DARPin-Assisted Crystallography of the CC2-LZ Domain of NEMO Reveals a Coupling between Dimerization and Ubiquitin Binding

Nuclear initiated NF-κB signaling: NEMO and ATM take center stage

“…Recent structural analyses of NEMO subdomains with or without different binding partners [85][86][87][88][89][90] suggest that recombinant NEMO forms an elongated parallel coiled-coil dimer that is predicted to elute in gel filtration with an apparent value of about 270 kDa [90]. Experimental evidence provided by Agou et al [91] showed that the elution profile of recombinant NEMO protein in gel filtration chromatography corresponds to a 500-kDa globular protein with a 73 Å Stokes radius.…”

Kaposi's Sarcoma-Associated Herpesvirus vFLIP and Human T Cell Lymphotropic Virus Type 1 Tax Oncogenic Proteins Activate IκB Kinase Subunit γ by Different Mechanisms Independent of the Physiological Cytokine-Induced Pathways

“…This response was heterogeneous, however, as some cells ( (1). Cytokine activation of IKK involves the fragment of IKK␥ spanning residues 254 to 337, which also forms a parallel coiled coil and changes conformation upon binding linear diubiquitin (8,14,17). Thus, the simplest model for activation of IKK is that much of IKK␥ forms a parallel coiled coil that changes conformation upon vFLIP, Tax, or ubiquitin binding, leading to a change in proximity or conformation of the IKK␣ and ␤ kinase subunits.…”

Two-sided Ubiquitin Binding of NF-κB Essential Modulator (NEMO) Zinc Finger Unveiled by a Mutation Associated with Anhidrotic Ectodermal Dysplasia with Immunodeficiency Syndrome

“…None of these mutations leading to defects of NF-B activation affects NEMO expression. We showed that A288G and Glu-315 EDA-ID mutations as well as A323P incontinentia pigmenti mutation in CC2-LZ, whether or not in the NOA/ UBAN binding site, strongly reduce folding and stability of NEMO oligomers (dimers and higher order oligomers), thereby leading to a defect in its ubiquitin binding activity (22) as well as in its TRAF6-induced Lys-63 ubiquitination (23). In the case of the C417F EDA-ID mutation in ZF domain, the solution structure of the mutant by NMR revealed a disordered region with an important loss of stability, providing a structural basis to explain the EDA-ID phenotype (14).…”

“…Plasmids, ZF Peptides, Antibodies, and Reagents-pcDNA3 plasmids (Invitrogen) encoding human FLAG-WT NEMO and its mutants ⌬ZF (ZF deletion), D406V, F395S, and F395W and the pGEX4T2-di-Ub plasmid (GE Healthcare) encoding linear di-ubiquitin were prepared using conventional molecular cloning and PCR mutagenesis methods as described previously (22,26). pLenti6.4/R4R2/V5-DEST-based plasmids (Invitrogen) encoding FLAG-WT and FLAG-D406V NEMO and a pET52b3C/LIC plasmid (Merck Millipore) encoding StrepTag IItagged human ubiquitin-conjugating enzyme E2-25K-C170S were prepared using Gateway Technology and Ligation Inde-pendent Cloning methods, respectively, following the manufacturer's instructions.…”

“…Homemade antibodies against NEMO (B24) and nucleoside diphosphate kinase B (NDPK-B) were previously described in Ref. 14 (27) were grown in DMEM, and Jurkat T cells (JM4.5.2; Ref 28) were cultured in RPMI supplemented with 10% fetal bovine serum, penicillin (50 units/ml), and streptomycin (50 g/ml) as previously described (22,29). NEMO-deficient Jurkat T and 293T cells were transiently transfected using FuGENE HD (Roche Applied Science) and Lipofectamine 2000 (Invitrogen), respectively, according to the manufacturer's instructions.…”