UC San Diego

The NF-κB signaling pathway plays a central role in immune response and normal cell function. A key regulator of the pathway is the IKK complex. Currently, understanding of the exact mechanism by which the IKK complex assembles remains incomplete. Linear ubiquitin chains alongside the IKK1, IKK2, and NEMO subunits of the complex have been well established as necessary for pathway activation. Phosphorylation of Ser177 and Ser181 on IKK2 subunits is a critical event in the activation of the complex. In efforts to understand the recruitment of each subunit in forming an active kinase, HDX-MS was used to characterize the dynamics of a model NEMO:IKK2+Ub4 complex. We confirmed previously established binding regions in both NEMO and IKK2. Linear Ub4 was found to enhance NEMO:IKK2 inter-subunit interactions and induced long-range conformational changes in IKK2. One alteration to the dynamics of the kinase domain’s activation loop exposed its critical serine residues. These changes support the model that activation of IKK2 can occur via trans-autophosphorylation, which requires both IKK2 and Ub4-bound NEMO.