The C helix is a central regulator of PKR activation.

Protein kinase R (PKR) functions in the eukaryotic innate immune system as a first-line defense against viral infections. PKR binds viral dsRNA, leading to autophosphorylation and activation. In its active state, PKR can phosphorylate its primary substrate, eIF2 , which blocks initiation of translation in the infected cell. It has been established that PKR activation occurs when the kinase domain dimerizes in a back-to-back configuration. However, the mechanism by which dimerization leads to enzymatic activation is not fully understood. Here, we investigate the structural mechanistic basis and energy landscape for PKR activation, with a focus on the C helix - a kinase activation and signal integration hub - using all-atom equilibrium and enhanced sampling molecular dynamics simulations. By employing window-exchange umbrella sampling, we compute free energy profiles of activation which show that back-to-back dimerization stabilizes a catalytically competent conformation of PKR. Key hydrophobic residues in the homodimer interface contribute to stabilization of the C helix in an active conformation and the position of its glutamate residue. Using linear mutual information analysis, we analyze allosteric communication connecting the protomers' N-lobes and the C helix dimer interface with the C helix.