Protein Kinase: Primary Structure and cAMP Interactions

描述（由申请人提供）：在本优异奖的任期内，我们已经建立了camp依赖性蛋白激酶（PKA）的催化(C)亚基的分子理解。以晶体结构为出发点，我们定义了酶在催化循环中穿梭时的子结构域和构象状态。我们将生物化学与结构研究相结合，特别是在过去的五年中，我们开发了一些方法，使我们能够监测溶液中c -亚基的动态特性。由此产生的PKA描述是所有蛋白激酶中最全面的，并将PKA建立为了解整个蛋白激酶超家族的原型。我们还认识到，催化亚基不仅是催化剂，而且是其他蛋白质停靠的支架。激酶家族的进化非常先进，不仅包括活性位点裂缝的保存，而且包括家族成员表现出显著多样性的表面。PKA如何识别其抑制剂以及如何被cAMP激活是了解其生物学功能的基础。在下一个授权期内，我们刚刚解决了rα:C复合物的结构，我们将首次了解PKA的cAMP激活的分子基础。它反映了两种主要信号通路的趋同。我们的注意力将集中在大叶的表面，在那里我们将尝试实现对如何实现分子识别以及cAMP如何使用广泛的rα:C界面作为模板激活PKA的全面和定量理解。此外，我们将绘制激活环的功能角色和动态状态，特别关注Thr197磷酸化的结构和功能重要性以及Cys199作为氧化传感器的作用。在进行生化研究的同时，我们将采用溶液法，特别是荧光法和氢/氘交换结合质谱法（H/DMS）来监测酶的动态特征并全面绘制表面图。最后，我们将继续使用晶体学来获得我们通过生物学研究定义的各种构象状态的高分辨率结构。

DESCRIPTION (provided by applicant): Over the tenure of this Merit Award we have built a molecular understanding of the catalytic (C) subunit of cAMP-dependent protein kinase (PKA). Using the crystal structure as a starting point, we have defined the subdomains and conformational states as the enzyme shuttles through its catalytic cycle. We have combined biochemistry with structural studies and over the past five years, in particular, have developed methods that allow us to monitor the dynamic properties of the C-subunit in solution. The resulting description of PKA is the most comprehensive for any protein kinase and has established PKA as a prototype for understanding the entire protein kinase superfamily. We have also come to appreciate that the catalytic subunit is not simply a catalyst but also a scaffold on which other proteins dock. The evolution of the kinase family is very advanced and includes not only conservation of the active site cleft but also the surface which is where the family members display remarkable diversity. How PKA recognizes its inhibitors and how it is activated by cAMP is fundamental to understanding its biological function. During this next granting period, having just solved the structure of an Rlalpha:C complex, we are poised for the first time to understand the molecular basis for cAMP activation of PKA. It reflects the convergence of two major signaling pathways. Our attention will focus on the surface of the large lobe where we will try to achieve a comprehensive and quantitative understanding of how molecular recognition is achieved and how cAMP activates PKA using the extensive Rlalpha:C interface as a template. In addition, we shall map the functional roles and dynamic states of the activation loop, focusing specifically on the structural and functional importance of Thr197 phosphorylation and on the role of Cys199 as a sensor for oxidation. In parallel with our biochemical studies, we shall use solution methods, specifically fluorescence approaches and hydrogen/deuterium exchange coupled with mass spectrometry (H/DMS) to monitor the dynamic features of the enzyme and to comprehensively map the surface. Finally, we shall continue to use crystallography to obtain high resolution structures of the various conformational states that we define by our biological studies.