Mapping of the auto-inhibitory interactions of protein kinase R by nuclear magnetic resonance

The dsRNA-dependent protein kinase (PKR) is a key mediator of the antiviral and anti-proliferative effects of interferon. Unphosphorylated PKR is characterized by inhibitory interactions between the kinase and RNA binding domains (RBDs), but the structural details of the latent state and its unraveling during activation are not well understood. To study PKR regulation by NMR we assigned a large portion of the backbone resonances of the catalytically inactive K296R kinase domain, and performed N-15-heteronuclear sin le quantum coherence (HSQC) titrations; of this kinase domain with the RBDs. Chemical shift perturbations in the kinase indicate that RBD2 binds to the substrate eIF2 alpha docking site in the kinase C-lobe. Consistent with these results, a mutation in the eIF2 alpha docking site, F495A, displays weaker interactions with the RBD. The full-length RBD1 + 2 binds more strongly to the kinase domain than RBD2 alone. The observed chemical shift changes extend from the eIF2 alpha binding site into the kinase N-lobe and inside the active site, consistent with weak interactions between the N-terminal part of the RBD and the kinase. (c) 2006 Elsevier Ltd. All rights reserved.