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EMBO J. Sep 15, 1998; 17(18): 5458–5465.
PMCID: PMC1170871

Structure of the double-stranded RNA-binding domain of the protein kinase PKR reveals the molecular basis of its dsRNA-mediated activation.

Abstract

Protein kinase PKR is an interferon-induced enzyme that plays a key role in the control of viral infections and cellular homeostasis. Compared with other known kinases, PKR is activated by a distinct mechanism that involves double-stranded RNA (dsRNA) binding in its N-terminal region in an RNA sequence-independent fashion. We report here the solution structure of the 20 kDa dsRNA-binding domain (dsRBD) of human PKR, which provides the first three-dimensional insight into the mechanism of its dsRNA-mediated activation. The structure of dsRBD exhibits a dumb-bell shape comprising two tandem linked dsRNA-binding motifs (dsRBMs) both with an alpha-beta-beta-beta-alpha fold. The structure, combined with previous mutational and biochemical data, reveals a highly conserved RNA-binding site on each dsRBM and suggests a novel mode of protein-RNA recognition. The central linker is highly flexible, which may enable the two dsRBMs to wrap around the RNA duplex for cooperative and high-affinity binding, leading to the overall change of PKR conformation and its activation.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Hodgkin PD, Bond MW, O'Garra A, Frank G, Lee F, Coffman RL, Zlotnik A, Howard M. Identification of IL-6 as a T cell-derived factor that enhances the proliferative response of thymocytes to IL-4 and phorbol myristate acetate. J Immunol. 1988 Jul 1;141(1):151–157. [PubMed]
  • Beretta L, Gabbay M, Berger R, Hanash SM, Sonenberg N. Expression of the protein kinase PKR in modulated by IRF-1 and is reduced in 5q- associated leukemias. Oncogene. 1996 Apr 4;12(7):1593–1596. [PubMed]
  • Bevilacqua PC, Cech TR. Minor-groove recognition of double-stranded RNA by the double-stranded RNA-binding domain from the RNA-activated protein kinase PKR. Biochemistry. 1996 Aug 6;35(31):9983–9994. [PubMed]
  • Brand SR, Kobayashi R, Mathews MB. The Tat protein of human immunodeficiency virus type 1 is a substrate and inhibitor of the interferon-induced, virally activated protein kinase, PKR. J Biol Chem. 1997 Mar 28;272(13):8388–8395. [PubMed]
  • Bycroft M, Grünert S, Murzin AG, Proctor M, St Johnston D. NMR solution structure of a dsRNA binding domain from Drosophila staufen protein reveals homology to the N-terminal domain of ribosomal protein S5. EMBO J. 1995 Jul 17;14(14):3563–3571. [PMC free article] [PubMed]
  • Carpick BW, Graziano V, Schneider D, Maitra RK, Lee X, Williams BR. Characterization of the solution complex between the interferon-induced, double-stranded RNA-activated protein kinase and HIV-I trans-activating region RNA. J Biol Chem. 1997 Apr 4;272(14):9510–9516. [PubMed]
  • Chong KL, Feng L, Schappert K, Meurs E, Donahue TF, Friesen JD, Hovanessian AG, Williams BR. Human p68 kinase exhibits growth suppression in yeast and homology to the translational regulator GCN2. EMBO J. 1992 Apr;11(4):1553–1562. [PMC free article] [PubMed]
  • Clarke PA, Mathews MB. Interactions between the double-stranded RNA binding motif and RNA: definition of the binding site for the interferon-induced protein kinase DAI (PKR) on adenovirus VA RNA. RNA. 1995 Mar;1(1):7–20. [PMC free article] [PubMed]
  • Clemens MJ. PKR--a protein kinase regulated by double-stranded RNA. Int J Biochem Cell Biol. 1997 Jul;29(7):945–949. [PubMed]
  • Cosentino GP, Venkatesan S, Serluca FC, Green SR, Mathews MB, Sonenberg N. Double-stranded-RNA-dependent protein kinase and TAR RNA-binding protein form homo- and heterodimers in vivo. Proc Natl Acad Sci U S A. 1995 Oct 10;92(21):9445–9449. [PMC free article] [PubMed]
  • Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR. 1995 Nov;6(3):277–293. [PubMed]
  • Der SD, Yang YL, Weissmann C, Williams BR. A double-stranded RNA-activated protein kinase-dependent pathway mediating stress-induced apoptosis. Proc Natl Acad Sci U S A. 1997 Apr 1;94(7):3279–3283. [PMC free article] [PubMed]
  • Donzé O, Jagus R, Koromilas AE, Hershey JW, Sonenberg N. Abrogation of translation initiation factor eIF-2 phosphorylation causes malignant transformation of NIH 3T3 cells. EMBO J. 1995 Aug 1;14(15):3828–3834. [PMC free article] [PubMed]
  • Galabru J, Hovanessian AG. Two interferon-induced proteins are involved in the protein kinase complex dependent on double-stranded RNA. Cell. 1985 Dec;43(3 Pt 2):685–694. [PubMed]
  • Galabru J, Katze MG, Robert N, Hovanessian AG. The binding of double-stranded RNA and adenovirus VAI RNA to the interferon-induced protein kinase. Eur J Biochem. 1989 Jan 2;178(3):581–589. [PubMed]
  • Garrett DS, Kuszewski J, Hancock TJ, Lodi PJ, Vuister GW, Gronenborn AM, Clore GM. The impact of direct refinement against three-bond HN-C alpha H coupling constants on protein structure determination by NMR. J Magn Reson B. 1994 May;104(1):99–103. [PubMed]
  • Green SR, Mathews MB. Two RNA-binding motifs in the double-stranded RNA-activated protein kinase, DAI. Genes Dev. 1992 Dec;6(12B):2478–2490. [PubMed]
  • Hunter T, Hunt T, Jackson RJ, Robertson HD. The characteristics of inhibition of protein synthesis by double-stranded ribonucleic acid in reticulocyte lysates. J Biol Chem. 1975 Jan 25;250(2):409–417. [PubMed]
  • Kay LE. Field gradient techniques in NMR spectroscopy. Curr Opin Struct Biol. 1995 Oct;5(5):674–681. [PubMed]
  • Kharrat A, Macias MJ, Gibson TJ, Nilges M, Pastore A. Structure of the dsRNA binding domain of E. coli RNase III. EMBO J. 1995 Jul 17;14(14):3572–3584. [PMC free article] [PubMed]
  • Koromilas AE, Roy S, Barber GN, Katze MG, Sonenberg N. Malignant transformation by a mutant of the IFN-inducible dsRNA-dependent protein kinase. Science. 1992 Sep 18;257(5077):1685–1689. [PubMed]
  • Kumar A, Haque J, Lacoste J, Hiscott J, Williams BR. Double-stranded RNA-dependent protein kinase activates transcription factor NF-kappa B by phosphorylating I kappa B. Proc Natl Acad Sci U S A. 1994 Jul 5;91(14):6288–6292. [PMC free article] [PubMed]
  • Kumar A, Yang YL, Flati V, Der S, Kadereit S, Deb A, Haque J, Reis L, Weissmann C, Williams BR. Deficient cytokine signaling in mouse embryo fibroblasts with a targeted deletion in the PKR gene: role of IRF-1 and NF-kappaB. EMBO J. 1997 Jan 15;16(2):406–416. [PMC free article] [PubMed]
  • Kuszewski J, Qin J, Gronenborn AM, Clore GM. The impact of direct refinement against 13C alpha and 13C beta chemical shifts on protein structure determination by NMR. J Magn Reson B. 1995 Jan;106(1):92–96. [PubMed]
  • Manche L, Green SR, Schmedt C, Mathews MB. Interactions between double-stranded RNA regulators and the protein kinase DAI. Mol Cell Biol. 1992 Nov;12(11):5238–5248. [PMC free article] [PubMed]
  • Maran A, Maitra RK, Kumar A, Dong B, Xiao W, Li G, Williams BR, Torrence PF, Silverman RH. Blockage of NF-kappa B signaling by selective ablation of an mRNA target by 2-5A antisense chimeras. Science. 1994 Aug 5;265(5173):789–792. [PubMed]
  • McMillan NA, Carpick BW, Hollis B, Toone WM, Zamanian-Daryoush M, Williams BR. Mutational analysis of the double-stranded RNA (dsRNA) binding domain of the dsRNA-activated protein kinase, PKR. J Biol Chem. 1995 Feb 10;270(6):2601–2606. [PubMed]
  • Meurs E, Chong K, Galabru J, Thomas NS, Kerr IM, Williams BR, Hovanessian AG. Molecular cloning and characterization of the human double-stranded RNA-activated protein kinase induced by interferon. Cell. 1990 Jul 27;62(2):379–390. [PubMed]
  • Minks MA, West DK, Benvin S, Baglioni C. Structural requirements of double-stranded RNA for the activation of 2',5'-oligo(A) polymerase and protein kinase of interferon-treated HeLa cells. J Biol Chem. 1979 Oct 25;254(20):10180–10183. [PubMed]
  • Nilges M, Clore GM, Gronenborn AM. Determination of three-dimensional structures of proteins from interproton distance data by hybrid distance geometry-dynamical simulated annealing calculations. FEBS Lett. 1988 Mar 14;229(2):317–324. [PubMed]
  • Nilges M, Clore GM, Gronenborn AM. 1H-NMR stereospecific assignments by conformational data-base searches. Biopolymers. 1990 Mar-Apr;29(4-5):813–822. [PubMed]
  • Pavletich NP, Pabo CO. Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 A. Science. 1991 May 10;252(5007):809–817. [PubMed]
  • Patel RC, Sen GC. Identification of the double-stranded RNA-binding domain of the human interferon-inducible protein kinase. J Biol Chem. 1992 Apr 15;267(11):7671–7676. [PubMed]
  • Patel RC, Stanton P, Sen GC. Role of the amino-terminal residues of the interferon-induced protein kinase in its activation by double-stranded RNA and heparin. J Biol Chem. 1994 Jul 15;269(28):18593–18598. [PubMed]
  • Patel RC, Stanton P, McMillan NM, Williams BR, Sen GC. The interferon-inducible double-stranded RNA-activated protein kinase self-associates in vitro and in vivo. Proc Natl Acad Sci U S A. 1995 Aug 29;92(18):8283–8287. [PMC free article] [PubMed]
  • Patel RC, Stanton P, Sen GC. Specific mutations near the amino terminus of double-stranded RNA-dependent protein kinase (PKR) differentially affect its double-stranded RNA binding and dimerization properties. J Biol Chem. 1996 Oct 11;271(41):25657–25663. [PubMed]
  • Rice AP, Duncan R, Hershey JW, Kerr IM. Double-stranded RNA-dependent protein kinase and 2-5A system are both activated in interferon-treated, encephalomyocarditis virus-infected HeLa cells. J Virol. 1985 Jun;54(3):894–898. [PMC free article] [PubMed]
  • Romano PR, Green SR, Barber GN, Mathews MB, Hinnebusch AG. Structural requirements for double-stranded RNA binding, dimerization, and activation of the human eIF-2 alpha kinase DAI in Saccharomyces cerevisiae. Mol Cell Biol. 1995 Jan;15(1):365–378. [PMC free article] [PubMed]
  • Samuel CE, Duncan R, Knutson GS, Hershey JW. Mechanism of interferon action. Increased phosphorylation of protein synthesis initiation factor eIF-2 alpha in interferon-treated, reovirus-infected mouse L929 fibroblasts in vitro and in vivo. J Biol Chem. 1984 Nov 10;259(21):13451–13457. [PubMed]
  • Schmedt C, Green SR, Manche L, Taylor DR, Ma Y, Mathews MB. Functional characterization of the RNA-binding domain and motif of the double-stranded RNA-dependent protein kinase DAI (PKR). J Mol Biol. 1995 May 26;249(1):29–44. [PubMed]
  • Tan SL, Gale MJ, Jr, Katze MG. Double-stranded RNA-independent dimerization of interferon-induced protein kinase PKR and inhibition of dimerization by the cellular P58IPK inhibitor. Mol Cell Biol. 1998 May;18(5):2431–2443. [PMC free article] [PubMed]
  • Williams BR, Gilbert CS, Kerr IM. The respective roles of the protein kinase and pppA2' p5' A2' p5 A-activated endonuclease in the inhibition of protein synthesis by double stranded RNA in rabbit reticulocyte lysates. Nucleic Acids Res. 1979 Apr;6(4):1335–1350. [PMC free article] [PubMed]
  • Wong AH, Tam NW, Yang YL, Cuddihy AR, Li S, Kirchhoff S, Hauser H, Decker T, Koromilas AE. Physical association between STAT1 and the interferon-inducible protein kinase PKR and implications for interferon and double-stranded RNA signaling pathways. EMBO J. 1997 Mar 17;16(6):1291–1304. [PMC free article] [PubMed]
  • Wu S, Kaufman RJ. Double-stranded (ds) RNA binding and not dimerization correlates with the activation of the dsRNA-dependent protein kinase (PKR). J Biol Chem. 1996 Jan 19;271(3):1756–1763. [PubMed]
  • Wu S, Kaufman RJ. A model for the double-stranded RNA (dsRNA)-dependent dimerization and activation of the dsRNA-activated protein kinase PKR. J Biol Chem. 1997 Jan 10;272(2):1291–1296. [PubMed]
  • Zhu S, Romano PR, Wek RC. Ribosome targeting of PKR is mediated by two double-stranded RNA-binding domains and facilitates in vivo phosphorylation of eukaryotic initiation factor-2. J Biol Chem. 1997 May 30;272(22):14434–14441. [PubMed]

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