title: 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.
abstract: 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.
authors.affiliation: From the Department of Biological Sciences, Institute of Structural and Molecular Biology, Birkbeck College, University of London, London WC1E 7HX, United Kingdom.
authors.forename: Claire
authors.initials: C
authors.lastname: Bagnéris
authors.affiliation: 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.
authors.forename: Kacper B
authors.initials: KB
authors.lastname: Rogala
authors.affiliation: 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.
authors.forename: Mehdi
authors.initials: M
authors.lastname: Baratchian
authors.affiliation: 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.
authors.forename: Vlad
authors.initials: V
authors.lastname: Zamfir
authors.affiliation: Institute of Structural and Molecular Biology, Darwin Building, University College London, Gower Street, London WC1E 6BT, United Kingdom.
authors.forename: Micha B A
authors.initials: MB
authors.lastname: Kunze
authors.affiliation: Institute of Structural and Molecular Biology, Darwin Building, University College London, Gower Street, London WC1E 6BT, United Kingdom.
authors.forename: Selina
authors.initials: S
authors.lastname: Dagless
authors.affiliation: Institute of Structural and Molecular Biology, Darwin Building, University College London, Gower Street, London WC1E 6BT, United Kingdom.
authors.forename: Katharina F
authors.initials: KF
authors.lastname: Pirker
authors.affiliation: 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.
authors.forename: Mary K
authors.initials: MK
authors.lastname: Collins
authors.affiliation: MRC Cancer Unit, Hutchison/MRC Research Centre, Cambridge Biomedical Campus, University of Cambridge, Cambridge CB2 0XZ, United Kingdom, and bh418@mrc-cu.cam.ac.uk.
authors.forename: Benjamin A
authors.initials: BA
authors.lastname: Hall
authors.affiliation: From the Department of Biological Sciences, Institute of Structural and Molecular Biology, Birkbeck College, University of London, London WC1E 7HX, United Kingdom, t.barrett@mail.cryst.bbk.ac.uk.
authors.forename: Tracey E
authors.initials: TE
authors.lastname: Barrett
authors.affiliation: 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 c.kay@ucl.ac.uk.
authors.forename: Christopher W M
authors.identifier: http://orcid.org/0000-0002-5200-6004
authors.initials: CW
authors.lastname: Kay
mesh_terms: D020816:Amino Acid Motifs; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D019288:Herpesvirus 8, Human / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051550:I-kappa B Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012514:Sarcoma, Kaposi / Q000201:enzymology* / Q000235:genetics / Q000821:virology; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D014764:Viral Proteins; C481275:viral FLIP protein, Human herpesvirus 8; D051550:I-kappa B Kinase
keywords: IKKγ; NEMO; NF-kappa B (NF-κB); electron paramagnetic resonance (EPR); molecular dynamics; molecular modeling; protein structure; signalosome; vFLIP; virology