BIROn - Birkbeck Institutional Research Online

    Probing the solution structure of IκB Kinase (IKK) subunit γ and its interaction with Kaposi's Sarcoma Associated Herpes Virus Flice Interacting Protein and IKK Subunit β by EPR Spectroscopy

    Bagneris, C and Rogala, K and Baratchian, M. and Zamfir, V.S. and Kunze, M.B.A. and Dagles, S and Pirker, K.F. and Collins, M. and Hall, B.A. and Barrett, Tracey E. and Kay, C.W.M. (2015) Probing the solution structure of IκB Kinase (IKK) subunit γ and its interaction with Kaposi's Sarcoma Associated Herpes Virus Flice Interacting Protein and IKK Subunit β by EPR Spectroscopy. Journal of Biological Chemistry 290 , pp. 16539-16549. ISSN 0021-9258.

    [img]
    Preview
    Text
    jbc.M114.622928.full.pdf - Author's Accepted Manuscript

    Download (9MB) | Preview

    Abstract

    Viral flice interacting protein (vFLIP), encoded by the oncogenic Kaposi's sarcoma associated herpes virus (KSHV), constitutively activates the canonical nuclear factor kappa-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. Since 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 (EPR) spectroscopy combined with molecular modelling and molecular dynamics (MD) simulations revealed that IKKγ is a parallel coiled coil whose response to binding of vFLIP or IKKβ is localised 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.

    Metadata

    Item Type: Article
    Additional Information: This research was originally published in Journal of Biological Chemistry. © the American Society for Biochemistry and Molecular Biology
    Keyword(s) / Subject(s): electron paramagnetic resonance (EPR), molecular dynamics, molecular modeling, NF-kappa B (NF-KB), protein structure, virology, IKKgamma, NEMO, signalosome, vFLIP
    School: Birkbeck Schools and Departments > School of Science > Biological Sciences
    Research Centre: Structural Molecular Biology, Institute of (ISMB)
    Depositing User: Dr Tracey E Barrett
    Date Deposited: 17 Jun 2015 12:54
    Last Modified: 06 Dec 2016 10:41
    URI: http://eprints.bbk.ac.uk/id/eprint/12348

    Statistics

    Downloads
    Activity Overview
    47Downloads
    151Hits

    Additional statistics are available via IRStats2.

    Archive Staff Only (login required)

    Edit/View Item Edit/View Item