The structure of a NEMO construct engineered for screening reveals novel determinants of inhibition

Summary 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 design and characterization of novel engineered constructs of the IKK-binding domain of NEMO, programmed to render this difficult protein domain amenable to NMR and X-ray characterization, while preserving the biological function. ZipNEMO binds IKKβ with nanomolar affinity, is amenable to heteronuclear NMR techniques and structure determination by X-ray crystallography. We show that NMR spectra of zipNEMO allow to detect inhibitor binding in solution and resonance assignment. The X-ray structure of zipNEMO highlights a novel ligand binding motif and the adaptability of the binding pocket and inspired the design of new peptide inhibitors. Competing Interest Statement A my E. Kennedy: Oxford Oxford Cryosystems, Hanover, NH, US Adam H. Barczewski: Incyte, Wilmington, DE, US Gevorg Grigoryan Generate Biomedicines, Inc., Arlington, MA, US We, Pellegrini, Grigoryan, Barczewski, have a patent application related to this work "ENGINEERED NEMO AND USES THEREOF". U.S. Patent Application No. 63/196,217