Higher-Order Substrate Recognition of eIF2α by the RNA-Dependent Protein Kinase PKR

In response to binding viral double-stranded RNA byproducts within a cell, the RNA-dependent protein kinase PKR phosphorylates the alpha subunit of the translation initiation factor eIF2 on a regulatory site, Ser51. This triggers the general shutdown of protein synthesis and inhibition of viral propagation. To understand the basis for substrate recognition by and the regulation of PKR, we determined X-ray crystal structures of the catalytic domain of PKR in complex with eIF2alpha. The structures reveal that eIF2alpha binds to the C-terminal catalytic lobe while catalytic-domain dimerization is mediated by the N-terminal lobe. In addition to inducing a local unfolding of the Ser51 acceptor site in eIF2alpha, its mode of binding to PKR affords the Ser51 site full access to the catalytic cleft of PKR. The generality and implications of the structural mechanisms uncovered for PKR to the larger family of four human eIF2alpha protein kinases are discussed.

[1]  A. Hinnebusch,et al.  Binding of Double-stranded RNA to Protein Kinase PKR Is Required for Dimerization and Promotes Critical Autophosphorylation Events in the Activation Loop* , 2001, The Journal of Biological Chemistry.

[2]  Randy J Read,et al.  Electronic Reprint Biological Crystallography Likelihood-enhanced Fast Rotation Functions Biological Crystallography Likelihood-enhanced Fast Rotation Functions , 2003 .

[3]  T. E. Dever,et al.  Gene-Specific Regulation by General Translation Factors , 2002, Cell.

[4]  K. Ozato,et al.  Heterologous dimerization domains functionally substitute for the double‐stranded RNA binding domains of the kinase PKR , 2001, The EMBO journal.

[5]  L. Johnson,et al.  Active and Inactive Protein Kinases: Structural Basis for Regulation , 1996, Cell.

[6]  Tyson V. Sharp,et al.  Homologous regions of the alpha subunit of eukaryotic translational initiation factor 2 (eIF2alpha) and the vaccinia virus K3L gene product interact with the same domain within the dsRNA-activated protein kinase (PKR). , 1997, European journal of biochemistry.

[7]  J. Widom,et al.  Crystal Structure of the N-terminal Segment of Human Eukaryotic Translation Initiation Factor 2α* , 2002, The Journal of Biological Chemistry.

[8]  C. Samuel,et al.  Mechanism of interferon action: characterization of the intermolecular autophosphorylation of PKR, the interferon-inducible, RNA-dependent protein kinase , 1995, Journal of virology.

[9]  G. Murshudov,et al.  Refinement of macromolecular structures by the maximum-likelihood method. , 1997, Acta crystallographica. Section D, Biological crystallography.

[10]  A. Hinnebusch,et al.  The tRNA‐binding moiety in GCN2 contains a dimerization domain that interacts with the kinase domain and is required for tRNA binding and kinase activation , 2001, The EMBO journal.

[11]  T. Hunter,et al.  Dual-specificity protein kinases: will any hydroxyl do? , 1992, Trends in biochemical sciences.

[12]  C. Proud,et al.  A synthetic peptide substrate for initiation factor-2 kinases. , 1991, Biochemical and biophysical research communications.

[13]  L. Johnson,et al.  The crystal structure of a phosphorylase kinase peptide substrate complex: kinase substrate recognition , 1997, The EMBO journal.

[14]  Susan S. Taylor,et al.  Regulation of protein kinases; controlling activity through activation segment conformation. , 2004, Molecular cell.

[15]  A. Hinnebusch,et al.  Phosphorylation of initiation factor 2α by protein kinase GCN2 mediates gene-specific translational control of GCN4 in yeast , 1992, Cell.

[16]  Jane-Jane Chen,et al.  Multiple autophosphorylation is essential for the formation of the active and stable homodimer of heme-regulated eIF2alpha kinase. , 2001, Biochemistry.

[17]  J. Silverman,et al.  Regulation of the protein kinase PKR by the vaccinia virus pseudosubstrate inhibitor K3L is dependent on residues conserved between the K3L protein and the PKR substrate eIF2alpha , 1997, Molecular and cellular biology.

[18]  Giovanni Cesareni,et al.  Modular protein domains , 2005 .

[19]  G. Wagner,et al.  Solution structure of human initiation factor eIF2alpha reveals homology to the elongation factor eEF1B. , 2004, Structure.

[20]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

[21]  G. Lathrop,et al.  EIF2AK3, encoding translation initiation factor 2-α kinase 3, is mutated in patients with Wolcott-Rallison syndrome , 2000, Nature Genetics.

[22]  Stevan R. Hubbard,et al.  Juxtamembrane autoinhibition in receptor tyrosine kinases , 2004, Nature Reviews Molecular Cell Biology.

[23]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[24]  M. Clemens,et al.  Targets and mechanisms for the regulation of translation in malignant transformation , 2004, Oncogene.

[25]  J. Kuriyan,et al.  The Conformational Plasticity of Protein Kinases , 2002, Cell.

[26]  F. Sicheri,et al.  X-ray crystal structure and functional analysis of vaccinia virus K3L reveals molecular determinants for PKR subversion and substrate recognition. , 2002, Molecular cell.

[27]  S. Nanduri,et al.  A dynamically tuned double‐stranded RNA binding mechanism for the activation of antiviral kinase PKR , 2000, The EMBO journal.

[28]  Arvin C. Dar,et al.  Mechanistic Link between PKR Dimerization, Autophosphorylation, and eIF2α Substrate Recognition , 2005, Cell.

[29]  I. Kerr,et al.  Molecular cloning and characterization of the human double-stranded RNA-activated protein kinase induced by interferon , 1990, Cell.

[30]  P. Romano,et al.  The Interferon-induced Double-stranded RNA-activated Protein Kinase PKR Will Phosphorylate Serine, Threonine, or Tyrosine at Residue 51 in Eukaryotic Initiation Factor 2α* , 1999, The Journal of Biological Chemistry.

[31]  C. Proud.  eIF2 and the control of cell physiology. , 2005, Seminars in cell & developmental biology.

[32]  D. Hoffman,et al.  The crystal structure of the N-terminal region of the alpha subunit of translation initiation factor 2 (eIF2alpha) from Saccharomyces cerevisiae provides a view of the loop containing serine 51, the target of the eIF2alpha-specific kinases. , 2003, Journal of molecular biology.

[33]  Anne Bertolotti,et al.  Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response , 2000, Nature Cell Biology.

[34]  F. Sicheri,et al.  PKR and GCN2 Kinases and Guanine Nucleotide Exchange Factor Eukaryotic Translation Initiation Factor 2B (eIF2B) Recognize Overlapping Surfaces on eIF2α , 2005, Molecular and Cellular Biology.

[35]  Z. Otwinowski,et al.  Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.