The IKK-binding domain of NEMO is an irregular coiled coil with a dynamic binding interface

Area of Science:

• Molecular Biology
• Structural Biology
• Immunology

Background:

• NEMO is vital for activating the canonical NF-κB pathway by recruiting IκB kinases (IKK).
• Inhibiting the NEMO/IKKs interaction is a therapeutic strategy for NF-κB-related diseases.
• The extensive protein-protein interface presents challenges for inhibitor design.

Purpose of the Study:

• To determine the high-resolution structure of the unbound IKKβ-binding domain of NEMO.
• To facilitate the design of novel NEMO/IKK inhibitors.
• To engineer a NEMO construct with improved properties for structural studies.

Main Methods:

• High-resolution structural determination of the unbound IKKβ-binding domain of NEMO.
• Analysis of NEMO conformation and potential ligand-binding states.
• Protein engineering by fusing coiled-coil adaptors to the NEMO IKKβ-binding domain.

Main Results:

• The structure of unbound NEMO reveals a closed conformation that partially hides binding sites.
• This conformation suggests an accessible transition to an open state for ligand binding.
• Engineered NEMO protein exhibits enhanced solution behavior, IKKβ-binding affinity, and crystallization compatibility.

Conclusions:

• The determined NEMO structure provides a crucial template for designing NEMO/IKK inhibitors.
• The engineered NEMO construct enables future structural characterization of NEMO/inhibitor complexes.
• This work advances therapeutic strategies for NF-κB pathway mis-regulation.