The prerequisites and mechanism in the binding and induced folding of pKID to KIX

描述（由申请人提供）：蛋白质中普遍存在的，但知之甚少的内在紊乱现象对分子生物学的基本问题和新型小分子药物的设计具有广泛的影响。内在无序的蛋白质在生理条件下固有地缺乏二级和/或三级结构；它们将折叠与结合结合到它们的相互作用伙伴上，具有高特异性，而且通常具有非凡的多功能性。在转录调控、蛋白质相互作用网络和许多疾病的核心发现了紊乱的特征。迫切需要新的方法来靶向和操纵这些蛋白质-蛋白质相互作用。详细了解从选择或相遇到中间体到结合形式的机制，将在有效设计小分子以控制这些相互作用方面取得重大进展。此外，解决这些小的，高度特异性的蛋白质的功能原理将进一步重新设计肽和模拟物，这是一个日益增长的制药目标。这项工作提出了核磁共振波谱，分子动力学和分子生物学的结合，以获得耦合折叠和结合的独特结构见解。具体来说，这项工作将利用CREB的磷酸化激酶诱导转激活结构域（pKID）与CBP的KIX结构域的复合物作为模型系统，以产生沿着诱导折叠途径的相遇复合物和中间体的详细视图。具体而言，本提案旨在(1)定义二级结构的演化，使其区别于诱导折叠途径中的分子间相互作用，(2)将对接过程中飞行铸造的影响与静电转向的影响分开，以及(3)生成结合中间体的详细模型。对于目标1，该工作将利用弛豫色散核磁共振光谱和羰基碳和α碳对二级结构的敏感性来量化中间体中残留的特定螺旋含量。在目的2中，不同长度的pKID结构体将被系统地突变，并通过等温量热法测量，以分离电荷和结构体长度对结合热力学的影响。对于目标3，该工作将利用核磁共振衍生的约束来限制结合途径中中间体的分子动力学模拟中的扭转角和相对位置。这些研究的结合将极大地促进我们对内在无序蛋白关联的基本机制和指导原则的理解。这种独特的结构见解将大大提高我们设计小分子和肽的能力，以结合和破坏蛋白质之间的相互作用。

DESCRIPTION (provided by applicant): The ubiquitous, yet poorly understood, phenomenon of intrinsic disorder in proteins has wide-ranging implications for fundamental questions in molecular biology and the design of novel small molecule pharmaceuticals. Intrinsically disordered proteins inherently lack secondary and/or tertiary structure under physiological conditions; they couple folding with binding to their interaction partners with high specificity and, often, extraordinary versatility. The signatures of disorder are found at the heart of transcriptional regulation, protein interaction networks, and a number of diseases. New methods to target and manipulate these protein-protein interactions are urgently required. Understanding the mechanisms in detail, from selection or encounter through intermediates to the bound form, will lead to significant advances in the efficient design of small molecules to control these interactions. Additionally, resolving the principles governing the function of these small, highly specific proteins will further de novo design of peptides and mimics, a growing pharmaceutical aim. This work proposes a combination of NMR spectroscopy, molecular dynamics and molecular biology to gain unique structural insights into coupled folding and binding. Specifically, this work will utilize the complex of the phosphorylated kinase inducible transactivation domain (pKID) of CREB with the KIX domain of CBP as a model system to generate detailed views of the encounter complex and intermediate(s) along an induced folding pathway. Specifically, this proposal aims to (1) define the evolution of secondary structure as distinct from intermolecular interactions in the induced folding pathway, (2) separate the effect of fly-casting from electrostatic steering in docking, and (3) generate detailed models of the binding intermediate(s). For aim 1, the work will utilize relaxation dispersion NMR spectroscopy and the sensitivity of the carbonyl and alpha carbons to secondary structure to quantify the residue specific helical content in the intermediate(s). For aim 2, pKID constructs of varied lengths will be systematically mutated and measured by isothermal calorimetry to separate the effects of charge and construct length on binding thermodynamics. For aim 3, the work will utilize NMR-derived restraints to limit the torsion angles and relative positions in molecular dynamic simulations of the intermediate(s) in the binding pathway. The combination of these studies will significantly advance our understanding of the fundamental mechanisms and guiding principles in the association of intrinsically disordered proteins. The unique structural insights will greatly increase our ability to design small molecules and peptides to bind and disrupt protein-protein interactions. ) PUBLIC HEALTH RELEVANCE: Proteins that inherently lack ordered structure are central to both healthy biological functions and a number of diseases. Disordered proteins often form ordered structure only upon binding an interaction partner forming unique surfaces difficult to target with traditional pharmaceutical design methods. The proposed work will greatly advance our understanding of the mechanisms of disordered proteins as well as our ability to effectively design new pharmaceuticals to target and disrupt protein-protein interactions. )