(B) Anti-NEMO (top) and anti-phospho-IκBα (bottom) Western blots of 293T NEMO KO cells transfected with the indicated FLAG-tagged NEMO expression vectors or an empty vector plasmid (–). Light blue residues indicate conservative amino acid substitutions, while orange residues indicate non-conservative substitutions. The sequence alignment was visualized and colored according to residue conservation using MView (EMBL-EBI) (56). Sequences were aligned with Clustal Omega (EMBL-EBI) using WT human NEMO as a reference sequence.

(D) Multiple sequence alignment for the IVD core region of NEMO and OPTN, from a broad spread of vertebrate proteins. (C) Structural comparison of NEMO and OPTN protein domain structures, with the black dotted line indicating a gap in the alignment. The red bars denote the location of the hot spot regions of the NEMO-IKKβ binding interaction (Fig. (B) Plot of the sequence conservation obtained using Rate4Site and the residue hydropathy calculated using the Kyte & Doolittle scale for human NEMO, represented by a moving average. HLX1/KBD, Helix 1/Kinase Binding Domain CC1/IVD, Coiled Coil 1/Intervening Domain HLX2, Helix 2 CC2, Coiled Coil 2 LZ, Leucine Zipper UBAN, Ubiquitin Binding in ABIN and NEMO domain ZF, Zinc Finger. (A) NEMO domain structure, with colored areas corresponding to the functional domains of NEMO and proposed unstructured regions represented by black lines. Overall, these results support a model in which the IVD of NEMO participates in signal-induced activation of the IKK/NF-κB pathway by acting as a mediator of conformational changes in NEMO. This conformational strain in the IVD mediates allosteric communication between N- and C-terminal regions of NEMO. Thermal and chemical denaturation studies of truncated NEMO variants indicate that the IVD, while not intrinsically destabilizing, can reduce the stability of surrounding regions of NEMO, due to the conflicting structural demands imparted on this region by flanking upstream and downstream domains. Moreover, inactivating mutations in this core region abrogate the ability of NEMO to form ubiquitin-induced liquid-liquid phase separation droplets in vitro and signal-induced puncta in vivo. We also show that an intact IVD is required for the formation of disulfide-bonded dimers of NEMO. We show that the analogous region of optineurin can functionally replace the core region of the NEMO IVD. Previous studies have shown that this central core region of the IVD is required for cytokine-induced activation of IκB kinase (IKK). Comparison of NEMO and the related protein optineurin from a variety of evolutionarily distant organisms revealed that a central region of NEMO, called the Intervening Domain (IVD), is conserved between NEMO and optineurin. Herein, we use comparative, biochemical, biophysical, molecular, and cellular approaches to investigate how the scaffold protein NEMO contributes to signaling in the NF-κB pathway. Scaffold proteins help mediate interactions between protein partners, often to optimize intracellular signaling.