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bioRxiv · 10.1101/2024.07.18.604176

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

Kenedy, A. E.·Barczewski, A. H.·Arnoldy, C. R.·Pennington, J. P.·Tiernan, K. A.·Hidalgo, M. B.·Reilly, C. C.·Ragusa, M. J.·Grigoryan, G.·Mierke, D. F.·Pellegrini, M.
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Abstract

NEMO is an essential component in the activation of the canonical NF-{kappa}B pathway and exerts its function by recruiting the I{kappa}B kinases (IKK) to the IKK complex. Inhibition of the NEMO/IKKs interaction is an attractive therapeutic paradigm for diseases related to NF-{kappa}B 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 IKK{beta} 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.

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Kenedy, A. E., Barczewski, A. H., Arnoldy, C. R., Pennington, J. P., Tiernan, K. A., Hidalgo, M. B., Reilly, C. C., Ragusa, M. J., Grigoryan, G., Mierke, D. F., Pellegrini, M.. 2024-07-22. The structure of a NEMO construct engineered for screening reveals novel determinants of inhibition. https://doi.org/10.1101/2024.07.18.604176

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