Crystal structure of inhibitor of κB kinase β (IKKβ)

Inhibitor of κB (IκB) kinase (IKK) phosphorylates IκB proteins, leading to their degradation and the liberation of nuclear factor κB for gene transcription. Here we report the crystal structure of IKKβ in complex with an inhibitor, at a resolution of 3.6 Å. The structure reveals a trimodular architecture comprising the kinase domain, a ubiquitin-like domain (ULD) and an elongated, α-helical scaffold/dimerization domain (SDD). Unexpectedly, the predicted leucine zipper and helix–loop–helix motifs do not form these structures but are part of the SDD. The ULD and SDD mediate a critical interaction with IκBα that restricts substrate specificity, and the ULD is also required for catalytic activity. The SDD mediates IKKβ dimerization, but dimerization per se is not important for maintaining IKKβ activity and instead is required for IKKβ activation. Other IKK family members, IKKα, TBK1 and IKK-i, may have a similar trimodular architecture and function.

[1]  Radha Akella,et al.  Substrate and docking interactions in serine/threonine protein kinases. , 2007, Chemical reviews.

[2]  Jae-Hyuck Shim,et al.  A Novel Ubiquitin-like Domain in IκB Kinase β Is Required for Functional Activity of the Kinase* , 2004, Journal of Biological Chemistry.

[3]  A. Hoffmann,et al.  CK2 Is a C-Terminal IkappaB Kinase Responsible for NF-kappaB Activation during the UV Response. , 2003, Molecular cell.

[4]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[5]  T. Huxford,et al.  The human IKKbeta subunit kinase domain displays CK2-like phosphorylation specificity. , 2008, Biochemical and biophysical research communications.

[6]  L. Johnson,et al.  Protein Kinase Inhibitors: Insights into Drug Design from Structure , 2004, Science.

[7]  Masahiko Hibi,et al.  c-Jun Can Recruit JNK to Phosphorylate Dimerization Partners via Specific Docking Interactions , 1996, Cell.

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

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

[10]  Michael Karin,et al.  Regulation and Function of IKK and IKK-Related Kinases , 2006, Science's STKE.

[11]  M. Karin.  Nuclear factor-kappaB in cancer development and progression. , 2006, Nature.

[12]  E. Zandi,et al.  A cytokine-responsive IkappaB kinase that activates the transcription factor NF-kappaB. , 1997, Nature.

[13]  C. Boshoff,et al.  Crystal structure of a vFlip-IKKgamma complex: insights into viral activation of the IKK signalosome. , 2008, Molecular cell.

[14]  W. Lim,et al.  Docking interactions in protein kinase and phosphatase networks. , 2006, Current opinion in structural biology.

[15]  S. Akira,et al.  Involvement of the ubiquitin‐like domain of TBK1/IKK‐i kinases in regulation of IFN‐inducible genes , 2007, The EMBO journal.

[16]  E. Zandi,et al.  The IkappaB kinase complex (IKK) contains two kinase subunits, IKKalpha and IKKbeta, necessary for IkappaB phosphorylation and NF-kappaB activation. , 1997, Cell.

[17]  Scott Bowes,et al.  Structure of a NEMO/IKK-associating domain reveals architecture of the interaction site. , 2008, Structure.

[18]  Soichi Wakatsuki,et al.  Ubiquitin-binding domains — from structures to functions , 2009, Nature Reviews Molecular Cell Biology.

[19]  T. Maniatis,et al.  Site-specific phosphorylation of IkappaBalpha by a novel ubiquitination-dependent protein kinase activity. , 1996, Cell.

[20]  Gabriel Waksman,et al.  Supplemental Data Crystal Structure of a vFlip-IKK γ Complex : Insights into Viral Activation of the IKK Signalosome , 2008 .

[21]  Nobuhiro Suzuki,et al.  Specific Recognition of Linear Ubiquitin Chains by NEMO Is Important for NF-κB Activation , 2009, Cell.

[22]  G. Courtois,et al.  Complementation cloning of NEMO, a component of the IkappaB kinase complex essential for NF-kappaB activation. , 1998, Cell.

[23]  G Bricogne,et al.  Generation, representation and flow of phase information in structure determination: recent developments in and around SHARP 2.0. , 2003, Acta crystallographica. Section D, Biological crystallography.

[24]  K. Guan,et al.  Roles for homotypic interactions and transautophosphorylation in IkappaB kinase beta IKKbeta) activation [corrected]. , 2003, The Journal of biological chemistry.

[25]  Ebrahim Zandi,et al.  Direct Phosphorylation of IκB by IKKα and IKKβ: Discrimination Between Free and NF-κB-Bound Substrate , 1998 .

[26]  M. Delepierre,et al.  Solution structure of NEMO zinc finger and impact of an anhidrotic ectodermal dysplasia with immunodeficiency-related point mutation. , 2008, Journal of molecular biology.

[27]  Kornelia Polyak,et al.  Mechanism of CDK activation revealed by the structure of a cyclinA-CDK2 complex , 1995, Nature.

[28]  J. Kuret,et al.  Structural basis for selectivity of the isoquinoline sulfonamide family of protein kinase inhibitors. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[29]  G. Courtois,et al.  Complementation Cloning of NEMO, a Component of the IκB Kinase Complex Essential for NF-κB Activation , 1998, Cell.

[30]  Martyn D Winn,et al.  Macromolecular TLS refinement in REFMAC at moderate resolutions. , 2003, Methods in enzymology.

[31]  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.

[32]  Matthias Mann,et al.  IKK-1 and IKK-2: Cytokine-Activated IκB Kinases Essential for NF-κB Activation , 1997 .

[33]  D. Goeddel,et al.  IkappaB kinase-beta: NF-kappaB activation and complex formation with IkappaB kinase-alpha and NIK. , 1997, Science.

[34]  E. Zandi,et al.  IKK-gamma is an essential regulatory subunit of the IkappaB kinase complex. , 1998, Nature.

[35]  A. Kikuchi,et al.  Axin, a negative regulator of the Wnt signaling pathway, forms a complex with GSK-3beta and beta-catenin and promotes GSK-3beta-dependent phosphorylation of beta-catenin. , 1998, The EMBO journal.

[36]  S. Ghosh,et al.  Shared Principles in NF-κB Signaling , 2008, Cell.

[37]  M. Karin,et al.  Regulation and function of NF-kappaB transcription factors in the immune system. , 2009, Annual review of immunology.

[38]  Geng Wu,et al.  Structure of a beta-TrCP1-Skp1-beta-catenin complex: destruction motif binding and lysine specificity of the SCF(beta-TrCP1) ubiquitin ligase. , 2003, Molecular cell.

[39]  E. Zandi,et al.  IKK-γ is an essential regulatory subunit of the IκB kinase complex , 1998, Nature.

[40]  D B Young,et al.  IKK-1 and IKK-2: cytokine-activated IkappaB kinases essential for NF-kappaB activation. , 1997, Science.

[41]  Paul Polakis,et al.  Downregulation of β-catenin by human Axin and its association with the APC tumor suppressor, β-catenin and GSK3β , 1998, Current Biology.

[42]  Susan S. Taylor,et al.  2.2 A refined crystal structure of the catalytic subunit of cAMP-dependent protein kinase complexed with MnATP and a peptide inhibitor. , 1993, Acta crystallographica. Section D, Biological crystallography.

[43]  Geng Wu,et al.  Structure of a -TrCP1-Skp1--Catenin Complex , 2003 .

[44]  Claus Scheidereit,et al.  IκB kinase complexes: gateways to NF-κB activation and transcription , 2006, Oncogene.

[45]  P. Polakis,et al.  Downregulation of beta-catenin by human Axin and its association with the APC tumor suppressor, beta-catenin and GSK3 beta. , 1998, Current biology : CB.

[46]  John Kuriyan,et al.  Crystal structure of the Src family tyrosine kinase Hck , 1997, Nature.

[47]  David M. Rothwarf,et al.  A cytokine-responsive IκB kinase that activates the transcription factor NF-κB , 1997, Nature.

[48]  A. Hoffmann,et al.  CK2 Is a C-Terminal IκB Kinase Responsible for NF-κB Activation during the UV Response , 2003 .

[49]  Michael D. Schneider,et al.  Essential role of TAK1 in thymocyte development and activation. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[50]  I. Verma,et al.  Constitutive phosphorylation of I kappa B alpha by casein kinase II. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[51]  M. Karin.  Nuclear factor-κB in cancer development and progression , 2006, Nature.

[52]  Akira Kikuchi,et al.  Axin, a negative regulator of the Wnt signaling pathway, forms a complex with GSK‐3β and β‐catenin and promotes GSK‐3β‐dependent phosphorylation of β‐catenin , 1998 .

[53]  K. Guan,et al.  Roles for Homotypic Interactions and Transautophosphorylation in IκB Kinase (IKKβ) Activation* , 2003, Journal of Biological Chemistry.

[54]  J. Zheng,et al.  Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. , 1991, Science.

[55]  T. Maniatis,et al.  Site-Specific Phosphorylation of IκBα by a Novel Ubiquitination-Dependent Protein Kinase Activity , 1996, Cell.

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

[57]  Hao Wu,et al.  Structural basis for recognition of diubiquitins by NEMO. , 2009, Molecular cell.

[58]  E. Zandi,et al.  The IκB Kinase Complex (IKK) Contains Two Kinase Subunits, IKKα and IKKβ, Necessary for IκB Phosphorylation and NF-κB Activation , 1997, Cell.

[59]  G. Cheetham,et al.  Structural basis for the interaction of TAK1 kinase with its activating protein TAB1. , 2005, Journal of molecular biology.

[60]  S. Akira,et al.  Essential function for the kinase TAK1 in innate and adaptive immune responses , 2005, Nature Immunology.

[61]  Jae-Hyuck Shim,et al.  A novel ubiquitin-like domain in IkappaB kinase beta is required for functional activity of the kinase. , 2004, The Journal of biological chemistry.

[62]  C. Scheidereit.  IkappaB kinase complexes: gateways to NF-kappaB activation and transcription. , 2006, Oncogene.

[63]  E. Zandi,et al.  Direct phosphorylation of IkappaB by IKKalpha and IKKbeta: discrimination between free and NF-kappaB-bound substrate. , 1998, Science.

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

[65]  R. Huber,et al.  Phosphotransferase and substrate binding mechanism of the cAMP‐dependent protein kinase catalytic subunit from porcine heart as deduced from the 2.0 A structure of the complex with Mn2+ adenylyl imidodiphosphate and inhibitor peptide PKI(5‐24). , 1993, The EMBO journal.

[66]  Mike Rothe,et al.  IκB Kinase-β: NF-κB Activation and Complex Formation with IκB Kinase-α and NIK , 1997 .