HDX-MS Analysis of Catalytic Activation of IKK2 in the IκB Kinase Complex

The IκB Kinase (IKK) complex,
containing catalytic
IKK2 and
noncatalytic NEMO subunits, plays essential roles in the induction
of transcription factors of the NF-κB family. Catalytic activation
of IKK2 via phosphorylation of its activation loop is promoted upon
noncovalent association of linear or K63-linked polyubiquitin chains
to NEMO within the IKK complex. The mechanisms of this activation
remain speculative. To investigate interaction dynamics within the
IKK complex during activation of IKK2, we conducted hydrogen–deuterium
exchange coupled with mass spectrometry (HDX-MS) on NEMO and IKK2
proteins in their free and complex-bound states. Altered proton exchange
profiles were observed in both IKK2 and NEMO upon complex formation,
and changes were consistent with the involvement of distinct regions
throughout the entire length of both proteins, including previously
uncharacterized segments, in direct or allosteric interactions. Association
with linear tetraubiquitin (Ub4) affected multiple regions
of the IKK2:NEMO complex, in addition to previously identified interaction
sites on NEMO. Intriguingly, observed enhanced solvent accessibility
of the IKK2 activation loop within the IKK2:NEMO:Ub4 complex,
coupled with contrasting protection of surrounding segments of the
catalytic subunit, suggests an allosteric role for NEMO:Ub4 in priming IKK2 for phosphorylation-dependent catalytic activation.