Mapping Allosteric Cooperativity in Protein Kinases

描述（由申请人提供）：蛋白激酶是参与细胞信号传导途径（包括成熟、分化和代谢）和许多病理性疾病（癌症、糖尿病、类风湿性关节炎、心肌病等）的变构酶。在这个计划中，我们将集中研究蛋白激酶A（PKA），激酶超家族的原型酶的变构结合过程。虽然PKA的研究已经有几十年的历史，但其底物识别的分子机制以及内源性和外源性抑制剂仍然是难以捉摸的。有令人信服的证据表明，这些现象是由酶的内部动力学介导的。对于PKA，配体结合（核苷酸，底物和抑制剂）调节酶的动态状态，直接影响催化营业额。在这里，我们将使用高分辨率技术（X射线，NMR和MD计算机模拟）以及生物物理方法（热量热法，动力学测定和H/D耦合到质谱法）的组合来跟踪PKA中的变构协同性。在AIM 1中，我们将使用功能失调的突变体来剖析变构通路。在AIM 2中，我们将研究核苷酸和ATP竞争性抑制剂对激酶的动态激活和失活。最后，在AIM 3中，我们将关注PKI（蛋白激酶抑制剂）的内源性抑制及其对酶内部动力学的影响。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（通过抑制或调节）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.