Structure of the Dual Enzyme Ire1 Reveals the Basis for Catalysis and Regulation in Nonconventional RNA Splicing

Ire1 is an ancient transmembrane sensor of ER stress with dual protein kinase and ribonuclease activities. In response to ER stress, Ire1 catalyzes the splicing of target mRNAs in a spliceosome-independent manner. We have determined the crystal structure of the dual catalytic region of Ire1at 2.4 A resolution, revealing the fusion of a domain, which we term the KEN domain, to the protein kinase domain. Dimerization of the kinase domain composes a large catalytic surface on the KEN domain which carries out ribonuclease function. We further show that signal induced trans-autophosphorylation of the kinase domain permits unfettered binding of nucleotide, which in turn promotes dimerization to compose the ribonuclease active site. Comparison of Ire1 to a topologically disparate ribonuclease reveals the convergent evolution of their catalytic mechanism. These findings provide a basis for understanding the mechanism of action of RNaseL and other pseudokinases, which represent 10% of the human kinome.

[1]  P. Walter,et al.  Oligomerization and phosphorylation of the Ire1p kinase during intracellular signaling from the endoplasmic reticulum to the nucleus. , 1996, The EMBO journal.

[2]  P. Walter,et al.  Signal integration in the endoplasmic reticulum unfolded protein response , 2007, Nature Reviews Molecular Cell Biology.

[3]  P. Walter,et al.  tRNA Ligase Is Required for Regulated mRNA Splicing in the Unfolded Protein Response , 1996, Cell.

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

[5]  Stevan R. Hubbard,et al.  IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA , 2002, Nature.

[6]  G. Sheldrick,et al.  In-house phase determination of the lima bean trypsin inhibitor: a low-resolution sulfur-SAD case. , 2003, Acta crystallographica. Section D, Biological crystallography.

[7]  Thomas C Terwilliger,et al.  SOLVE and RESOLVE: automated structure solution and density modification. , 2003, Methods in enzymology.

[8]  Hiderou Yoshida,et al.  IRE1-mediated unconventional mRNA splicing and S2P-mediated ATF6 cleavage merge to regulate XBP1 in signaling the unfolded protein response. , 2002, Genes & development.

[9]  S. Hubbard,et al.  Crystal structure of the tyrosine kinase domain of the human insulin receptor , 1994, Nature.

[10]  R. Silverman,et al.  Intrinsic molecular activities of the interferon-induced 2-5A-dependent RNase. , 1994, The Journal of biological chemistry.

[11]  P. Walter,et al.  Mechanism of non‐spliceosomal mRNA splicing in the unfolded protein response pathway , 1999, The EMBO journal.

[12]  C. Sander,et al.  Dali: a network tool for protein structure comparison. , 1995, Trends in biochemical sciences.

[13]  Hong Li,et al.  RNA Recognition and Cleavage by a Splicing Endonuclease , 2006, Science.

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

[15]  Peter Walter,et al.  On the mechanism of sensing unfolded protein in the endoplasmic reticulum. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[16]  J. Sambrook,et al.  A transmembrane protein with a cdc2+ CDC28 -related kinase activity is required for signaling from the ER to the nucleus , 1993, Cell.

[17]  Peter Walter,et al.  The Transmembrane Kinase Ire1p Is a Site-Specific Endonuclease That Initiates mRNA Splicing in the Unfolded Protein Response , 1997, Cell.

[18]  N. Echols,et al.  Allosteric Activation by Dimerization of the PknD Receptor Ser/Thr Protein Kinase from Mycobacterium tuberculosis* , 2007, Journal of Biological Chemistry.

[19]  Peter Walter,et al.  Transcriptional induction of genes encoding endoplasmic reticulum resident proteins requires a transmembrane protein kinase , 1993, Cell.

[20]  K. Mori,et al.  XBP1 mRNA Is Induced by ATF6 and Spliced by IRE1 in Response to ER Stress to Produce a Highly Active Transcription Factor , 2001, Cell.

[21]  Tom Alber,et al.  A conserved dimer and global conformational changes in the structure of apo-PknE Ser/Thr protein kinase from Mycobacterium tuberculosis. , 2006, Journal of molecular biology.

[22]  E. Birney,et al.  Patterns of somatic mutation in human cancer genomes , 2007, Nature.

[23]  Chao Zhang,et al.  Bypassing a Kinase Activity with an ATP-Competitive Drug , 2003, Science.

[24]  P. Alzari,et al.  Crystal Structure of the Catalytic Domain of the PknB Serine/Threonine Kinase from Mycobacterium tuberculosis * , 2003, The Journal of Biological Chemistry.

[25]  Zhaohui Xu,et al.  The crystal structure of human IRE1 luminal domain reveals a conserved dimerization interface required for activation of the unfolded protein response , 2006, Proceedings of the National Academy of Sciences.

[26]  P. Walter,et al.  Basis for regulated RNA cleavage by functional analysis of RNase L and Ire1p. , 2001, RNA.

[27]  S. Bryant,et al.  Threading a database of protein cores , 1995, Proteins.

[28]  A. Protopopov,et al.  The Differentiation and Stress Response Factor XBP-1 Drives Multiple Myeloma Pathogenesis , 2007, Cancer cell.

[29]  R. Silverman,et al.  2-5A-dependent RNase Molecules Dimerize during Activation by 2-5A (*) , 1995, The Journal of Biological Chemistry.

[30]  Fei Long,et al.  REFMAC5 dictionary: organization of prior chemical knowledge and guidelines for its use. , 2004, Acta crystallographica. Section D, Biological crystallography.

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

[32]  George M Sheldrick,et al.  Substructure solution with SHELXD. , 2002, Acta crystallographica. Section D, Biological crystallography.

[33]  Peter Walter,et al.  Inaugural Article: On the mechanism of sensing unfolded protein in the endoplasmic reticulum , 2005 .

[34]  Tom Alber,et al.  Structure of Mycobacterium tuberculosis PknB supports a universal activation mechanism for Ser/Thr protein kinases , 2003, Nature Structural Biology.

[35]  E. Birney,et al.  Patterns of somatic mutation in human cancer genomes , 2007, Nature.

[36]  P. Lengyel,et al.  Interferon action: RNA cleavage pattern of a (2'-5')oligoadenylate--dependent endonuclease. , 1981, Science.

[37]  Daisuke Oikawa,et al.  A role for BiP as an adjustor for the endoplasmic reticulum stress-sensing protein Ire1 , 2004, The Journal of cell biology.

[38]  G. Barton,et al.  Emerging roles of pseudokinases. , 2006, Trends in cell biology.

[39]  P. Walter,et al.  Intracellular signaling by the unfolded protein response. , 2006, Annual review of cell and developmental biology.

[40]  Peter Walter,et al.  Functional and Genomic Analyses Reveal an Essential Coordination between the Unfolded Protein Response and ER-Associated Degradation , 2000, Cell.

[41]  K. Mori.  Frame Switch Splicing and Regulated Intramembrane Proteolysis: Key Words to Understand the Unfolded Protein Response , 2003, Traffic.

[42]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .