A Central Region of NF-κB Essential Modulator Is Required for IKKβ-Induced Conformational Change and for Signal Propagation

Abstract

NF-κB essential modulator (NEMO) regulates NF-κB signaling by acting as a scaffold for the kinase IKKβ to direct its activity toward the NF-κB inhibitor, IκBα. Here, we show that a highly conserved central region of NEMO termed the intervening domain (IVD, amino acids 112−195) plays a key role in NEMO function. We determined a structural model of full-length NEMO by small-angle X-ray scattering and show that full-length, wild-type NEMO becomes more compact upon binding of a peptide comprising the NEMO binding domain of IKKβ (amino acids 701−745). Mutation of conserved IVD residues (9SG-NEMO) disrupts this conformational change in NEMO and abolishes the ability of NEMO to propagate NF-κB signaling in cells, although the affinity of 9SG-NEMO for IKKβ compared to that of the wild type is unchanged. On the basis of these results, we propose a model in which the IVD is required for a conformational change in NEMO that is necessary for its ability to direct phosphorylation of IκBα by IKKβ. Our findings suggest a molecular explanation for certain disease-associated mutations within the IVD and provide insight into the role of conformational change in signaling scaffold proteins.

CRISPR/Cas9-based editing of a sensitive transcriptional regulatory element to achieve cell type-specific knockdown of the NEMO scaffold protein

“…Re-expression of NEMO proteins with mutations that are found in some human disease patients has variable abilities to restore TNFα-induced phosphorylation of IκBα: mutants D113N, R123W, and Q183H were not statistically different than wild-type NEMO, whereas mutants L153R, R173G and R175P had statistically reduced activity as compared to wild-type NEMO (Fig 3C). Consistent with our previous results [22], expression of a NEMO mutant (9-SG) with an insertion in a core domain of NEMO did not restore TNFα-induced phosphorylation of IκBα (Fig 3C). Taken together, these results indicate that clone 1 cells are specifically defective for stimulus-based activation of canonical NF-κB signaling, and that TNFα-induced activation of NF-κB signaling in clone 1 cells can be restored by re-expression of wild-type NEMO.…”

“…Moreover, the ability of transfected NEMO mutants to support TNFα-induced phosphorylation of IκBα was consistent with their reported activities from previous papers. That is, mutants 9-SG, L153R, R173G, and R175P showed limited activity in 1.1 cells and in previous reports [22, 36, 37, 38], whereas mutants D113N, R123W, and Q183H were nearly as active as wild-type NEMO in both 1.1 cells and previous reports [39].…”

Regulatory subunit NEMO promotes polyubiquitin-dependent induction of NF-κB through a targetable second interaction with upstream activator IKK2

“…For example, the N-terminal KBD of NEMO binds the IKK2 NBD to form IKK 2 –NEMO 2 heterotetramers in solution. The central Ub-binding CC2 domain of NEMO, which spans residues 259 to 360, can form homodimers on its own, though a NEMO fragment containing both the CC2 and contiguous regions destabilizes CC2 homodimerization suggesting that neighboring portions of NEMO may antagonize dimerization of one another ( 40 , 41 ). Located C-terminal to the CC2 domain of NEMO is the polypeptide sequence QRRSPP (amino acid residues 384–389) that we have identified as mediating the linear poly-Ub–dependent second interaction between NEMO and IKK2.…”

Scaffold proteins as dynamic integrators of biological processes

“…Meanwhile, ubiquitin (primarily M1-linear ubiquitin) binds to the UBAN domain of NEMO (aa 289–320) ( 39 ). Due to its propensity to aggregate when expressed in bacteria, a crystal structure of full-length NEMO has yet to be solved; however, analytical ultracentrifugation of NEMO aa 1 to 355 ( 40 ) and SAXS of full-length NEMO in solution ( 41 ) have shown that unliganded NEMO is an extended coiled coil ( Fig. 3 , A and B ), suggesting that NEMO must undergo structural rearrangement to bring N-terminally bound IKK and C-terminally bound IκB into proximity for the phosphorylation of IκB.…”

“…3 A ). For example, biophysical studies of full-length NEMO indicate that NEMO undergoes conformational changes following binding to IKKβ or linear ubiquitin ( 41 , 42 , 43 ). Specifically, it has been shown through 8-anilinonaphthalene-1-sulfonic acid/tryptophan fluorescence emission that incubation of NEMO with IκB and long ubiquitin chains induces a shift in emission of NEMO that is consistent with solvent exposure of hydrophobic residues ( 43 ).…”