Molecular Insights into the Regulation of 3-Phosphoinositide-Dependent Protein Kinase 1: Modeling the Interaction between the Kinase and the Pleckstrin Homology Domains

[Image: see text] The 3-phosphoinositide-dependent protein kinase 1 (PDK1) K465E mutant kinase can still activate protein kinase B (PKB) at the membrane in a phosphatidylinositol-3,4,5-trisphosphate (PIP(3), PtdIns(3,4,5)P(3)) independent manner. To understand this new PDK1 regulatory mechanism, docking and molecular dynamics calculations were performed for the first time to simulate the wild-type kinase domain–pleckstrin homology (PH) domain complex with PH-in and PH-out conformations. These simulations were then compared to the PH-in model of the KD–PH(mutant K465E) PDK1 complex. Additionally, three KD–PH complexes were simulated, including a substrate analogue bound to a hydrophobic pocket (denominated the PIF-pocket) substrate-docking site. We find that only the PH-out conformation, with the PH domain well-oriented to interact with the cellular membrane, is active for wild-type PDK1. In contrast, the active conformation of the PDK1 K465E mutant is PH-in, being ATP-stable at the active site while the PIF-pocket is more accessible to the peptide substrate. We corroborate that both the docking-site binding and the catalytic activity are in fact enhanced in knock-in mouse samples expressing the PDK1 K465E protein, enabling the phosphorylation of PKB in the absence of PIP(3) binding.