The Zinc Finger of NEMO Is a Functional Ubiquitin-binding Domain

NEMO (NF-κB essential modulator) is a regulatory protein essential to the canonical NF-κB signaling pathway, notably involved in immune and inflammatory responses, apoptosis, and oncogenesis. Here, we report that the zinc finger (ZF) motif, located in the regulatory C-terminal half of NEMO, forms a specific complex with ubiquitin. We have investigated the NEMO ZF-ubiquitin interaction and proposed a structural model of the complex based on NMR, fluorescence, and mutagenesis data and on the sequence homology with the polymerase η ubiquitin-binding zinc finger involved in DNA repair. Functional complementation assays and in vivo pull-down experiments further show that ZF residues involved in ubiquitin binding are functionally important and required for NF-κB signaling in response to tumor necrosis factor-α. Thus, our findings indicate that NEMOZFisa bona fide ubiquitin-binding domain of the ubiquitin-binding zinc finger type. NEMO (NF-κB essential modulator) is a regulatory protein essential to the canonical NF-κB signaling pathway, notably involved in immune and inflammatory responses, apoptosis, and oncogenesis. Here, we report that the zinc finger (ZF) motif, located in the regulatory C-terminal half of NEMO, forms a specific complex with ubiquitin. We have investigated the NEMO ZF-ubiquitin interaction and proposed a structural model of the complex based on NMR, fluorescence, and mutagenesis data and on the sequence homology with the polymerase η ubiquitin-binding zinc finger involved in DNA repair. Functional complementation assays and in vivo pull-down experiments further show that ZF residues involved in ubiquitin binding are functionally important and required for NF-κB signaling in response to tumor necrosis factor-α. Thus, our findings indicate that NEMOZFisa bona fide ubiquitin-binding domain of the ubiquitin-binding zinc finger type. The NF-κB transcription factors regulate the expression of genes involved in inflammation, immunity, cell proliferation, apoptosis, and oncogenesis (1Hayden M.S. Ghosh S. Genes Dev... 2004; 18: 2195-2224Google Scholar). The classical NF-κB signaling pathway is induced in response to proinflammatory cytokines (interleukin-1, TNF-α), 5The abbreviations used are: TNF-α, tumor necrosis factor α; IKK, IκB kinase; WT, wild type; NEMO, NF-κB essential modulator; ZF, zinc finger; LZ, leucine zipper; UBZ, ubiquitin-binding ZF; UBD, ubiquitin-binding domain; UIM, ubiquitin-interacting motif; polyUb, polyubiquitin; pol, polymerase; NOE, nuclear Overhauser effect; CC, coiled-coil. antigens, and endotoxins. Although different receptors and downstream molecules are engaged depending on the stimulus, they all transduce signals through the IKK complex. These active complexes comprise two catalytic kinase subunits, IKKα and/or IKKβ, and a dimer of regulatory subunits termed NEMO, which is required for IKK complex activity and subsequent NF-κB activation (2Fontan E. Traincard F. Levy S.G. Yamaoka S. Veron M. Agou F. FEBS J.. 2007; 274: 2540-2551Google Scholar, 3Miller B.S. Zandi E. J. Biol. Chem... 2001; 276: 36320-36326Google Scholar). The N-terminal part of NEMO interacts with the IKK kinases, whereas the regulatory C-terminal half is involved in signal recognition (4Hacker H. Karin M. Science's STKE.. 2006; 2006: re13Google Scholar, 5Sebban H. Yamaoka S. Courtois G. Trends Cell Biol... 2006; 16: 569-577Google Scholar). The latter comprises the CC2-LZ domain, including a coiled-coil (CC2) and a leucine zipper (LZ) motif and a CCHC-type zinc finger (ZF) domain (Fig. 1). The CC2-LZ domain is required for NEMO oligomerization (6Agou F. Traincard F. Vinolo E. Courtois G. Yamaoka S. Israel A. Veron M. J. Biol. Chem... 2004; 279: 27861-27869Google Scholar, 7Tegethoff S. Behlke J. Scheidereit C. Mol. Cell. Biol... 2003; 23: 2029-2041Google Scholar) and contains a ubiquitin-binding domain (UBD) that preferentially interacts with Lys-63-polyubiquitin (Lys-63-polyUb) chains (8Ea C.K. Deng L. Xia Z.P. Pineda G. Chen Z.J. Mol. Cell.. 2006; 22: 245-257Google Scholar, 9Wu C.J. Conze D.B. Li T. Srinivasula S.M. Ashwell J.D. Nat. Cell Biol... 2006; 8: 398-406Google Scholar). Lys-63-polyUb binding through the CC2-LZ domain of NEMO is required for NF-κB signaling from several different receptors. In the case of TNF-α receptor 1 (TNFR1), the IKK complex seems to be recruited to the TNFR1 receptor via interaction with Lys-63-polyubiquitinated RIP1 protein (8Ea C.K. Deng L. Xia Z.P. Pineda G. Chen Z.J. Mol. Cell.. 2006; 22: 245-257Google Scholar, 9Wu C.J. Conze D.B. Li T. Srinivasula S.M. Ashwell J.D. Nat. Cell Biol... 2006; 8: 398-406Google Scholar). In addition to binding Lys-63-polyUb, a minor fraction of NEMO undergoes ubiquitination (post-translational modification via the covalent attachment of ubiquitin) within the regulatory portion of NEMO. Several monoubiquitination and Lys-63-polyubiquitination sites on NEMO have been identified, including a Lys-399 residue in the ZF domain (10Zhou H. Wertz I. O'Rourke K. Ultsch M. Seshagiri S. Eby M. Xiao W. Dixit V.M. Nature.. 2004; 427: 167-171Google Scholar), and the site of modification and its impact on IKK activity appear to be signal-dependent (11Abbott D.W. Wilkins A. Asara J.M. Cantley L.C. Curr. Biol... 2004; 14: 2217-2227Google Scholar, 12Huang T.T. Wuerzberger-Davis S.M. Wu Z.H. Miyamoto S. Cell.. 2003; 115: 565-576Google Scholar, 13Tang E.D. Wang C.Y. Xiong Y. Guan K.L. J. Biol. Chem... 2003; 278: 37297-37305Google Scholar). Although the CC2-LZ domain of NEMO is required for a wide range of NF-κB-inducing signals, the role of the NEMO ZF domain remains controversial (14Temmerman S.T. Ma C.A. Borges L. Kubin M. Liu S. Derry J.M. Jain A. Blood.. 2006; 108: 2324-2331Google Scholar, 15Yang F. Yamashita J. Tang E. Wang H.L. Guan K. Wang C.Y. J. Immunol... 2004; 172: 2446-2452Google Scholar) and appears to depend on the stimulus. Several studies reported that NEMO ZF is required for interleukin-1 and TNF-α but not lipopolysaccharide-induced signaling (16Huang T.T. Feinberg S.L. Suryanarayanan S. Miyamoto S. Mol. Cell. Biol... 2002; 22: 5813-5825Google Scholar, 17Makris C. Roberts J.L. Karin M. Mol. Cell. Biol... 2002; 22: 6573-6581Google Scholar). On the other hand, a clear requirement for ZF in the genotoxic stress response has been established (12Huang T.T. Wuerzberger-Davis S.M. Wu Z.H. Miyamoto S. Cell.. 2003; 115: 565-576Google Scholar, 18Wu Z.H. Miyamoto S. J. Mol. Med... 2007; 85: 1187-1202Google Scholar). This distinct pathway is activated by UV radiation and other DNA-damaging agents and involves import of NEMO into the nucleus, where it interacts with the ataxia telangiectasia mutated (ATM) checkpoint kinase. The importance of NEMO ZF is also emphasized by the fact that (i) several mutations in the ZF are linked to the human diseases, anhidrotic ectodermal dysplasia with immunodeficiency and incontinentia pigmenti (19Fusco F. Pescatore A. Bal E. Ghoul A. Paciolla M. Lioi M.B. D'Urso M. Rabia S.H. Bodemer C. Bonnefont J.P. Munnich A. Miano M.G. Smahi A. Ursini M.V. Hum. Mutat... 2008; 29: 595-604Google Scholar), and that (ii) mice lacking the NEMO ZF die during embryonic development (20Nenci A. Pasparakis M. Israel A. Baltimore D. Ben-Neriah Y.M. NF-κB: 20 Years on the Road from Biochemistry to Pathology. Keystone Symposia, Banff, Canada2006: 84Google Scholar). We recently determined the solution structure of NEMO ZF (21Cordier F. Vinolo E. Veron M. Delepierre M. Agou F. J. Mol. Biol... 2008; 377: 1419-1432Google Scholar), which highlighted the importance of a large hydrophobic cluster sharing similarities with the ubiquitin-interacting region of the ubiquitin-binding ZF (UBZ) of the human DNA Y-polymerase (pol) η (22Bomar M.G. Pai M.T. Tzeng S.R. Li S.S. Zhou P. EMBO Rep... 2007; 8: 247-251Google Scholar). Because UBDs are usually found in multiple copies in proteins, the question arises whether NEMO ZF could actually act as a second ubiquitin-binding site, in addition to the CC2-LZ domain. We show here, using NMR, fluorescence, mutagenesis, and in vivo pull-down experiments, that NEMO ZF is a bona fide UBD of the UBZ type. We have investigated the ZF-ubiquitin interaction, proposed a model of the complex, and further showed that ZF residues involved in ubiquitin binding are functionally important and required for NF-κB signaling in response to TNF-α. Sample Preparation—NEMO ZF synthetic peptides (>98% purity) termini-blocked by N-acetyl and C-amide groups were purchased from Biopeptide Co. (San Diego, CA). Five different peptides were used in this study; ZF corresponds to 2 Ser residues followed by residues 394–419 of human NEMO. In ZF(W), Phe-395 is replaced by a tryptophan. ZF(W)-M407V, ZF(W)-V414S, and ZF(W)-M415S are three point mutant peptides of ZF(W). The sequences are given in supplemental Fig. S1. For binding assays, each of the four ZF(W) peptides was solubilized in a buffer of 0.6 mm ZnCl2, 1 mm tris(2-carboxyethyl)phosphine, and 50 mm Tris-HCl (pH 7.5) to a stock concentration of ∼0.2 mm. Recombinant human ubiquitin and the I44A ubiquitin mutant were purchased from Boston Biochem at >95% purity. Ubiquitin was further purified by cation exchange column, dialyzed in water, lyophilized, and solubilized in 50 mm Tris-HCl (pH 7.5). For NMR, 1.7 mm of the ZF was prepared by dissolving 2.8 mg of peptide in 500 μl of 3.4 mm ZnCl2, 3.4 mm tris(2-carboxyethyl)phosphine, 20 mm Tris-HCl, 90% H2O, 10% D2O (pH 6.2). The U-15N-labeled human ubiquitin was purchased from VLI Research Inc. (Malvern, PA), and a 1 mm stock solution was prepared in exactly the same buffer as the ZF. Three NMR samples of ZF-ubiquitin complex were successively prepared at concentrations (ratio) of 1.45:0.15 mm (r = 9.7), 0.87:0.15 mm (r = 5.8), and 0.24:0.15 mm (r = 1.6). NMR Spectroscopy—NMR experiments were carried out at 25 °C on a Varian Inova 600-MHz spectrometer equipped with a cryoprobe. 1H-15N heteronuclear single quantum correlation spectra were recorded with 200 (90 ms) and 1024 (122 ms) complex points (acquisition times) in 15N and 1H, respectively. Average (1H, 15N) chemical shift changes upon binding were calculated as Δδav = [(ΔδH)2 + (ΔδN/5)2]½. The binding affinity (KD) was estimated by fitting the ZF titration data, assuming a simple complex formation model and using nonlinear regression. 15N relaxation data (T1, T2, and {1H}-15N NOE) were acquired by standard methods (23Kay L.E. Nicholson L.K. Delaglio F. Bax A. Torchia D.A. J. Magn. Reson... 1992; 97: 359-375Google Scholar). Docking—The docking was performed with HADDOCK 1.3 (24Dominguez C. Boelens R. Bonvin A.M. J. Am. Chem. Soc... 2003; 125: 1731-1737Google Scholar), with initial coordinates of NEMO ZF and ubiquitin taken from their structure in free form (Protein Data Bank entries 2JVX and 1D3Z, respectively) and by using 16 ambiguous interaction restraints. The latter were defined on the basis of the NMR chemical shift mapping, surface accessibility data, and the homology with the pol η UBZ signature motif. Binding Affinity—Tryptophan fluorescence measurements were recorded on a PTI QuantaMaster spectrofluorimeter (PTI, Lawrenceville, NJ) at 22 °C. The excitation wavelength was set to 295 nm to minimize the contribution of the tyrosyl group. The fluorescence emission at 352 nm was measured using 20 μm peptide and 20–500 μm ubiquitin. The observed fluorescence (Fobs) was corrected for dilution and for fluorescence from ubiquitin alone. Data for the ZF(W) peptide were fitted by nonlinear regression using the equation (Fobs - F0) = (Fmax - F0) [L]/(KD + [L]) to solve the maximum fluorescence (Fmax) and the dissociation constant (KD), given [L] (ligand concentration), F0 (fluorescence in the absence of ligand), and Fobs. The KD for ZF(W) mutant peptides was estimated using the Fmax value obtained for ZF(W). For ZF peptide folding experiments, ZF peptide stocks, prepared in the presence or absence of ZnCl2, were diluted to 20 μm in 50 mm Tris-HCl (pH 7.5). The emission spectra (305–415 nm) for these peptides were recorded, the buffer contribution was subtracted, and the fluorescence quantum yields were determined using N-acetyl tryptophanamide (Sigma) as standard as described previously (25Deville-Bonne D. Sellam O. Merola F. Lascu I. Desmadril M. Veron M. Biochemistry.. 1996; 35: 14643-14650Google Scholar). Plasmid Construction—A pcDNA3 vector encoding FLAG-tagged human NEMO protein (residues 2–419) was used to generate five NEMO mutant constructs, including point mutants M407V, Q411R, V414S, and M415S and a NEMO ZF deletion (ΔZF). Substitutions were chosen conservatively, so as not to perturb the overall structure of the ZF domain. The point mutations were generated using overlap PCR. The primer sequences used for PCR are available upon request. The final PCR product was digested with EcoRI and XhoI and ligated into the pcDNA3-FLAG vector. A NEMO construct lacking 25 C-terminal residues (ΔZF) was generated using PCR introducing a stop codon after residue 394. The PCR product was digested and cloned as described above. Functional Complementation Experiments—4 × 106 NEMO-/- Jurkat T cells (JM4.5.2) (26Harhaj E.W. Good L. Xiao G. Uhlik M. Cvijic M.E. Rivera-Walsh I. Sun S.C. Oncogene.. 2000; 19: 1448-1456Google Scholar) were transfected with 3 μg of pcDNA3-FLAG (empty or with various NEMO constructs), 1 μg of Igκ-luciferase, and 300 ng of β-galactosidase plasmids using DEAE-dextran as described previously (27Vinolo E. Sebban H. Chaffotte A. Israel A. Courtois G. Veron M. Agou F. J. Biol. Chem... 2006; 281: 6334-6348Google Scholar). After 24 h, cells were treated with TNF-α (10 ng/ml) for 4 h and then harvested. Luciferase activity in soluble cell extracts was measured as described (27Vinolo E. Sebban H. Chaffotte A. Israel A. Courtois G. Veron M. Agou F. J. Biol. Chem... 2006; 281: 6334-6348Google Scholar). β-galactosidase activity was measured using the luminescent β-galactosidase detection kit (Clontech) according to manufacturer's instructions. The level of luciferase, representing NF-κB activity, was normalized to the level of β-galactosidase, representing transfection efficiency. Immunoprecipitation and Western Blot Analysis—293T cells, grown in were transfected with μl of and 3 μg of (empty or with various NEMO and 24 h after were in 25 mm (pH 1 mm and 10% Western or in 50 mm Tris-HCl (pH mm and was using the kit (Sigma) according to the manufacturer's were using (Sigma) or and ubiquitin was using NEMO ZF binding of a NEMO ZF synthetic peptide the sequence in supplemental Fig. to ubiquitin was by NMR chemical shift The and 15N chemical of several ubiquitin residues from the free (Fig. to the upon the addition of ZF, as observed for a complex in exchange on the NMR chemical shift changes or of are observed for a of residues (Fig. the binding surface on ubiquitin. of these residues are located and on the hydrophobic region of the of ubiquitin by UBDs L. H.L. Nat. Mol. Cell Biol... Scholar) Fig. from 15N relaxation measurements (T1, T2, and {1H}-15N NOE) were for the free and of ubiquitin (Fig. correlation estimated from the of are and for the free and respectively. the these are in with of and for molecules with of and the formation of a complex. the of ubiquitin is not by peptide with and T2, and {1H}-15N This the formation of a complex, exchange be on the to for residues and in the as from their in the of the NEMO ZF-ubiquitin complex. of the NEMO ZF sequence with the signature of pol η UBZ, and NEMO is in The in to as is replaced by a in NEMO ZF. hydrophobic residues are in and residues are in and large hydrophobic and respectively. are in NEMO mutations in this are by Q411R, V414S, and and of NEMO ZF with the ubiquitin-binding surface as in A. and of ubiquitin the surface as by the NMR chemical shift Δδav in and Δδav in with but in the form are in (Fig. is in NEMO ZF in our docking model is in docking model proposed for the NEMO ZF-ubiquitin complex with the ZF in and ubiquitin in The residues are as and and to and and is for the ZF The surface is The binding affinity (KD) of the ZF-ubiquitin complex was estimated to be μm by Because we observed the presence of peptide at ZF concentration (21Cordier F. Vinolo E. Veron M. Delepierre M. Agou F. J. Mol. Biol... 2008; 377: 1419-1432Google Scholar), we this affinity by fluorescence using a synthetic peptide the as ZF(W) the sequence in supplemental Fig. This not the of ZF(W) to upon the addition of zinc (Fig. in Fig. in fluorescence signal is observed during ubiquitin titration to a KD of which is within the range reported for other UBDs L. H.L. Nat. Mol. Cell Biol... Scholar). that the KD value from fluorescence data is in with the estimated by NMR, that the not the Ubiquitin the I44A not ZF(W) that the in fluorescence is to the formation of a specific NEMO ZF-ubiquitin complex and that residue is for binding to NEMO ZF. of the NEMO the structure of NEMO ZF, a large hydrophobic cluster the hydrophobic of the was to be a surface (21Cordier F. Vinolo E. Veron M. Delepierre M. Agou F. J. Mol. Biol... 2008; 377: 1419-1432Google Scholar). on Fig. this region similarities with other ubiquitin-binding notably with that of the pol η UBZ domain (22Bomar M.G. Pai M.T. Tzeng S.R. Li S.S. Zhou P. EMBO Rep... 2007; 8: 247-251Google Scholar) to a with the ubiquitin-interacting motif of S. R. M.S. A.M. Nat. Mol. Biol... 2006; Scholar, L. M. S. L. A. S. Cell.. 2006; Scholar). residues at and that are important for binding are in NEMO ZF, whereas the is replaced by a The binding on NEMO ZF (Fig. and on ubiquitin (Fig. were defined according to the sequence homology with the UBZ motif and to the NMR chemical shift data, respectively. On the basis of these two we determined a docking model of the ZF-ubiquitin complex (Fig. using the HADDOCK (24Dominguez C. Boelens R. Bonvin A.M. J. Am. Chem. Soc... 2003; 125: 1731-1737Google Scholar). We found that the hydrophobic of the ZF interacts with the hydrophobic region of ubiquitin in a as the UBZ Fig. of M.G. Pai M.T. Tzeng S.R. Li S.S. Zhou P. EMBO Rep... 2007; 8: 247-251Google Scholar). in the overall binding of the zinc the shift observed from the of the 2 residues in the sequence into a shift of the NEMO by into the ubiquitin and to a different of the finger with to ubiquitin. These are in with the fact that the in NEMO ZF is in the pol η UBZ by The interaction ubiquitin and NEMO ZF to a surface of and is by hydrophobic all or part of the chains of and of the ZF and notably the ubiquitin residues and In the pol η UBZ, the of the into a was to in NEMO ZF, the residue at the into the hydrophobic by and the pol η UBZ and the NEMO ZF this In addition to these hydrophobic the chains of and at and to the binding by the formation of with the or groups of the residues to of in a as the and residues in the pol η our model of the NEMO ZF-ubiquitin complex, we the of three ZF(W) peptides to with ubiquitin using In addition to the these ZF(W) peptides the point in a with the incontinentia pigmenti A. Courtois G. P. Yamaoka S. S. Munnich A. Israel A. S.M. P. S. A. T. T. F. A. D'Urso M. H. T. D. H. S. T. Levy M. Nature.. 2000; V414S, or The and ZF(W)-M415S peptides show in binding and the with a in ubiquitin binding (Fig. that the fluorescence quantum yields in the presence of zinc are for all that the binding affinity of these peptides is not to a of the NEMO ZF (Fig. and supplemental These mutagenesis are with our that residues and which on the hydrophobic part of the are for ubiquitin the binding affinity observed for the mutant from the of hydrophobic the and (Fig. of the NEMO in we the question whether the interaction NEMO ZF and ubiquitin is important for NF-κB the we investigated whether ZF mutations located at the binding Q411R, V414S, and are to NF-κB activation in using of plasmids not to a in activity in the absence of TNF-α not On the other hand, with TNF-α to a in activity in NEMO cells at as with (Fig. NEMO mutant a in NF-κB signaling to the activity as with NEMO point mutations and M415S are as in the construct The to NF-κB signaling in these cells is not to NEMO protein expression or all NEMO are at upon transfection (Fig. mutations of ZF residues that a in ubiquitin binding NF-κB signaling in T the importance of ubiquitin recognition by the ZF motif, via hydrophobic We whether the M415S which the in NF-κB the of NEMO to in of FLAG-tagged NEMO protein after transfection in cells to of a of proteins, whereas with NEMO M415S (Fig. a in the ubiquitin-binding surface of the ZF the of NEMO to proteins, that NEMO ZF is involved in ubiquitin binding in our the role of the ZF as a ubiquitin-binding domain in NEMO, of the UBZ into the of ubiquitin recognition by NEMO to the NF-κB The ZF affinity for complex be in the of the which contains UBD in the CC2-LZ domain. We that the ZF in a with the CC2-LZ domain to ubiquitin recognition and to preferentially chains our other ectodermal dysplasia with immunodeficiency and incontinentia pigmenti mutations located in the CC2-LZ UBD, mutations in NEMO ZF NF-κB activation by the binding to ubiquitin. We and for and for and S. C. Sun for with

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.