Protein Kinase: Primary Structure and cAMP Interactions

描述（由申请人提供）：在此优异奖的任期内，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。使用晶体结构作为出发点，我们已经定义了子域和构象状态的酶穿梭通过其催化循环。我们结合了生物化学与结构研究，并在过去的五年中，特别是，已经开发出的方法，使我们能够监测溶液中的C-亚基的动态特性。由此产生的PKA的描述是最全面的任何蛋白激酶，并建立了PKA作为一个原型，了解整个蛋白激酶超家族。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隙的保守，还包括家族成员显示出显著多样性的表面。PKA如何识别其抑制剂以及它如何被cAMP激活是理解其生物学功能的基础。在下一个授权期间，刚刚解决了R1 α：C复合物的结构，我们准备第一次了解cAMP激活PKA的分子基础。它反映了两个主要信号通路的融合。我们的注意力将集中在大叶的表面上，在那里我们将尝试实现一个全面的和定量的了解如何实现分子识别和cAMP如何激活PKA使用广泛的Rl α：C接口作为模板。此外，我们将映射的功能作用和激活环的动态状态，特别是在结构和功能的重要性，Thr 197磷酸化和Cys 199作为氧化传感器的作用。在我们的生化研究的同时，我们将使用溶液方法，特别是荧光方法和氢/氘交换与质谱（H/DMS）相结合，以监测酶的动态特征，并全面绘制表面。最后，我们将继续使用晶体学来获得我们在生物学研究中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.