The α 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 α , 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 α 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 α 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 α C helix dimer interface with the α C helix.

Structural Basis of Protein Kinase R Autophosphorylation.

The RNA-activated protein kinase, PKR, is a key mediator of the innate immunity response to viral infection. Viral double-stranded RNAs induce PKR dimerization and autophosphorylation. The PKR kinase domain forms a back-to-back dimer. However, intermolecular ( trans) autophosphorylation is not feasible in this arrangement. We have obtained PKR kinase structures that resolves this dilemma. The kinase protomers interact via the known back-to-back interface as well as a front-to-front interface that is formed by exchange of activation segments. Mutational analysis of the front-to-front interface support a functional role in PKR activation. Molecular dynamics simulations reveal that the activation segment is highly dynamic in the front-to-front dimer and can adopt conformations conducive to phosphoryl transfer. We propose a mechanism where back-to-back dimerization induces a conformational change that activates PKR to phosphorylate a "substrate" kinase docked in a front-to-front geometry. This mechanism may be relevant to related kinases that phosphorylate the eukaryotic initiation factor eIF2α.