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

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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.).