Abstract
Modulating the transitions between active and inactive conformations of protein kinases is the primary means of regulating their catalytic activity, achieved by phosphorylation of the activation loop (A-loop). To elucidate the mechanism of this conformational activation, we applied the string method to determine the conformational transition path of insulin receptor kinase between the active and inactive conformations and the corresponding free-energy profiles with and without A-loop phosphorylation. The conformational change was found to proceed in three sequential steps: first, the flipping of the DFG motif of the active site; second, rotation of the A-loop; finally, the inward movement of the αC helix. The main energetic bottleneck corresponds to the conformational change in the A-loop, while changes in the DFG motif and αC helix occur before and after A-loop conformational change, respectively. In accordance with this, two intermediate states are identified, the first state just after the DFG flipping and the second state after the A-loop rotation. These intermediates exhibit structural features characteristic of the corresponding inactive and active conformations of other protein kinases. To understand the impact of A-loop phosphorylation on kinase conformation, the free energies of A-loop phosphorylation were determined at several states along the conformational transition path using the free-energy perturbation simulations. The calculated free energies reveal that while the unphosphorylated kinase interconverts between the inactive and active conformations, A-loop phosphorylation restricts access to the inactive conformation, thereby increasing the active conformation population. Overall, this study suggests a consensus mechanism of conformational activation between different protein kinases.