Conformational change in human IKK2 permits dimers to form higher-order oligomers that support interaction between kinase domains and promote activation through trans auto-phosphorylation.

Summary Besides activating NFκB by phosphorylating IκBs, IKKα/IKKβ kinases are also involved in regulating metabolic insulin signaling, the mTOR pathway, Wnt signaling, and autophagy. How IKKβ enzymatic activity is targeted to stimulus-specific substrates has remained unclear. We show here that NEMO, known to be essential for IKKβ activation by inflammatory stimuli, is also a specificity factor that directs IKKβ activity towards IκBα. Physical interaction and functional competition studies with mutant NEMO and IκB proteins indicate that NEMO functions as a scaffold to recruit IκBα to IKKβ. Interestingly, expression of NEMO mutants that allow for IKKβ activation by the cytokine IL-1 but fail to recruit IκBs, results in hyperphosphorylation of alternative IKKβ substrates. Furthermore IKK's function in autophagy, which is independent of NFκB is significantly enhanced without NEMO as IκB scaffold. Our work establishes a role for scaffolds such as NEMO in determining stimulus-specific signal transduction via the pleiotropic signaling hub IKK.

Highlights d A 3.5-Å cryo-EM structure of the filamentous form of DNAbound SgrAI d Conformational changes play a role in catalytic Mg 2+ stabilization d New configuration explains enzyme activation in filamentous state d Indirect readout of DNA structure associated with SgrAI filamentation and activity

SummaryDistinct signaling pathways activate the NF-κB family of transcription factors. The canonical NF-κB-signaling pathway is mediated by IκB kinase 2/β (IKK2/β), while the non-canonical pathway depends on IKK1/α. The structural and biochemical bases for distinct signaling by these otherwise highly similar IKKs are unclear. We report single-particle cryoelectron microscopy (cryo-EM) and X-ray crystal structures of human IKK1 in dimeric (∼150 kDa) and hexameric (∼450 kDa) forms. The hexamer, which is the representative form in the crystal but comprises only ∼2% of the particles in solution by cryo-EM, is atrimer of IKK1 dimers. While IKK1 hexamers are not detectable in cells, the surface that supports hexamer formation is critical for IKK1-dependent cellular processing of p100 to p52, the hallmark of non-canonical NF-κB signaling. Comparison of this surface to that in IKK2 indicates significant divergence, and it suggests a fundamental role for this surface in signaling by these kinases through distinct pathways.