Blueprint of the Intramolecular Regulatory Mechanism of Eukaryotic Protein Kinases
- Author(s): Meharena, Hiruy Sibhatu
- et al.
Eukaryotic protein kinases (EPKs) regulate numerous signaling processes by phosphorylating targeted substrates through a highly conserved catalytic domain. Previous computational studies proposed a model stating that a properly assembled non-linear motif termed the Regulatory (R) spine is essential for catalytic activity of EPKs. Here we define the required intramolecular interactions and biochemical properties of the R-spine and the newly identified "Shell" that surrounds the R-spine using site- directed mutagenesis and various in vitro phosphoryl transfer assays using cyclic AMP-dependent protein kinase as a representative of the entire kinome. Analysis of the 172 available Apo EPK structures in the protein data bank (PDB) revealed 4 unique structural conformations of the R- spine that correspond with catalytic inactivation of various EPKs. Elucidating the molecular entities required for the catalytic activation of EPKs and the identification of these inactive conformations opens new avenues for the design of efficient therapeutic EPK inhibitors. The catalytic core of EPKs oscillates between inactive and active states as well as toggling between open and closed conformations when active. Currently, the intramolecular interactions that regulate this dynamic behavior are not well understood. Here, we show that there are at least two possible mechanisms regulating this dynamics. The first mechanism involves the highly conserved salt bridge between a lysine from the [Beta]3- strand and a glutamate from the [alpha]C-helix as well as a hydrogen bond that only forms when the activation loop is phosphorylated. The second mechanism involves an ensemble of hydrophobic interactions within the nonlinear motifs known as the Regulatory spine and Shell. Our findings also show that the highly conserved [Beta]3- lysine serves as a "catalytic synchronization hub" that aligns and positions the dynamic components required for catalysis