Mapping Allosteric Cooperativity in Protein Kinases

描述(申请人提供)：蛋白激酶是一种变构酶，参与细胞信号通路(包括成熟、分化和代谢)和许多病理疾病(癌症、糖尿病、类风湿性关节炎、心肌病等)。在这个提案中，我们将重点研究蛋白激酶A(PKA)的变构结合过程，PKA是蛋白激酶超家族的典型酶。尽管PKA已经被研究了几十年，但底物识别的分子机制以及内源和外源抑制剂仍然难以捉摸。有令人信服的证据表明，这些现象是由酶的内部动力学调节的。对于PKA，配体结合(核苷酸、底物和抑制剂)调节酶的动态，直接影响催化周转。在这里，我们将使用高分辨率技术(X射线、核磁共振和MD计算机模拟)以及生物物理方法(热量热法、动力学分析和H/D耦合质谱学)来追踪PKA中的变构协作性。在目标1中，我们将使用功能失调的突变体来剖析变构途径。在AIM 2中，我们将研究核苷酸和ATP竞争性抑制剂对该激酶的动态激活和失活。最后，在AIM 3中，我们将重点讨论蛋白激酶抑制物(PKI)的内源性抑制及其对酶的内部动力学的影响。了解变构信号如何传播和触发结合协同性，将有助于设计新的策略来控制(通过抑制或调整)激酶活性，以用于治疗疾病的创新疗法。

DESCRIPTION (provided by applicant): Protein kinases are allosteric enzymes involved in cell signaling pathways (including maturation, differentiation, and metabolism) and many pathological diseases (cancer, diabetes, rheumatoid arthritis, cardiomyopathy, and others). In this proposal, we will focus on the study of allosteric binding processes of protein kinase A (PKA), a prototypical enzyme for the kinase superfamily. Although PKA has been studied for several decades, the molecular mechanisms for substrate recognition, as well as endogenous and exogenous inhibitors are still elusive. There is compelling evidence that these phenomena are mediated by the enzyme's internal dynamics. For PKA, ligand binding (nucleotides, substrates, and inhibitors) modulates the dynamic state of the enzyme, with direct repercussions on the catalytic turnover. Here, we will use a combination of high-resolution techniques (X-ray, NMR, and MD computer simulations) as well as biophysical approaches (thermocalorimetry, kinetic assays, and H/D coupled to mass spectrometry) to trace the allosteric cooperativity in PKA. In AIM 1, we will dissect the allosteric pathways using dysfunctional mutants. In the AIM 2, we will study the dynamic activation and deactivation of the kinase by nucleotides and ATP-competitive inhibitors. Finally, in AIM 3, we will focus on the endogenous inhibition of PKI (protein kinase inhibitor) and its effects on the enzyme's internal dynamics. Understanding how allosteric signals propagate and trigger binding cooperativity will help in designing new strategies to control (by inhibiting or tuning) kinase activity for innovative therapies to treat disease.