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NEMO is an essential component in the activation of the canonical nuclear factor κB (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 measurements and crystallization, while preserving its biological function. ZipNEMO binds IKKβ with nanomolar affinity, is amenable to heteronuclear nuclear magnetic resonance (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 crystal structure of zipNEMO reveals a novel ligand binding motif and the adaptability of the binding pocket and inspired the design of new peptide inhibitors., Competing Interests: Declaration of interests A.E.K. is affiliated to Oxford Cryosystems, Hanover, NH, US. A.H.B. is affiliated to Incyte, Wilmington, DE, US. G.G. is affiliated to Generate Biomedicines, Inc., Arlington, MA, US. M.P., G.G., and A.H.B. have a patent application related to this work “ENGINEERED NEMO AND USES THEREOF” US Patent Application No. 63/196,217., (Copyright © 2025 Elsevier Inc. All rights reserved.)

A de novo lanthanide-binding coiled-coil polypeptide (MB1-2) was previously reported to self-assemble into a trimeric complex upon addition of Tb 3+ with a micromolar range dissociation constant. This study examines the effect of substitution of hydrophobic residues in heptad repeats of MB1-2 on the thermodynamic stability of the resulting Tb-peptide complex. Substitution of isoleucine to norleucine in each heptad repeat was assessed considering the greater accessible surface area of the latter and predicted increased hydrophobic interaction. Job's method of continuous variation using circular dichroism spectroscopy suggests a trimeric structure for the analog complex equivalent to that formed by MB1-2. The dissociation constant and CD spectra suggest that complex formation in the analog is more favorable as a result of ligand preorganization. In addition, thermal denaturation suggests greater stability of the Tb-MB1-2 Nle complex in comparison to the parent Tb-MB1-2. These results indicate improved stability of the complex class can be achieved through heptad repeat amino acid substitutions that increase peptide interchain interaction., (© 2024 European Peptide Society and John Wiley & Sons Ltd.)