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

Adam H Barczewski,Michael J Ragusa,Maria Pellegrini,Dale F Mierke

doi:10.1038/s41598-019-39588-2

Adam H Barczewski, Michael J Ragusa + Show 2 more

Open Access

https://doi.org/10.1038/s41598-019-39588-2

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Journal: Scientific Reports	Publication Date: Feb 27, 2019
Citations: 22	License type: open-access

Affiliation: Dartmouth College

Abstract

NEMO is an essential component in the activation of the canonical NF-κB pathway and exerts its function by recruiting the IκB kinases (IKK) to the IKK complex. Inhibition of the NEMO/IKKs interaction is an attractive therapeutic paradigm for diseases related to NF-κB mis-regulation, but a difficult endeavor because of the extensive protein-protein interface. Here we report the high-resolution structure of the unbound IKKβ-binding domain of NEMO that will greatly facilitate the design of NEMO/IKK inhibitors. The structures of unbound NEMO show a closed conformation that partially occludes the three binding hot-spots and suggest a facile transition to an open state that can accommodate ligand binding. By fusing coiled-coil adaptors to the IKKβ-binding domain of NEMO, we succeeded in creating a protein with improved solution behavior, IKKβ-binding affinity and crystallization compatibility, which will enable the structural characterization of new NEMO/inhibitor complexes.

Highlights

The nuclear factor κB (NF-κB) transcription factor is key to the regulation of multiple cellular processes, including cell proliferation and survival, B-cell and T-cell maturation, and inflammatory response[1]
We started from our previously reported construct of the minimal IKKβ binding domain of NF-κB essential modulator (NEMO), NEMO (44–111), fused to a homodimeric coiled-coil adaptor sequence based on GCN4 at both the N- and C-terminus[22]
We propose that the GCN4 adaptors stabilize the native structure of the NEMO IKKβ-binding domain to what the full-length protein does, while providing a construct amenable to crystallization

Summary

Introduction

The nuclear factor κB (NF-κB) transcription factor is key to the regulation of multiple cellular processes, including cell proliferation and survival, B-cell and T-cell maturation, and inflammatory response[1]. The NF-κB pathway presents multiple possible levels of intervention for inhibition, among which targeting the NF-κB inducers TNFα, IL-1 and IL-610,11, inhibition of cell surface receptors (e.g., TNFR, IL-1R)[12,13], inhibition of IKKβ, inhibition of IκBα degradation[14,15] or further downstream inhibition of NF-κB nuclear translocation or DNA binding[16]. The structure displays a four helical bundle in which the two helices of the NEMO (44–111) dimer are intercalated by the two helices of IKKβ with an extensive interaction interface. Analysis of this structure coupled with Ala-scanning mutagenesis identified three hot-spot regions for binding within IKKβ, with the strongest interaction occurring at the very C-terminus of IKKβ (residues 734–742)[20].

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