Protein Kinase: Primary Structure and cAMP Interactions

描述(由申请人提供)：在这个荣誉奖的任期内，我们已经建立了对cAMP依赖的蛋白激酶(PKA)催化(C)亚单位的分子理解。以晶体结构为起点，我们定义了酶在其催化循环中穿梭时的亚域和构象状态。我们将生物化学与结构研究结合起来，特别是在过去的五年里，我们开发了一些方法，使我们能够监测溶液中C亚基的动态性质。由此得到的对PKA的描述是所有蛋白激酶中最全面的，并建立了PKA作为理解整个蛋白激酶超家族的原型。我们也逐渐认识到，催化亚基不仅是一个催化剂，也是其他蛋白质停靠的支架。激酶家族的进化是非常先进的，不仅包括活性部位裂解的保守，而且还包括家族成员表现出显着多样性的表面。PKA如何识别其抑制物以及如何被cAMP激活是理解其生物学功能的基础。在接下来的授权期内，在刚刚解决了Rlpha：C复合体的结构之后，我们第一次准备好了解PKA激活cAMP的分子基础。它反映了两条主要信号通路的汇聚。我们的注意力将集中在大叶的表面，在那里我们将试图实现对分子识别如何实现以及cAMP如何利用广泛的Rlpha：C界面作为模板激活PKA的全面和定量的了解。此外，我们将绘制激活环的功能角色和动态图，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/氢交换-质谱仪(H/DMS)来监测酶的动态特征，并全面绘制表面图。最后，我们将继续使用结晶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.