Molecular mechanisms of allosteric activation and inhibition by the PIF-pocket in AGC protein kinases

Allostery是一个最初用来解释多聚体蛋白质对称性调节的概念，今天被认为是蛋白质-蛋白质相互作用和调节细胞信号传导的二级修饰的基本机制。15年前，我率先发现了蛋白激酶催化结构域上的调节位点，多年来，我专注于其中一个位点的机制的详细表征，即“PIF口袋”，它存在于AGC组的大量蛋白激酶中。在PDK 1中，PIF-口袋起两个作用，1-它是底物的疏水基序（HM）的对接位点，所述疏水基序是底物磷酸化所需的，2-HM与PIF-口袋的结合增加PDK 1的激酶活性。在过去的10年里，我们率先发现和开发了与PIF口袋结合的变构化合物、PDK 1和其他AGC激酶的变构激活剂和抑制剂。然而，对靶变构蛋白的分子要求是未知的，并且在PDK 1和其他变构模型蛋白中还未被探索。我们现在呈现了与HM-多肽复合的PDK 1的晶体结构，该HM-多肽来自PIF-口袋处的底物对接。此外，我们显示了一种小化合物PS 653的鉴定，该化合物取代了PDK 1和与PDK 1的PIF口袋结合的底物衍生HM多肽之间的相互作用。我们还提供了晶体结构信息，显示PS 653与PDK 1的ATP结合位点结合。总之，我们以前的工作确定了与PIF口袋结合并变构影响PDK 1活性位点的化合物;我们现在显示了相反的效果：PS 653与ATP结合位点结合并变构影响PIF口袋，共同提供了蛋白激酶中正构和变构位点之间双向药理学变构通信的第一个例子。在目前的应用中，我们将采用分子生物学，生物化学，晶体学，并将与分子动力学模拟专家合作，研究主要的剩余问题：-化合物的直接和反向变构转换的分子要求是什么？- PDK 1与其生理底物之间的对接相互作用的分子细节是什么？- 反向变构途径在生理上是否有用？该工作计划将阐明PDK 1与底物对接的分子细节以及PDK 1的药理学双向变构调节。由于变构广泛存在于健康和疾病的信号转导中，因此该研究有望对未来变构药物的开发产生广泛的影响。作为补助金的一部分，我们将培养两名医学生沿着他们的博士论文（博士）。

Allostery, a concept originally developed to explain the regulation of multimeric proteins with symmetry, is considered today the fundamental mechanism of protein-protein interactions and secondary modifications that regulate cellular signaling. Fifteen years ago, I pioneered the discovery of regulatory sites on the catalytic domain of protein kinases, and throughout the years, I focused on the detailed characterization of the mechanisms of one of those sites, the "PIF-pocket", which is present in a large number of protein kinases from the AGC group. In PDK1 the PIF-pocket plays two roles, 1- it is a docking site for the hydrophobic motif (HM) of substrates required for their phosphorylation and 2- binding of the HM to the PIF-pocket increases the kinase activity of PDK1. Over the last 10 years, we pioneered the discovery and development of allosteric compounds binding to the PIF-pocket, allosteric activators and inhibitors of PDK1 and other AGC kinases. However, the molecular requirements on the target allosteric proteins are unknown and vastly unexplored in PDK1 and other allosteric model proteins. We now present the crystal structure of PDK1 in complex with the HM- polypeptide from a substrate docking at the PIF-pocket. In addition, we show the identification of a small compound, PS653, which displaces the interaction between PDK1 and the substrate-derived HM-polypeptide that binds to the PIF-pocket of PDK1. We further provide crystal structure information showing that PS653 binds to the ATP-binding site of PDK1. Together, our previous work identified compounds that bind to the PIF-pocket and allosterically affect the active site of PDK1; we now show the reverse effect: PS653 binds to the ATP-binding site and allosterically affects the PIF-pocket, providing together the first example of a bi-directional pharmacological allosteric communication between orthosteric and allosteric sites in protein kinases. In the present, application we will employ molecular biology, biochemistry, crystallography and will collaborate with experts in molecular dynamics simulations to investigate major remaining questions: - which are the molecular requirements for the direct and reverse allosteric transitions by compounds?, - what are the molecular details of the docking interaction between PDK1 and its physiological substrates? - Is the reverse allosteric path used physiologically? The work program will shed light on the molecular details of PDK1 docking with substrates and on the pharmacological bi-directional allosteric modulation of PDK1. Since allostery is widely present in signal transduction in health and disease, the research is expected to have broad implications for future developments of allosteric drugs. As part of the grant, we will train two medical students along their doctoral thesis (Dr. med.).