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Updated: Feb 19, 2026
Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A
Published on: June 10, 2020
Evidence for M1-Linked Polyubiquitin-Mediated Conformational Change in NEMO
Arthur V Hauenstein1, Guozhou Xu1, Venkataraman Kabaleeswaran1
1Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, and Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA 02115, United States.
Insights
NF-κB essential modulator (NEMO) adopts a compact structure, suggesting auto-inhibition. Long M1-linked polyubiquitin chains may allosterically activate NEMO during inflammatory signaling.
Area of Science:
- Molecular Biology
- Structural Biology
- Immunology
Background:
- NF-κB essential modulator (NEMO) is crucial for inflammatory responses.
- NEMO is the scaffolding subunit of the IKK holocomplex, activating IKKα and IKKβ.
- Full-length NEMO structure is challenging due to conformational plasticity.
Purpose of the Study:
- To determine the overall dimensions of full-length NEMO.
- To investigate the mechanism of ubiquitin binding to NEMO.
- To explore the regulation of NEMO activity in the resting and signaling states.
Main Methods:
- In-line size exclusion chromatography-small-angle X-ray scattering (SEC-SAXS) for structural analysis.
- Mapping of ubiquitin binding interfaces on NEMO.
- Testing NEMO binding with different polyubiquitin chain types and lengths.
Main Results:
- Full-length NEMO exhibits a compact conformation (Dmax=320Å), contrary to predictions for an extended coiled-coil.
- A specific region (residues 112-150) in NEMO's coiled-coil 1 domain inhibits linear di-ubiquitin binding.
- Binding of longer M1-linked polyubiquitin chains, but not K63-linked chains, overcomes this inhibition.
Conclusions:
- NEMO may exist in an auto-inhibited state via intramolecular interactions.
- Allosteric activation of NEMO by long M1-linked polyubiquitin chains is proposed for signaling.
- These findings offer insights into the regulation of canonical inflammatory pathways.
Abstract:
The NF-κB essential modulator (NEMO) is the scaffolding subunit of the inhibitor of κB kinase (IKK) holocomplex and is required for the activation of the catalytic IKK subunits, IKKα and IKKβ, during the canonical inflammatory response. Although structures of shorter constructs of NEMO have been solved, efforts to elucidate the full-length structure of NEMO have proved difficult due to its apparent high conformational plasticity. To better characterize the gross dimensions of full-length NEMO, we employed in-line size exclusion chromatography-small-angle X-ray scattering. We show that NEMO adopts a more compact conformation (Dmax=320Å) than predicted for a fully extended coiled-coil structure (>500Å). In addition, we map a region of NEMO (residues 112-150) in its coiled-coil 1 domain that impedes the binding of linear (M1-linked) di-ubiquitin to its coiled-coil 2-leucine zipper ubiquitin binding domain. This ubiquitin binding inhibition can be overcome by a longer chain of linear, but not K63-linked polyubiquitin. Collectively, these observations suggest that NEMO may be auto-inhibited in the resting state by intramolecular interactions and that during signaling, NEMO may be allosterically activated by binding to long M1-linked polyubiquitin chains.
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