TheαC helix is a central regulator of PKR activation

Abstract

AbstractProtein 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, eIF2α, 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 theαC 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 theαC 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 theαC helix dimer interface with theαC helix.