Download A low resolution structure of a component of the Cytokine responsive

A Low Resolution Structure of a Component of the Cytokine
Responsive Kinase Complex IKappaB Kinase (IKK) using SANS
Joseph DiDonato
Cleveland Clinic, Ohio, USA
A Low Resolution Structure of a Component of the Cytokine Responsive Kinase Complex
IKappaB Kinase (IKK) using SANS Thomas Tallant1, Xavier Lee1, Joe Zaccai2, Roland
May2, Vitaliy Pipich3, Joseph DiDonato1,4* 1Department of Cell Biology, Lerner Research
Institute, Cleveland Clinic, Cleveland, Ohio 44195 USA 2Institute Laue-Langevin and EMBL
Grenoble, France 38042 3Munich Outstation of Juelich Research Center, Germany
4Department of Cardiovascular Medicine, Cleveland Clinic, Cleveland, Ohio 44195 The
IKappaB kinase (IKK) is a key regulator of the proinflammatory signaling cascade. It controls
the release of the transcription factor NF-kB from its inhibitor molecule IkB via
phosphorylation of conserved serine residues in IkB preferentially by the IKKbeta catalytic
subunit triggering IkB’s polyubiquitination and degradation by the 26S proteasome. Once NFkB is freed from IkB, it translocates into the nucleus where it activates expression of its target
genes acting as a key mediator of the proinflammatory gene response. IKK consist of two
catalytic components, IKKalpha and IKKbeta, and a third structural component, IKKgamma.
IKKgamma links upstream activation signals from the outer and inner cell membranes via
signal-induced site-specific phosphorylation and/or K63-linked ubiquitination events on
IKKgamma, leading to activation of the IKK complex. To date, there has been no
experimentally-derived high or low-resolution structural information on the IKK complex or
any of its components presented in the literature. Here we present for the first time structural
information on the isolated recombinant IKKgamma subunit using SANS. The recombinant
IKKgamma exists as a compact trimer which can be broken into a monomeric form using
antibodies directed against its amino-terminus. Insight into the potential structure of
IKKgamma in its “active” state was determined by SANS interrogation of two subdomains of
IKKgamma that when expressed transiently together in trans in IKKgamma deficient cells
restored basal IKK activation in the absence of stimulatory signals. These two subdomains
interact strongly with each other in vitro as determined by surface plasmon resonance (SPR)
using Biacore. Each subdomain individually forms a homo-dimer with itself and when mixed
with each other preferentially forms a hetero-dimer. SANS-determined shape of the
heterodimeric IKKgamma subdomains reveals an open extended structure compared to the
compact structure of the IKKgamma subunits present either in the IKK trimer or the
monomeric IKKgamma subunit in the monomeric IKKgamma-antibody complex. This closed
to open conformation of IKKgamma may indicate how posttranslational modifications of
IKKgamma in response to cell signals may trigger structural conformational changes in
IKKgamma that lead to IKK complex activation of the catalytic subunits IKKbeta and
IKKalpha.