A Conserved Core Region of the Scaffold NEMO is Essential for Signal-induced Conformational Change and Liquid-liquid Phase Separation

Scaffold proteins help mediate interactions between protein partners, often to optimize intracellular signaling. 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. 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. Previous studies have shown that this central core region of the IVD is required for cytokine-induced activation of IκB kinase (IKK). We show that the analogous region of optineurin can functionally replace the core region of the NEMO IVD. We also show that an intact IVD is required for the formation of disulfide-bonded dimers of NEMO. 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. 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. This conformational strain in the IVD mediates allosteric communication between N- and C-terminal regions of NEMO. 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.

. NEMO/IKK interaction hot spots. The crystal structure of the NEMO/IKK interface (PDB ID 3brv). IKK is in red, with the residues found to be involved in hot spot interactions highlighted in bright red and labelled. NEMO is in blue, with regions involved in the hot spot interactions highlighted in teal.  Figure S4. Comparison of the 7XAla and 9SG-7XAla NEMO proteins. Domain maps of 7XAla-NEMO and 9SG (7XAla background) constructs used in this study. Orange numbers represent the seven native cysteines that were changed to alanine (11,54,76,95,131,167,347) in both constructs. Orange letters indicate the mutation of residues 145-153 to the Ser-Gly repeat SGSGSGSGS.  to a concentration of 0.5%. Samples were then vortexed, and pelleted at 800 x g for 5 min at 4°C. The cytosolic fraction was collected from the supernatant, while the nuclear pellet was washed with hypotonic buffer and re-pelleted. The nuclear pellet was re-suspended in hypertonic buffer (20 mM HEPES pH 7.9, 1.5 mM MgCl2, 0.2 mM EDTA, 420 mM NaCl, 25% v/v glycerol) and rocked for 1 h. The nuclear extract was clarified by centrifugation at 13,000 rpm for 30 min.

Figure S8. Constructs displaying no visible cooperative chemical denaturation event, as monitored via CD.
Shown is the CD-monitored chemical denaturation using urea as the denaturant, measuring the loss in secondary structure of the indicated NEMO constructs as an increase in the 222 nm signal.
Has a 5' EcoRI and 3' BamHI sites for excision. Synthesized by GenScript.

pET-SUMO-NEMO-310-419
TA overhang PCR fragment containing amino acids 310-419 of human NEMO using primers 310-F and 419-R. Fragment was then subcloned into compatible TA linearized pET SUMO vector.

pET-SUMO-NEMO-344-419
TA overhang PCR fragment containing amino acids 344-419 of human NEMO using primers 344-F and 419-R. Fragment was then subcloned into compatible TA linearized pET SUMO vector.