Advertisement

Probing the Solution Structure of IκB Kinase (IKK) Subunit γ and Its Interaction with Kaposi Sarcoma-associated Herpes Virus Flice-interacting Protein and IKK Subunit β by EPR Spectroscopy*

  • Claire Bagnéris
    Affiliations
    From the Department of Biological Sciences, Institute of Structural and Molecular Biology, Birkbeck College, University of London, London WC1E 7HX, United Kingdom
    Search for articles by this author
  • Kacper B. Rogala
    Footnotes
    Affiliations
    From the Department of Biological Sciences, Institute of Structural and Molecular Biology, Birkbeck College, University of London, London WC1E 7HX, United Kingdom

    Institute of Structural and Molecular Biology, Darwin Building, University College London, Gower Street, London WC1E 6BT, United Kingdom
    Search for articles by this author
  • Mehdi Baratchian
    Affiliations
    MRC Centre for Medical Molecular Virology, UCL Cancer Institute and National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Herts EN6 3QG, United Kingdom
    Search for articles by this author
  • Vlad Zamfir
    Affiliations
    From the Department of Biological Sciences, Institute of Structural and Molecular Biology, Birkbeck College, University of London, London WC1E 7HX, United Kingdom

    Institute of Structural and Molecular Biology, Darwin Building, University College London, Gower Street, London WC1E 6BT, United Kingdom
    Search for articles by this author
  • Micha B.A. Kunze
    Footnotes
    Affiliations
    Institute of Structural and Molecular Biology, Darwin Building, University College London, Gower Street, London WC1E 6BT, United Kingdom
    Search for articles by this author
  • Selina Dagless
    Affiliations
    Institute of Structural and Molecular Biology, Darwin Building, University College London, Gower Street, London WC1E 6BT, United Kingdom
    Search for articles by this author
  • Katharina F. Pirker
    Footnotes
    Affiliations
    Institute of Structural and Molecular Biology, Darwin Building, University College London, Gower Street, London WC1E 6BT, United Kingdom
    Search for articles by this author
  • Mary K. Collins
    Affiliations
    MRC Centre for Medical Molecular Virology, UCL Cancer Institute and National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Herts EN6 3QG, United Kingdom
    Search for articles by this author
  • Benjamin A. Hall
    Correspondence
    To whom correspondence may be addressed: MRC Cancer Unit, University of Cambridge, Hutchison/MRC Research Centre, Box 197, Cambridge Biomedical Campus, Cambridge, United Kingdom CB2 0XZ. Tel.: +44-0-1223-763268; Fax: 44-0-1223-763241
    Affiliations
    MRC Cancer Unit, Hutchison/MRC Research Centre, Cambridge Biomedical Campus, University of Cambridge, Cambridge CB2 0XZ, United Kingdom, and
    Search for articles by this author
  • Tracey E. Barrett
    Correspondence
    To whom correspondence may be addressed: Dept. of Biological Sciences, Institute of Structural and Molecular Biology, Birkbeck College, University of London, London WC1E 7HX, UK. Tel.: +44-0-207-631-6822; Fax: +44-0-207-631-6803
    Affiliations
    From the Department of Biological Sciences, Institute of Structural and Molecular Biology, Birkbeck College, University of London, London WC1E 7HX, United Kingdom
    Search for articles by this author
  • Christopher W.M. Kay
    Correspondence
    To whom correspondence may be addressed: Institute of Structural and Molecular Biology, Darwin Bldg, University College London, Gower St., London WC1E 6BT, UK. Tel.: +44-0-207-679-7312; Fax: +44-0-207-679-7096
    Affiliations
    Institute of Structural and Molecular Biology, Darwin Building, University College London, Gower Street, London WC1E 6BT, United Kingdom

    London Centre for Nanotechnology, University College London, 17-19 Gordon Street, London WC1H 0AH, United Kingdom
    Search for articles by this author
  • Author Footnotes
    * This work was funded by a Medical Research Council Grant (ID18218) (to T. E. B.), a Cancer Research UK Grant (A12595) and a UCL Cancer Institute grant (to M. K. C.), a Wellcome Trust PhD Studentship (to M. B. A. K.), and a Royal Society Fellowship (to B. A. H.). We declare that no conflict of interest arises from the work presented here.
    This article contains supplemental Table 1 and supplemental Movie 1.
    1 Current address: Dept. of Biochemistry, University of Oxford, South Parks Road, Oxford, UK.
    2 Current address: Structural Biology and NMR Laboratory, Dept. of Biology, University of Copenhagen, Copenhagen, Denmark
    3 Current address: Div. of Biochemistry, Dept. of Chemistry, BOKU - University of Natural Resources and Life Sciences, Muthgasse 18, Vienna, Austria.
Open AccessPublished:July 03, 2015DOI:https://doi.org/10.1074/jbc.M114.622928
      Viral flice-interacting protein (vFLIP), encoded by the oncogenic Kaposi sarcoma-associated herpes virus (KSHV), constitutively activates the canonical nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) pathway. This is achieved through subversion of the IκB kinase (IKK) complex (or signalosome), which involves a physical interaction between vFLIP and the modulatory subunit IKKγ. Although this interaction has been examined both in vivo and in vitro, the mechanism by which vFLIP activates the kinase remains to be determined. Because IKKγ functions as a scaffold, recruiting both vFLIP and the IKKα/β subunits, it has been proposed that binding of vFLIP could trigger a structural rearrangement in IKKγ conducive to activation. To investigate this hypothesis we engineered a series of mutants along the length of the IKKγ molecule that could be individually modified with nitroxide spin labels. Subsequent distance measurements using electron paramagnetic resonance spectroscopy combined with molecular modeling and molecular dynamics simulations revealed that IKKγ is a parallel coiled-coil whose response to binding of vFLIP or IKKβ is localized twisting/stiffening and not large-scale rearrangements. The coiled-coil comprises N- and C-terminal regions with distinct registers accommodated by a twist: this structural motif is exploited by vFLIP, allowing it to bind and subsequently activate the NF-κB pathway. In vivo assays confirm that NF-κB activation by vFLIP only requires the N-terminal region up to the transition between the registers, which is located directly C-terminal of the vFLIP binding site.

      Introduction

      Activation of the canonical nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)
      The abbreviations used are: NF-κB
      nuclear factor kappa-light-chain-enhancer of activated B cells
      vFLIP
      viral Flice-interacting protein
      KSHV
      Kaposi sarcoma-associated herpes virus
      IKK
      IκB kinase
      NEMO
      NF-κB essential modulator
      EPR
      electron paramagnetic resonance
      MD
      molecular dynamics
      TNF
      tumor necrosis factor
      TRAF
      TNF receptor-associated factors
      TEV
      tobacco etch virus
      IPTG
      isopropyl 1-thio-β-d-galactopyranoside
      CVs
      column volumes
      LV
      lentiviral vector
      RMSDs
      root mean square deviations.
      transcriptional pathway occurs in response to a wide variety of cellular stimuli, including cell differentiation, infection, and stress responses (
      • Scheidereit C.
      IκB kinase complexes: gateways to NF-κB activation and transcription.
      ). It is normally tightly regulated since constitutive activation can lead to prolonged cellular survival and the production of inflammatory cytokines both of which have been directly linked to cancer and inflammatory diseases. The pathway converges on a set of NF-κB transcription factors that in resting cells are localized to the cytoplasm because of their association with inhibitory IκB proteins (
      • DiDonato J.A.
      • Hayakawa M.
      • Rothwarf D.M.
      • Zandi E.
      • Karin M.
      A cytokine-responsive IκB kinase that activates the transcription factor NF-κB.
      ,
      • Mercurio F.
      • Zhu H.
      • Murray B.W.
      • Shevchenko A.
      • Bennett B.L.
      • Li J.
      • Young D.B.
      • Barbosa M.
      • Mann M.
      • Manning A.
      • Rao A.
      IKK-1 and IKK-2: cytokine-activated IκB kinases essential for NF-κB activation.
      • Israël A.
      The IKK complex, a central regulator of NF-κB activation.
      ). Liberation of the NF-κB transcription factors and their transition to the nucleus requires degradation of the IκBs that are first phosphorylated and subsequently targeted for proteolysis by the 26S proteasome following Lys-48 ubiquitination. Phosphorylation is facilitated by IκB kinase (IKK) or signalosome that minimally comprises the kinase subunits IKKα and/or IKKβ together with a modulatory element IKKγ (also known as NEMO: NF-κB essential modulator). This assembly is the target for several viruses since its constitutive activation results in the downstream overproduction of proteins that indirectly promote viral propagation and proliferation through their capacity to prolong cellular survival (
      • Hiscott J.
      • Nguyen T.L.
      • Arguello M.
      • Nakhaei P.
      • Paz S.
      Manipulation of the NF-κB pathway and the innate immune response by viruses.
      ,
      • Keller S.A.
      • Hernandez-Hopkins D.
      • Vider J.
      • Ponomarev V.
      • Hyjek E.
      • Schattner E.J.
      • Cesarman E.
      NF-κB is essential for the progression of KSHV- and EBV-infected lymphomas in vivo.
      ). One such virus is Kaposi sarcoma-associated herpes virus (KSHV) that during its latent phase encodes vFLIP (
      • Jenner R.G.
      • Albà M.M.
      • Boshoff C.
      • Kellam P.
      Kaposi's sarcoma-associated herpesvirus latent and lytic gene expression as revealed by DNA arrays.
      ). The pathogenicity of vFLIP appears to derive from its capacity to render IKK constitutively active by associating with its modulatory element IKKγ (
      • Field N.
      • Low W.
      • Daniels M.
      • Howell S.
      • Daviet L.
      • Boshoff C.
      • Collins M.
      KSHV vFLIP binds to IKKγ to activate IKK.
      ,
      • Matta H.
      • Sun Q.
      • Moses G.
      • Chaudhary P.M.
      Molecular genetic analysis of human herpes virus 8-encoded viral FLICE inhibitory protein-induced NF-κB activation.
      ). This prolonged activation has been directly linked to Kaposi sarcoma (KS) and other KSHV associated malignancies that includes primary effusion lymphoma (PELs) and multicentric Castleman disease where knockdown of vFLIP alone is sufficient to arrest the growth of KS tumors and kill PELs cells (
      • Guasparri I.
      • Keller S.A.
      • Cesarman E.
      KSHV vFLIP is essential for the survival of infected lymphoma cells.
      ,
      • Grossmann C.
      • Podgrabinska S.
      • Skobe M.
      • Ganem D.
      Activation of NF-κB by the latent vFLIP gene of Kaposi's sarcoma-associated herpesvirus is required for the spindle shape of virus-infected endothelial cells and contributes to their proinflammatory phenotype.
      ). The crystal structure of the vFLIP·IKKγ complex (
      • Bagnéris C.
      • Ageichik A.V.
      • Cronin N.
      • Wallace B.
      • Collins M.
      • Boshoff C.
      • Waksman G.
      • Barrett T.
      Crystal structure of a vFlip-IKKγ complex: insights into viral activation of the IKK signalosome.
      ) has revealed the nature of this interaction at the atomic level, which was later verified in vivo (
      • Shimizu A.
      • Baratchian M.
      • Takeuchi Y.
      • Escors D.
      • Macdonald D.
      • Barrett T.
      • Bagneris C.
      • Collins M.
      • Noursadeghi M.
      Kaposi's sarcoma-associated herpesvirus vFLIP and human T cell lymphotropic virus type 1 Tax oncogenic proteins activate IκB kinase subunit gamma by different mechanisms independent of the physiological cytokine-induced pathways.
      ). However, the mechanism by which vFLIP is able to activate the IKKα and IKKβ kinases remained unclear, especially because they associate with the N terminus of IKKγ, which is more than 200 residues from the vFLIP binding site. Furthermore, recent studies have shown that the ability of vFLIP to activate the canonical NF-κB pathway appears to be independent of up and downstream effectors such as: tumor necrosis factor (TNF) receptor-associated factors (TRAF)-2, TRAF-3, TRAF-6; linear ubiquitin chain assembly complex (LUBAC); and transforming growth factor (TGF)-β-activated kinase 1 (TAK1) that are all essential for the mechanisms utilized by pro-inflammatory cytokines (
      • Matta H.
      • Gopalakrishnan R.
      • Graham C.
      • Tolani B.
      • Khanna A.
      • Yi H.
      • Suo Y.
      • Chaudhary P.M.
      Kaposi's sarcoma associated herpesvirus encoded viral FLICE inhibitory protein K13 activates NF-κB pathway independent of TRAF6, TAK1 and LUBAC.
      ).
      It has been proposed that vFLIP activation of IKK involves conformational changes within the IKKγ molecule that effectively switches the assembly from an inactive to an active state that would favorably juxtapose IKKα/β for either trans or autophosphorylation (
      • Bagnéris C.
      • Ageichik A.V.
      • Cronin N.
      • Wallace B.
      • Collins M.
      • Boshoff C.
      • Waksman G.
      • Barrett T.
      Crystal structure of a vFlip-IKKγ complex: insights into viral activation of the IKK signalosome.
      ). Because there is currently neither a crystal structure of full-length IKKγ nor a fragment that encompasses both the kinase and vFLIP binding sites (either alone or in the relevant complexes) to allow direct testing of this hypothesis in terms of both local and global transitions, we used electron paramagnetic resonance (EPR) spectroscopy to determine distances between spin-labeled cysteine residues introduced at intervals along IKKγ. These allowed in silico models of IKKγ to be validated, enabling a solution structure of IKKγ to be obtained for the first time. Measurements were also performed in the presence of vFLIP and separately an IKKβ fragment comprising the IKKγ binding site to provide readout of any induced conformational changes.

      Discussion

      It has been established that constitutive activation of the canonical NF-κB pathway by the KSHV is pivotal to viral pathogenesis having been directly linked to KS and the other lymphoproliferative disorders. Although key to this process is a physical interaction between virally encoded vFLIP and IKK modulatory subunit IKKγ that has been extensively characterized, it remains unclear how persistent activation is achieved. It has been proposed that IKKγ is held in a configuration that promotes phosphorylation of the kinases (either through autophosphorylation or recruitment of upstream kinases) distinct to that of its unbound state in response to vFLIP binding (
      • Bagnéris C.
      • Ageichik A.V.
      • Cronin N.
      • Wallace B.
      • Collins M.
      • Boshoff C.
      • Waksman G.
      • Barrett T.
      Crystal structure of a vFlip-IKKγ complex: insights into viral activation of the IKK signalosome.
      ). This mechanism of IKK activation was first put forward for the T-cell leukemia virus oncoprotein TAX (
      • Hong S.
      • Wang L.C.
      • Gao X.
      • Kuo Y.L.
      • Liu B.
      • Merling R.
      • Kung H.J.
      • Shih H.M.
      • Giam C.Z.
      Heptad repeats regulate protein phosphatase 2a recruitment to I-κB kinase γ/NF-κB essential modulator and are targeted by human T-lymphotropic virus type 1 tax.
      ), a functional analogue of vFLIP.
      To test this hypothesis given the absence of a full-length structure of IKKγ (either in isolation or in complex with vFLIP and IKKα/β simultaneously), we used EPR spectroscopy to obtain distances between pairs and quartets of spin-labeled cysteines positioned at intervals along the length of the IKKγ molecule. The inter-label distance distributions observed for 56–95, 95–133, 133–169, and 158–186 toward the N terminus and 265–297 and 297–331 toward the C terminus are narrow, indicating a relatively rigid structure, while those observed for 186–218, 218–250, and 230–265 in the central region of the coiled-coil are broad indicating a more flexible structure. The distance distributions were subsequently used to generate a model of the entire IKKγ molecule as well as being analyzed for changes indicative of conformational rearrangements following incubation with either vFLIP or IKKβ. Apart from stiffening of the coiled-coil observed in the vicinity of the vFLIP binding site, our results demonstrate that IKKγ does not undergo gross structural reorganization in response to binding. Furthermore, although the twisting observed for 133–169 (and the denaturation found for 95–133) indicates that subtle changes appear to be transmitted toward the N terminus of the molecule, no effect is observed for 56–96 in the vicinity of the IKKβ binding site.
      The main structural insight gained is that to accommodate the change in register between the N and C termini, the region between them contains a twist located around residues Lys-246 to Ser-248. This builds tension into the structure as neither region can find a low energy conformation. As observed by x-ray crystallography, this region is essential for formation of the IKKγ·vFLIP complex (
      • Bagnéris C.
      • Ageichik A.V.
      • Cronin N.
      • Wallace B.
      • Collins M.
      • Boshoff C.
      • Waksman G.
      • Barrett T.
      Crystal structure of a vFlip-IKKγ complex: insights into viral activation of the IKK signalosome.
      ), which in turn leads to activation of the canonical NF-κB pathway. The D242R mutation in this region appears to mimic the effect of vFLIP binding: formation of a salt-bridge to Glu-240 might induce a subtle change in structure that results in constitutive activation. The EPR data also demonstrate that binding of vFLIP stiffens this region of IKKγ.
      Thus, it appears that although for the human cell, IKKγ is simply designed with different registers so that the N and C termini can perform their separate roles, namely kinase activity and regulation, respectively, the Kaposi sarcoma associated herpes virus has managed to exploit the structure of the transition between them in order to hijack the canonical NF-κB pathway.
      We note that bacterial chemoreceptors, another class of coiled-coil signaling proteins have been proposed to signal through subtle modifications of a frustrated domain (
      • Hall B.A.
      • Armitage J.P.
      • Sansom M.S.P.
      Mechanism of bacterial signal transduction revealed by molecular dynamics of Tsr dimers and trimers of dimers in lipid vesicles.
      ). Given the importance of this motif in signaling proteins, it is intriguing to speculate that this might be a common mechanism within the wider superfold family.
      Since the induction of conformational changes can now be excluded as a potential mechanism for vFLIP-mediated NF-κB activation, alternatives need to be considered. Potentially, vFLIP may function by recruiting as yet unknown cofactors to IKKγ or through blocking those that are known to down-regulate the pathway in pro-inflammatory cytokine induced mechanisms, for example phosphatases (
      • Hong S.
      • Wang L.C.
      • Gao X.
      • Kuo Y.L.
      • Liu B.
      • Merling R.
      • Kung H.J.
      • Shih H.M.
      • Giam C.Z.
      Heptad repeats regulate protein phosphatase 2a recruitment to I-κB kinase γ/NF-κB essential modulator and are targeted by human T-lymphotropic virus type 1 tax.
      ). The former seems less likely considering recent reports where the absence of several factors essential to cytokine-induced activation failed to diminish vFLIP's capacity to activate the pathway (
      • Matta H.
      • Gopalakrishnan R.
      • Graham C.
      • Tolani B.
      • Khanna A.
      • Yi H.
      • Suo Y.
      • Chaudhary P.M.
      Kaposi's sarcoma associated herpesvirus encoded viral FLICE inhibitory protein K13 activates NF-κB pathway independent of TRAF6, TAK1 and LUBAC.
      ). In agreement with this, truncation of IKKγ at Arg-254 in the region of altered helical register directly C-terminal to the vFLIP binding site has little impact on NF-κB activation. Interestingly, a crystal structure of IKKγ has recently been reported in complex with Hoip, a component of the linear ubiquitin chain assembly ligase essential for IKKγ polyubiquitination (
      • Fujita H.
      • Rahighi S.
      • Akita M.
      • Kato R.
      • Sasaki Y.
      • Wakatsuki S.
      • Iwai K.
      Mechanism underlying IκB kinase activation mediated by the linear ubiquitin chain assembly complex.
      ). The Hoip-IKKγ interface encompasses residues in close proximity to this region which may therefore have an important role in NF-κB activation by pro-inflammatory cytokines in contrast to vFLIP. Although the mechanism by which vFLIP activates the IKK complex remains unclear, given its apparent failure to induce anything but subtle conformational changes within IKKγ, we note that oligomerization of IKKβ is key to autophosphorylation (
      • Polley S.
      • Huang D.B.
      • Hauenstein A.V.
      • Fusco A.J.
      • Zhong X.
      • Vu D.
      • Schröfelbauer B.
      • Kim Y.
      • Hoffmann A.
      • Verma I.M.
      • Ghosh G.
      • Huxford T.
      A structural basis for IκB kinase 2 activation via oligomerization-dependent trans auto-phosphorylation.
      ). This may suggest that vFLIP functions to promote oligomerization of the IKKβ·IKKγ assemblies and that the D242R mutation mimics this action of vFLIP.

      Author Contributions

      C.B. performed cloning and site-directed mutagenesis. C.B., K.R., V.Z., and S.D. carried out expression and spin-labeling of proteins. C.B., K.R., V.Z., M.K., K.P., and C.K. performed the EPR spectroscopy. B.H. carried out the molecular modelling. M.B. performed in vivo assays. The manuscript was written by C.B., B.H., T.B., and C.K. with contributions from all authors. The study was conceived and supervised by M.C., T.B., and C.K.

      Acknowledgments

      We thank Professors Fabrice Agou and Alain Israël (Institut Pasteur, Paris, France) for their generous gift of the pRC-actin IKKβ plasmid that incorporated the full human gene, Professor Chris Boshoff (UCL) for kindly providing the pGEX-KT IKKγ plasmid, and Dr. Enrico Salvadori and Karen Fung (UCL) for helpful discussions.

      References

        • Scheidereit C.
        IκB kinase complexes: gateways to NF-κB activation and transcription.
        Oncogene. 2006; 25: 6685-6705
        • DiDonato J.A.
        • Hayakawa M.
        • Rothwarf D.M.
        • Zandi E.
        • Karin M.
        A cytokine-responsive IκB kinase that activates the transcription factor NF-κB.
        Nature. 1997; 388: 548-554
        • Mercurio F.
        • Zhu H.
        • Murray B.W.
        • Shevchenko A.
        • Bennett B.L.
        • Li J.
        • Young D.B.
        • Barbosa M.
        • Mann M.
        • Manning A.
        • Rao A.
        IKK-1 and IKK-2: cytokine-activated IκB kinases essential for NF-κB activation.
        Science. 1997; 278: 860-866
        • Israël A.
        The IKK complex, a central regulator of NF-κB activation.
        Cold Spring Harb. Perspect. Biol. 2010; 2: a000158
        • Hiscott J.
        • Nguyen T.L.
        • Arguello M.
        • Nakhaei P.
        • Paz S.
        Manipulation of the NF-κB pathway and the innate immune response by viruses.
        Oncogene. 2006; 25: 6844-6867
        • Keller S.A.
        • Hernandez-Hopkins D.
        • Vider J.
        • Ponomarev V.
        • Hyjek E.
        • Schattner E.J.
        • Cesarman E.
        NF-κB is essential for the progression of KSHV- and EBV-infected lymphomas in vivo.
        Blood. 2006; 107: 3295-3302
        • Jenner R.G.
        • Albà M.M.
        • Boshoff C.
        • Kellam P.
        Kaposi's sarcoma-associated herpesvirus latent and lytic gene expression as revealed by DNA arrays.
        J. Virol. 2001; 75: 891-902
        • Field N.
        • Low W.
        • Daniels M.
        • Howell S.
        • Daviet L.
        • Boshoff C.
        • Collins M.
        KSHV vFLIP binds to IKKγ to activate IKK.
        J. Cell Sci. 2003; 116: 3721-3728
        • Matta H.
        • Sun Q.
        • Moses G.
        • Chaudhary P.M.
        Molecular genetic analysis of human herpes virus 8-encoded viral FLICE inhibitory protein-induced NF-κB activation.
        J. Biol. Chem. 2003; 278: 52406-52411
        • Guasparri I.
        • Keller S.A.
        • Cesarman E.
        KSHV vFLIP is essential for the survival of infected lymphoma cells.
        J. Exp. Med. 2004; 199: 993-1003
        • Grossmann C.
        • Podgrabinska S.
        • Skobe M.
        • Ganem D.
        Activation of NF-κB by the latent vFLIP gene of Kaposi's sarcoma-associated herpesvirus is required for the spindle shape of virus-infected endothelial cells and contributes to their proinflammatory phenotype.
        J. Virol. 2006; 80: 7179-7185
        • Bagnéris C.
        • Ageichik A.V.
        • Cronin N.
        • Wallace B.
        • Collins M.
        • Boshoff C.
        • Waksman G.
        • Barrett T.
        Crystal structure of a vFlip-IKKγ complex: insights into viral activation of the IKK signalosome.
        Mol. Cell. 2008; 30: 620-631
        • Shimizu A.
        • Baratchian M.
        • Takeuchi Y.
        • Escors D.
        • Macdonald D.
        • Barrett T.
        • Bagneris C.
        • Collins M.
        • Noursadeghi M.
        Kaposi's sarcoma-associated herpesvirus vFLIP and human T cell lymphotropic virus type 1 Tax oncogenic proteins activate IκB kinase subunit gamma by different mechanisms independent of the physiological cytokine-induced pathways.
        J. Virol. 2011; 85: 7444-7448
        • Matta H.
        • Gopalakrishnan R.
        • Graham C.
        • Tolani B.
        • Khanna A.
        • Yi H.
        • Suo Y.
        • Chaudhary P.M.
        Kaposi's sarcoma associated herpesvirus encoded viral FLICE inhibitory protein K13 activates NF-κB pathway independent of TRAF6, TAK1 and LUBAC.
        PLoS One. 2012; 7: e36601
        • Pannier M.
        • Veit S.
        • Godt A.
        • Jeschke G.
        • Spiess H.W.
        Dead-time free measurement of dipole–dipole interactions between electron spins.
        J. Magn. Reson. 2000; 142: 331-340
        • Jeschke G.
        • Chechik V.
        • Ionita P.
        • Godt A.
        • Zimmermann H.
        • Banham J.
        • Timmel C.R.
        • Hilger D.
        • Jung H.
        DeerAnalysis2006—a comprehensive software package for analyzing pulsed ELDOR data.
        Appl. Magn. Reson. 2006; 30: 473-498
        • Marrink S.-J.
        • de Vries A.H.
        • Mark A.E.
        Coarse grained model for semiquantitative lipid simulations.
        J. Phys. Chem. B. 2004; 108: 750-760
        • Monticelli L.
        • Kandasamy S.K.
        • Periole X.
        • Larson R.G.
        • Tieleman D.P.
        • Marrink S.-J.
        The MARTINI coarse-grained force field: extension to proteins.
        J. Chem. Theory. Comput. 2008: 819-834
        • Pronk S.
        • Páll S.
        • Schulz R.
        • Larsson P.
        • Bjelkmar P.
        • Apostolov R.
        • Shirts M.R.
        • Smith J.C.
        • Kasson P.M.
        • van der Spoel D.
        • Hess B.
        • Lindahl E.
        GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit.
        Bioinformatics. 2013; 29: 845-854
        • Polyhach Y.
        • Bordignon E.
        • Jeschke G.
        Rotamer libraries of spin labelled cysteines for protein studies.
        Phys. Chem. Chem. Phys. 2011; 13: 2356-2366
        • Stansfeld P.J.
        • Sansom M.S.P.
        From coarse grained to atomistic: a serial multiscale approach to membrane protein simulations.
        J. Chem. Theory Comput. 2011; 7: 1157-1166
        • Rushe M.
        • Silvian L.
        • Bixler S.
        • Chen L.L.
        • Cheung A.
        • Bowes S.
        • Cuervo H.
        • Berkowitz S.
        • Zheng T.
        • Guckian K.
        • Pellegrini M.
        • Lugovskoy A.
        Structure of a NEMO/IKK-associating domain reveals architecture of the interaction site.
        Structure. 2008; 16: 798-808
        • Lo Y.-C.
        • Lin S.-C.
        • Rospigliosi C.C.
        • Conze D.B.
        • Wu C.-J.
        • Ashwell J.D.
        • Eliezer D.
        • Wu H.
        Structural basis for recognition of diubiquitins by NEMO.
        Mol. Cell. 2009; 33: 602-615
        • Rahighi S.
        • Ikeda F.
        • Kawasaki M.
        • Akutsu M.
        • Suzuki N.
        • Kato R.
        • Kensche T.
        • Uejima T.
        • Bloor S.
        • Komander D.
        • Randow F.
        • Wakatsuki S.
        • Dikic I.
        Specific recognition of linear ubiquitin chains by NEMO is important for NF-κB activation.
        Cell. 2009; 136: 1098-1109
        • Fujita H.
        • Rahighi S.
        • Akita M.
        • Kato R.
        • Sasaki Y.
        • Wakatsuki S.
        • Iwai K.
        Mechanism underlying IκB kinase activation mediated by the linear ubiquitin chain assembly complex.
        Mol. Cell. Biol. 2014; 34: 1322-1335
        • Lupas A.
        • Van Dyke M.
        • Stock J.
        Predicting coiled coils from protein sequences.
        Science. 1991; 252: 1162-1164
        • McDonnell A.V.
        • Jiang T.
        • Keating A.E.
        • Berger B.
        Paircoil2: improved prediction of coiled coils from sequence.
        Bioinformatics. 2006; 22: 356-358
        • Trigg J.
        • Gutwin K.
        • Keating A.E.
        • Berger B.
        Multicoil2: predicting coiled coils and their oligomerization states from sequence in the twilight zone.
        PLoS ONE. 2011; 6: e23519
        • Delorenzi M.
        • Speed T.
        An HMM model for coiled-coil domains and a comparison with PSSM-based predictions.
        Bioinformatics. 2002; 18: 617-625
        • Stafford R.L.
        • Tang M.-Y.
        • Sawaya M.R.
        • Phillips M.L.
        • Bowie J.U.
        Crystal structure of the central coiled-coil domain from human liprin-β2.
        Biochemistry. 2011; 50: 3807-3815
        • Eswar N.
        • Marti-Renom M.A.
        • Webb B.
        • Madhusudhan M.S.
        • Eramian D.
        • Shen M.
        • Pieper U.
        • Sali A.
        Comparative protein structure modeling with MODELLER.
        Curr. Protoc. Bioinform. 2006; 15: 5.6.1-5.6.30
        • Tolani B.
        • Matta H.
        • Gopalakrishnan R.
        • Punj V.
        • Chaudhary P.M.
        NEMO is essential for Kaposi's sarcoma-associated herpesvirus-encoded vFLIP K13-induced gene expression and protection against death receptor-induced cell death, and its N-terminal 251 residues are sufficient for this process.
        J. Virol. 2014; 88: 6345-6354
        • Hong S.
        • Wang L.C.
        • Gao X.
        • Kuo Y.L.
        • Liu B.
        • Merling R.
        • Kung H.J.
        • Shih H.M.
        • Giam C.Z.
        Heptad repeats regulate protein phosphatase 2a recruitment to I-κB kinase γ/NF-κB essential modulator and are targeted by human T-lymphotropic virus type 1 tax.
        J. Biol. Chem. 2007; 282: 12119-12126
        • Hall B.A.
        • Armitage J.P.
        • Sansom M.S.P.
        Mechanism of bacterial signal transduction revealed by molecular dynamics of Tsr dimers and trimers of dimers in lipid vesicles.
        PLoS Comput. Biol. 2012; 8: e1002685
        • Polley S.
        • Huang D.B.
        • Hauenstein A.V.
        • Fusco A.J.
        • Zhong X.
        • Vu D.
        • Schröfelbauer B.
        • Kim Y.
        • Hoffmann A.
        • Verma I.M.
        • Ghosh G.
        • Huxford T.
        A structural basis for IκB kinase 2 activation via oligomerization-dependent trans auto-phosphorylation.
        PLoS Biol. 2013; 11: e1001581

      Linked Article