作用数据，并使用它来开发和测试各种预测相互作用的计算技术。计算方法也将开发用于设计盘绕状肽，并提出了实验来表征设计。这项建议的具体目标是：(1)测试和改进预测螺旋结构、能量和相互作用的方法；(2)扩展现有螺旋设计能力，以治疗更广泛的靶标；(3)在动物物种中广泛测量bZIP螺旋相互作用，并开发蛋白质-蛋白质相互作用特异性如何进化的模型。(4)确定2组分受体组氨酸激酶的相互作用特异性决定因素，并操纵这些决定因素产生异源特异性激酶。目标1和目标2的预测和设计方法将主要使用基于结构的技术。物理动机的能量函数将用于评估，也将与更多的经验方法一起进行测试。目标3涉及开发一种新的蛋白质-蛋白质相互作用测定方法及其用于测量bbb10 000种可能的关联。这些数据将用于分析bZIP转录因子相互作用的演变，也将对测试Aim 1的计算方法有价值。目的4验证了原核组氨酸激酶主要是同源结合的假设，并提出了确定其相互作用特异性决定因素的计算和实验技术。总之，这四个目标包括结构建模、生物物理和生物信息学分析以及旨在理解线圈相互作用特异性的实验测试的综合计划。

DESCRIPTION (provided by applicant): The broad, long-term objectives of the proposed work are to understand how proteins establish interaction specificity at the level of individual domains and to capture this in models that can be used for computational prediction and design. The problem is central to issues of human health. Improper interactions among mutated or mis-regulated proteins can lead to disease, and achieving a deeper understanding of how this occurs is important. Further, the design of novel proteins or peptides to specifically inhibit native protein complexes would provide a route to future therapies. The interaction specificity problem can be simplified by focusing on a ubiquitous yet structurally simple protein-protein interaction motif: the alpha-helical coiled coil. Coiled coils occur throughout the proteomes of all species and are associated with a wide variety of functions. Their sequence and structural properties make them tractable for computational analysis, and coiled coils are also convenient for experimental study using biophysical methods. The proposed research involves collecting systematic experimental interaction data and using it to develop and test diverse computational techniques for predicting interactions. Computational methods will also be developed for designing coiled-coil-like peptides, and experiments are proposed to characterize the designs. The specific aims of this proposal are: (1) To test and improve methods for predicting coiled-coil structures, energies and interactions, (2) To extend existing coiled-coil design capabilities to treat a broader range of targets, (3) To measure bZIP coiled-coil interactions broadly across animal species and to develop a model of how protein- protein interaction specificity can evolve, and (4) To identify interaction specificity determinants of 2- component receptor histidine kinases and manipulate these to generate heterospecific kinases. The prediction and design methods of Aims 1 and 2 will use primarily structure-based techniques. Physically motivated energy functions will be used for evaluation, and also tested in conjunction with more empirical approaches. Aim 3 involves the development of a new protein-protein interaction assay and its use to measure >10,000 possible associations. These data will be applied to analyze the evolution of bZIP transcription factor interactions and will also be valuable for testing the computational methods of Aim 1. Aim 4 tests the hypothesis that prokaryotic histidine kinases primarily homoassociate, and proposes computational and experimental techniques for identifying determinants of their interaction specificity. Together, the four aims comprise an integrated program of structural modeling, biophysical and bioinformatic analysis and experimental testing aimed at understanding coiled-coil interaction specificity. PUBLIC HEALTH RELEVANCE: Interactions between proteins are essential to the proper functioning of cells. It is important that the correct complexes form and the incorrect ones do not, even when many closely related possibilities exist. This project addresses how the sequences and structures of coiled-coil proteins determine their interaction properties, and proposes methods for predicting and modifying coiled-coil interactions using computational techniques.