title: Mapping of the auto-inhibitory interactions of protein kinase R by nuclear magnetic resonance.
abstract: The dsRNA-dependent protein kinase (PKR) is a key mediator of the anti-viral 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 (15)N-heteronuclear single quantum coherence (HSQC) titrations of this kinase domain with the RBDs. Chemical shift perturbations in the kinase indicate that RBD2 binds to the substrate eIF2alpha docking site in the kinase C-lobe. Consistent with these results, a mutation in the eIF2alpha 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 eIF2alpha 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.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Ave, Boston, MA 02115, USA.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Gelev
authors.forename: Huseyin
authors.initials: H
authors.lastname: Aktas
authors.forename: Assen
authors.initials: A
authors.lastname: Marintchev
authors.forename: Takuhiro
authors.initials: T
authors.lastname: Ito
authors.forename: Dominique
authors.initials: D
authors.lastname: Frueh
authors.forename: Michael
authors.initials: M
authors.lastname: Hemond
authors.forename: David
authors.initials: D
authors.lastname: Rovnyak
authors.forename: Mirijam
authors.initials: M
authors.lastname: Debus
authors.forename: Sven
authors.initials: S
authors.lastname: Hyberts
authors.forename: Anny
authors.initials: A
authors.lastname: Usheva
authors.forename: Jose
authors.initials: J
authors.lastname: Halperin
authors.forename: Gerhard
authors.initials: G
authors.lastname: Wagner
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D015852:Eukaryotic Initiation Factor-2; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D010766:Phosphorylation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry; D019892:eIF-2 Kinase / Q000737:chemistry* / Q000235:genetics
chemical_list: D015852:Eukaryotic Initiation Factor-2; D012313:RNA; D019892:eIF-2 Kinase