title: Conserved intermolecular salt bridge required for activation of protein kinases PKR, GCN2, and PERK.
abstract: The protein kinases PKR, GCN2, and PERK phosphorylate translation initiation factor eIF2alpha to regulate general and genespecific protein synthesis under various cellular stress conditions. Recent x-ray crystallographic structures of PKR and GCN2 revealed distinct dimeric configurations of the kinase domains. Whereas PKR kinase domains dimerized in a back-to-back and parallel orientation, the GCN2 kinase domains displayed an antiparallel orientation. The dimerization interfaces on PKR and GCN2 were localized to overlapping surfaces on the N-terminal lobes of the kinase domains but utilized different intermolecular contacts. A key feature of the PKR dimerization interface is a salt bridge interaction between Arg(262) from one protomer and Asp(266) from the second protomer. Interestingly, these two residues are conserved in all eIF2alpha kinases, although in the GCN2 structure, the two residues are too remote to interact. To test the importance of this potential salt bridge interaction in PKR, GCN2, and PERK, the residues constituting the salt bridge were mutated either independently or together to residues with the opposite charge. Single mutations of the Asp (or Glu) and Arg residues blocked kinase function both in yeast cells and in vitro. However, for all three kinases, the double mutation designed to restore the salt bridge interaction with opposite polarity resulted in a functional kinase. Thus, the salt bridge interaction and dimer interface observed in the PKR structure is critical for the activity of all three eIF2alpha kinases. These results are consistent with the notion that the PKR structure represents the active state of the eIF2alpha kinase domain, whereas the GCN2 structure may represent an inactive state of the kinase.
authors.affiliation: Laboratory of Gene Regulation and Development, NICHD, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Madhusudan
authors.initials: M
authors.lastname: Dey
authors.forename: Chune
authors.initials: C
authors.lastname: Cao
authors.forename: Frank
authors.initials: F
authors.lastname: Sicheri
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Dever
mesh_terms: D000818:Animals; D001120:Arginine; D001216:Asparagine; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry; D019281:Dimerization; D006801:Humans; D011494:Protein Kinases / Q000737:chemistry*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry; D017434:Protein Structure, Tertiary; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry; D055672:Static Electricity; D019892:eIF-2 Kinase / Q000737:chemistry
chemical_list: D001120:Arginine; D001216:Asparagine; D029742:Caenorhabditis elegans Proteins; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D029701:Saccharomyces cerevisiae Proteins; D019892:eIF-2 Kinase; C512912:GCN2 protein, S cerevisiae