Probing the Solution Structure of I Kappa B Kinase (IKK) Subunit gamma and Its Interaction with Kaposi Sarcoma-associated Herpes Virus Flice-interacting Protein and IKK Subunit beta by EPR Spectroscopy<inline-graphic xlink:href="sbox.jpg"/>

• Published in: The Journal of biological chemistry Vol. 290; no. 27; pp. 16539 - 16549
• Main Authors: Bagneris, Claire, Rogala, Kacper B, Baratchian, Mehdi, Zamfir, Vlad, Kunze, Micha BA, Dagless, Selina, Pirker, Katharina F, Collins, Mary K, Hall, Benjamin A, Barrett, Tracey E, Kay, Christopher WM
• Format: Journal Article
• Language: English
• Published: 04.05.2015
• Subjects: Herpesvirus; Kaposi's sarcoma-associated herpesvirus
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M114.622928

Background: The I Kappa B kinase subunit gamma (IKK gamma ) is an essential modulator of the IKK signalosome whose solution structure is unsolved. Results: IKK gamma is a parallel coiled-coil comprising two registers accommodated by a twist. Conclusion: The response of IKK gamma to binding of signaling partners is twisting and stiffening, rather than major rearrangement. Significance: Viral activation of IKK gamma exploits the structural motif of the twist. Viral flice-interacting protein (vFLIP), encoded by the oncogenic Kaposi sarcoma-associated herpes virus (KSHV), constitutively activates the canonical nuclear factor Kappa -light-chain-enhancer of activated B cells (NF- Kappa B) pathway. This is achieved through subversion of the I Kappa B kinase (IKK) complex (or signalosome), which involves a physical interaction between vFLIP and the modulatory subunit IKK gamma . 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 IKK gamma functions as a scaffold, recruiting both vFLIP and the IKK alpha / beta subunits, it has been proposed that binding of vFLIP could trigger a structural rearrangement in IKK gamma conducive to activation. To investigate this hypothesis we engineered a series of mutants along the length of the IKK gamma 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 IKK gamma is a parallel coiled-coil whose response to binding of vFLIP or IKK beta 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- Kappa B pathway. In vivo assays confirm that NF- Kappa B 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.