1. DARPin-assisted crystallography of the CC2-LZ domain of NEMO reveals a coupling between dimerization and ubiquitin binding
- Author
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Alain Israël, Muriel Delepierre, Ahmed Haouz, Jeanne Chiaravalli, Bertrand Raynal, Fabrice Agou, Elisabeth Fontan, Florence Cordier, Olivera Grubisha, Michel Véron, Stéphane Duquerroy, Monika Kaminska, Biochimie Structurale et Cellulaire, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Virologie Structurale, Université Paris-Sud - Paris 11 (UP11), Résonance Magnétique Nucléaire des Biomolécules, Cristallogenèse et Diffraction des Rayons X (Plate-forme/PF6), Biophysique des Macromolécules et de leurs Interactions, Signalisation Moléculaire et Activation Cellulaire (SMAC), and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
Protein Folding ,Ubiquitin binding ,Mutant ,MESH: NF-kappa B ,MESH: Amino Acid Sequence ,Crystallography, X-Ray ,MESH: Ankyrin Repeat ,Mice ,MESH: Mutant Proteins ,MESH: Protein Structure, Tertiary ,0302 clinical medicine ,Protein structure ,MESH: Structure-Activity Relationship ,Structural Biology ,MESH: Animals ,Polyubiquitin ,Peptide sequence ,0303 health sciences ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,Protein Stability ,Chemistry ,MESH: Protein Multimerization ,Intracellular Signaling Peptides and Proteins ,NF-kappa B ,Ankyrin Repeat ,I-kappa B Kinase ,DARPin ,Signal transduction ,Protein Binding ,MESH: Mutation ,MESH: Ubiquitin ,MESH: Protein Folding ,Molecular Sequence Data ,Cell Line ,Structure-Activity Relationship ,03 medical and health sciences ,MESH: Protein Stability ,MESH: Intracellular Signaling Peptides and Proteins ,Animals ,Humans ,Structure–activity relationship ,MESH: Protein Binding ,MESH: Lysine ,Amino Acid Sequence ,MESH: I-kappa B Kinase ,Molecular Biology ,MESH: Mice ,030304 developmental biology ,MESH: Molecular Sequence Data ,MESH: Humans ,Tumor Necrosis Factor-alpha ,Ubiquitin ,Lysine ,MESH: Polyubiquitin ,MESH: Crystallography, X-Ray ,Protein Structure, Tertiary ,MESH: Cell Line ,Crystallography ,MESH: Tumor Necrosis Factor-alpha ,Mutation ,Mutant Proteins ,Ankyrin repeat ,Protein Multimerization ,030217 neurology & neurosurgery - Abstract
International audience; NEMO is an integral part of the IkappaB kinase complex and serves as a molecular switch by which the NF-kappaB signaling pathway can be regulated. Oligomerization and polyubiquitin (poly-Ub) binding, mediated through the regulatory CC2-LZ domain, were shown to be key features governing NEMO function, but the relationship between these two activities remains unclear. In this study, we solved the structure of this domain in complex with a designed ankyrin repeat protein, which helps its crystallization. We generated several NEMO mutants in this domain, including those associated with human diseases incontinentia pigmenti and immunodeficiency with or without anhidrotic ectodermal dysplasia. Analytical ultracentrifugation and thermal denaturation experiments were used to evaluate the dimerization properties of these mutants. A fluorescence-based assay was developed, as well, to quantify the interaction to monoubiquitin and poly-Ub chains. Moreover, the effect of these mutations was investigated for the full-length protein. We show that a proper folding of the ubiquitin-binding domain, termed NOA/UBAN/NUB, into a stable coiled-coil dimer is required but not sufficient for efficient interaction with poly-Ub. In addition, we show that binding to poly-Ub and, to a lesser extent, to monoubiquitin increases the stability of the NOA coiled-coil dimer. Collectively, these data provide structural insights into how several pathological mutations within and outside of the CC2-LZ's NOA ubiquitin binding site affect IkappaB kinase activation in the NF-kappaB signaling pathway.
- Published
- 2010
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