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

IF 4.3 2区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Structure Pub Date : 2025-02-04 DOI:10.1016/j.str.2025.01.010

Amy E. Kennedy, Adam H. Barczewski, Christina R. Arnoldy, J. Pepper Pennington, Kelly A. Tiernan, M. Beatriz Hidalgo, Caroline C. Reilly, Tanyawan Wongsri, Michael J. Ragusa, Gevorg Grigoryan, Dale F. Mierke, Maria Pellegrini

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Abstract

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.

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用于筛选的NEMO结构揭示了抑制的新决定因素

NEMO是激活典型核因子κB （NF-κB）通路的重要组成部分，并通过向IKK复合体募集i -κB激酶（IKK）来发挥其功能。抑制NEMO/IKKs相互作用是治疗NF-κB错误调节相关疾病的一种有吸引力的治疗范例，但由于广泛的蛋白质-蛋白质界面，这是一项困难的努力。在这里，我们报告了NEMO的ikk结合结构域的新工程结构的设计和表征，编程使这个困难的蛋白质结构域适合核磁共振测量和结晶，同时保留其生物学功能。ZipNEMO结合IKKβ具有纳米摩尔亲和力，适用于异核核磁共振（NMR）技术和x射线晶体学结构测定。我们发现zipNEMO的核磁共振光谱允许检测抑制剂在溶液中的结合和共振分配。zipNEMO的晶体结构揭示了一种新的配体结合基序和结合口袋的适应性，并启发了新的肽抑制剂的设计。

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来源期刊

Structure 生物-生化与分子生物学

CiteScore

8.90

自引率

1.80%

发文量

155

审稿时长

3-8 weeks

期刊介绍： Structure aims to publish papers of exceptional interest in the field of structural biology. The journal strives to be essential reading for structural biologists, as well as biologists and biochemists that are interested in macromolecular structure and function. Structure strongly encourages the submission of manuscripts that present structural and molecular insights into biological function and mechanism. Other reports that address fundamental questions in structural biology, such as structure-based examinations of protein evolution, folding, and/or design, will also be considered. We will consider the application of any method, experimental or computational, at high or low resolution, to conduct structural investigations, as long as the method is appropriate for the biological, functional, and mechanistic question(s) being addressed. Likewise, reports describing single-molecule analysis of biological mechanisms are welcome. In general, the editors encourage submission of experimental structural studies that are enriched by an analysis of structure-activity relationships and will not consider studies that solely report structural information unless the structure or analysis is of exceptional and broad interest. Studies reporting only homology models, de novo models, or molecular dynamics simulations are also discouraged unless the models are informed by or validated by novel experimental data; rationalization of a large body of existing experimental evidence and making testable predictions based on a model or simulation is often not considered sufficient.