Capturing Autoinhibited PDK1 Reveals the Linker’s Regulatory Role, Informing Innovative Inhibitor Design

PDK1 is crucial for PI3K/AKT/mTOR and Ras/MAPK cancer signaling. It phosphorylates AKT in a PIP₃-dependent but S6K, SGK, and RSK kinases in a PIP₃-independent manner. Unlike its substrates, its autoinhibited monomeric state has been unclear, likely due to its low population time, and phosphorylation in the absence of PIP₃ has been puzzling too. Here, guided by experimental data, we constructed models and performed all-atom molecular dynamics simulations. In the autoinhibited PDK1 conformation that resembles autoinhibited AKT, binding of the linker between the kinase and PH domains to the PIF-binding pocket promotes the formation of the Glu¹³⁰-Lys¹¹¹ salt bridge and weakens the association of the kinase domain with the PH domain, shifting the population from the autoinhibited state to states accessible to the membrane and its kinase substrates. The interaction of the substrates' hydrophobic motif and the PDK1 PIF-binding pocket facilitates the release of the autoinhibition even in <i>the absence of PIP</i>_<i>3</i>. Phosphorylation of the serine-rich motif within the linker further attenuates the association of the PH domain with the kinase domain. These suggest that while the monomeric autoinhibited state is relatively stable, it can readily shift to its active, catalysis-prone state to phosphorylate its diverse substrates. Our findings reveal the PDK1 activation mechanism and discover the regulatory role of PDK1's linker, which lead to two innovative linker-based inhibitor strategies: (i) locking the autoinhibited PDK1 through optimization of the interactions of AKT inhibitors with the PH domain of PDK1 and (ii) analogs (small molecules or peptidomimetics) that mimic the linker interactions with the PIF-binding pocket.