Cell, Volume 136
Simin Rahighi, Fumiyo Ikeda, Masato Kawasaki, Masato Akutsu, Nobuhiro Suzuki, Ryuichi Kato, Tobias
Kensche, Tamami Uejima, Stuart Bloor, David Komander, Felix Randow, Soichi Wakatsuki, and Ivan
Dikic
Supplemental Experimental Procedures cDNAs, antibodies and reagents
M5P-AU1-muNEMO was previously described (Bloor et al., 2008). Amino acid numbering refers to murine NEMO, unless specified otherwise. Mutations in AU1-mouseNEMO, pGEX4T1-diubiquitin, pBabe-mouseNEMO were introduced by site directed mutagenesis.
Murine IL1β (PeproTech), murine TNFα (PeproTech), LPS E.coli
O127:B8 (Sigma), CpG
DNA ODN1668 (Operon) and Lys63-diUb chains (Boston Biochem) were purchased.
Antibodies against NEMO were from BD, Cell Signaling and Santa Cruz. Antibodies against
AU1, Tubulin and ubiquitin were from Covance, Sigma-Aldrich and SantaCruz, respectively.
Antibodies against I κ B α , p- I κ B α , p-p65, p65, p38, p-p38, JNK, p-JNK and Cleaved Caspase 3
(Asp175) were purchased from Cell Signalling and anti-IKK α and anti-IKK β antibodies from
Imgenex.
Cells
HEK293T cells were purchased from ATCC and cultured as suggested. Transfection into
HEK293T cells were done by Lipofectamine (Invitrogen) following manufacturer’s protocol.
Cells were harvested after 48 hours of transfection using lysis buffer (50 mM HEPES, 150 mM
NaCl, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1% Triton-X-100, 25 mM NaF, 10 μ M
ZnCl2, pH 7.5) containing protease inhibitors (aprotinin, leupeptin and PMSF).). Wild type and NEMO-deficient MEF cells were described previously (Schmidt-Supprian et al., 2000).
Different NEMO constructs were introduced into NEMO deficient MEF cells by retroviral infection. Reconstituted MEF cells were serum starved for 16 hours and stimulated by TNFα
(20 ng/ml) for indicated time. The NFκ B reporter B cell line GTPT3 and its NEMO-deficient subclone F40 have been described earlier (Bloor et al., 2008). Cells were transduced with
M6PhisD-Nemo, an M5P derivative carrying an IRES controlled hisD resistance, selected with histidinol and stimulated with agonists for 24h. NFκ B dependent GFP expression was measured by flow cytometry.
GST-pull down assay
Immunoblotting and GST-pull-downs were performed as previously described (Hoeller et al.,
2006). Briefly, pGex-diubiquitin, pGex-NEMO (250-339), pGex-ABIN1 (465-520) (Wagner et al., 2008), pGex-Pol ι -UBMs (Bienko et al., 2005), pGex-hRad23A-UBA1 and pGex-HDAC6-Cterm (Boyault et al., 2006) were transformed into the bacterial strain BL21 and protein productions were induced with IPTG at 37 o C for 4 hours. GST fusions were purified
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Cell, Volume 136 with Glutathione Sepharose 4B (GE Healthcare) according to the manufacturer’s instructions.
K63-linked ubiquitin chains used for GST-pull down experiments were produced as described in Komander et al. (2008).
Briefly, GST-immobilized ubiquitin binding domains (UBDs) were tested for ubiquitin-chain binding using Linear, Lys63 or Lys48-linked di or tetraubiquitin chains. 1.5micro g of Ub chains were incubated with GST-UBD in pull down buffer (150 mM NaCl, 50mM Tris-pH7.5,
5mM DTT, 0.1% NP-40, 0.25 mg/ml BSA) for 12 hours at +4C with rotation. Beads were washed for 3 times with pull down buffer (without BSA). Beads were then mixed with loading buffer and boiled briefly (1 minute). The samples were preceded for western blot using PVDF membrane. Loadings of GST-UBDs were confirmed by Ponceau S staining.
Surface Plasmon Resonance
GST-NEMO
250-339
and linear diubiquitin were purified as described below. K63-linked diubiquitin chains were produced as described in Komander et al. (2008). K48-linked diubiquitin was purchased from Boston Biochem. Experiments were conducted on a
BIAcore TM 2000 instrument (BIAcore TM ). The measurements were done using a sandwich assay with anti GST antibody to immobilize GST or GST fused protein to fix first ligands onto the Sensor Chip CM5 (BIAcore TM ). All data collection were performed using the HBS-EP buffer including 10mM-HEPES (pH 7.4), 150mM-NaCl, 3mM-EDTA and 0.005% surfactant
P20 (pH 7.4) (BIAcore
(K
D
TM ) and repeated three times for each sample. Dissociation constants
) were computed by fitting to a 1:1 interaction model using Biacore software (BIAcore TM ).
Protein expression and purification
For crystallization and surface Plasmon resonance (SPR) experiments, the CoZi region of mouse NEMO (250-339) and linear diubiquitin were cloned into pGEX-4T-1 (GE Healthcare) and overexpressed as GST fusion proteins in Escherichia coli BL21(DE3) cells in LB medium.
Site-directed mutagenesis of NEMO was performed using the QuickChange mutagenesis kit
(Stratagene). For incorporation of SeMet, proteins were expressed in E. coli DL41 cells grown in LeMaster medium supplemented with 25mg/L seleno-L-methionine (Wako Pure Chemical).
Expression was induced by addition of 0.5 mM IPTG and cells were incubated at 25 ˚ C overnight. Harvested cells were lysed using sonication in PBS buffer and the supernatant was applied to a glutathione sepharose 4B column (GE Healthcare). The GST tag was cleaved on the column using thrombin protease and cleaved proteins were eluted with PBS buffer. For SPR experiments, the GST-fusion protein was eluted using glutathione. Proteins were further purified by gel filtration chromatography using a Superdex 75 column (GE Healthcare), then dialyzed against a buffer of 20 mM Tris-HCl pH 8.0, 150 mM NaCl and concentrated.
Crystallization, data collection and structure determination of NEMO
A mutant of NEMO
250-339
CoZi region alone
, K285N, was used for crystallization as no diffraction quality crystals could be obtained for the wild type protein despite an extensive search of crystallization conditions using crystallization robotics (Hiraki et al., 2006). Crystals of the
SeMet NEMO
250-339
(K285N) were grown in a condition comprising 45% (v/v) ethylene glycol,
50mM acetate (pH 4.0) and 0.02% (v/v) polyoxyethylene(6)decyl ether in the reservoir solution.
Diffraction quality crystals were obtained in hanging drops after 1 day incubation at 16 ˚ C. Data for the SAD phasing were collected to 2.8 Å resolution at 100 K using beamline BL-17A of
Photon Factory, KEK (Tsukuba, Japan). Diffraction data were processed using HKL2000
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(Otwinowski and Minor, 1997) and Se atom positions were determined and phases were obtained using SOLVE (Terwilliger and Berendzen, 1999). Density modification and initial model building were done using RESOLVE (Terwilliger, 2003). The structure contains one coiled-coil homo dimer of NEMO in the asymmetric unit. Further manual model building and refinement were performed using COOT (Emsley and Cowtan, 2004), CNS (Brünger et al.,
1998) and REFMAC5 (Murshudov et al., 1997). Two residues from the C-terminus and 4 residues from the N-terminus were not visible in the electron density maps. Data collection and refinement statistics are summarized in Supplementary Table 1. All structure figures were prepared with Pymol (DeLano Scientific; http://www.pymol.org
).
Crystallization, data collection and structure determination of NEMO
CoZi
in complex with diubiquitin
NEMO
250-339
wild-type and K285N mutant were co-crystallized with linear diubiquitin, however the crystals displayed a diffraction limit of 3.0 Å resolution and molecular replacement (MR) or SAD/MAD phasing were unsuccessful for solving the complex structure.
We then incorporated E282A/K285A double mutations of NEMO to reduce the surface entropy of the protein (Longenecker et al., 2001; Derewenda and Vekilov, 2006). The mutation sites were selected from the region upstream to the putative ubiquitin binding site. Binding assays were performed to ensure that the mutant retained the ability to bind to linear diubiquitin and tetraubiquitin (data not shown).
NEMO
250-339
(E282A, K285A) was mixed with SeMet-substituted diubiquitin in 1:1 molar ratio and crystallized using the sitting drop method. This molar ratio corresponds to one NEMO dimer to two diubiquitin molecules. Complex crystals were obtained after 5 days at 20 ˚ C. The reservoir contained 16% (w/v) polyethylene glycol 3350, 50 mM bis-tris propane (pH 5.0) and
50 mM citric acid. X-ray diffraction data to 2.7 Å resolution were collected at beamline
BL-17A of Photon Factory at 100 K using a wavelength of 0.9790 Å, and the data were processed with HKL2000 (Otwinowski and Minor, 1997). The crystal belonged to the space group C2. Although SeMet-labeled diubiquitin was used for crystallization, the anomalous signal from the Se atoms was not strong enough for phasing, and instead MOLREP (Vagin and
Teplyakov, 1997) from the CCP4 package (Collaborative Computational Project, Number 4.,
1994) was applied to solve the crystal structure of the complex protein by molecular replacement, using monoubiquitin (PDB entry 1UBQ) and the NEMO
CoZi dimer structure described above as search models. Four monoubiquitin molecules and one NEMO
CoZi
dimer were found in the asymmetric unit. The model was further built and refined using COOT
(Emsley and Cowtan, 2004) and REFMAC5 (Murshudov et al., 1997). Data collection and refinement statistics are summarized in Supplementary Table 1. All structure figures were prepared with Pymol (DeLano Scientific; http://www.pymol.org).
Using a slightly shorter construct of wild-type NEMO
CoZi
(residues 253-337), co-crystals with diubiquitin were obtained in a different setting (space group
P
2
1
2
1
2
1
), using hanging drop with a reservoir solution comprising 20% (w/v) polyethylene glycol 3350 and 0.2 M di-ammonium tartrate (pH 6.6). Crystals appeared after 10 days of incubation at 16 ˚ C. The X-ray diffraction data was collected to 3.0 Å resolution at PF beamline BL-17A and processed with HKL2000
(Otwinowski and Minor, 1997). The previously determined tetrameric NEMO
CoZi
● diubiquitin structure in the C2 space group was used as a search model for MR by MOLREP29 which readily found two tetrameric complexes in the asymmetric unit.
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The tetrameric NEMO
CoZi
● diubiquitin complex structures of C 2 and P 2
1
2
1
2
1
space groups can be superimposed with RMS deviation of 1.69 Å for the C α positions of the NEMO dimer
(residues 255-335), and 1.97 Å for the C α atoms of the NEMO
CoZi
● diubiquitin tetramer
(255-335 residues of NEMO and 1-146 of diubiquitin). In the C 2 crystal form, the C-terminal tails of NEMO dimers from two neighboring complexes are close to each other making the
C-termini of the NEMO coiled-coil unwind slightly, which is not observed in the
P
2
1
2
1
2
1 structure. Despite these small differences due to crystal packing, the NEMO-diubiquitin interactions are highly conserved and the RMS deviation of the UBAN motif of NEMO
(residues 289-320) between the C 2 and the P 2
1
2
1
2
1
structure is 0.47 Å, indicating the intrinsic nature of the double-sided diubiquitin binding. In the manuscript, the slightly longer
C
2 crystal structure is described.
Supplemental References
Brünger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse-Kunstleve, R.W.,
Jiang, J.S., Kuszewski, J., Nilges, M., Pannu, N.S., et al . (1998). Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta. Cryatallogr
.
D
54
, 905-921.
Derewenda, Z.S. and Vekilov, P.G. (2006). Entropy and surface engineering in protein crystallization. Acta. Crystallogr. D
62
116-124.
Emsley, P. and Cowtan, K. (2004). Coot: model-building tools for molecular graphics. Acta
Crystallogr. D 60 , 2126-2132.
Hiraki, M., Kato, R., Nagai, M., Satoh, T., Hirano, S., Ihara, K., Kudo, N., Nagae, M.,
Kobayashi, M., Inoue, M., et al . (2006). Development of an automated large-scale protein-crystallization and monitoring system for high-throughput protein-structure analyses.
Acta. Crystallogr. D
62
, 1058-1065.
Hoeller, D., Crosetto, N., Blagoev, B., Raiborg, C., Tikkanen, R., Wagner, S., Kowanetz, K.,
Breitling, R., Mann, M., Stenmark, H. et al . (2006). Regulation of ubiquitin-binding proteins by monoubiquitination. Nat. Cell Biol.
8,
163-169
Longenecker, K. L., Garrard, S. M., Sheffield, P. J. and Derewenda, Z. S. (2001). Protein crystallization by rational mutagenesis of surface residues: Lys to Ala mutations promote crystallization of RhoGDI. Acta Crystallogr
.
D
57
, 679-688 .
Murshudov, G.,N., Vagin, A.,A., and Dodson, E.J. (1997). Refinement of macromolecular structures by the maximum-likelihood method. Acta. Crystallogr. D. 53 , 240-255.
Otwinowski, Z., and Minor, W. (1997). Processing of X-ray diffraction data collected in oscillation mode. Methods. Enzymol.
276
, 307-326.
Terwilliger, T.C. and Berendzen, J. (1999). Automated MAD and MIR structure solution. Acta.
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Crystallogr.
D. 55 , 849-861.
Terwilliger, T.C. (2003). Automated main-chain model building by template matching and iterative fragment extension. Acta. Crystallogr. D.
59
, 38-44.
Figure S1.
Ubiquitin binding region of NEMO
(A) Schematic domain organization of mouse NEMO (mNEMO) and the construct used in this study. HLX, helix; CC, coiled-coil; LZ, leucine zipper; ZF, zinc finger. (B) Binding of
Lys63-linked diUb molecules (Boston Biochem) to GST (empty), GST-NEMO CoZi region
(NEMO 250-339), and its mutants as indicated were examined by immunoblotting using an anti-ubiquitin antibody. Loading of GST fusion proteins was determined by Ponceau S staining.
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(C) Bindings of linear, Lys63 or Lys48-linked diUb to GST-NEMO CoZi (NEMO 250-339) were examined. Panels are showing the blots with a very long exposure time.
Figure S2
. Sequence alignment of the UBAN motif of NEMO, ABINs and Optineurin Fully and partially conserved amino acid residues are coloured in red and orange, respectively. The arrowheads indicate NEMO residues that interact with diubiquitin. NEMO residues, which are involved in dimer formation, are highlighted in yellow. Letters “a” and “d” above the mNEMO sequence indicate their position in the coiled-coil heptad repeats (a-b-c-d-e-f-g). The three residues of mNEMO that do not fit the heptad repeats are marked by a box.
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Figure S3.
Binding surface of linear diUb and NEMO-UBAN
(A) Mapping of the NEMO-UBAN interaction surface onto one ubiquitin molecule showing the mutually exclusive binding surfaces for distal and proximal ubiquitin interactions. Residues involved in the interaction are labeled in the left panel. In the right panel, the bound linear diubiquitin are drawn as transparent ribbon models; Ub in orange and Ub proximal distal
in gold.
(B and C) Electron density of the distal (B) and proximal (C) Ub interactions with NEMO
(corresponding to Figures 3G and 3I, respectively). Final 2Fo - Fc electron density map is contoured at 1.0 σ . (D) Bindings of full length AU1-NEMO and GST (empty), GST-diUb,
GST-diUb mutant R72A L73A R74A in distal Ub and GST-diUb mutant Q2A in proximal ubiquitin were examined by immunoblotting using anti-AU1 antibody. The loading of GST were determined by Ponceau S staingin.
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Figure S4. Recognition of Lys63-linked tetraubiquitin by UBAN domain of NEMO and effects of NEMO single point mutations on activation of NFκ B signaling
(A) The bindings of Lys63-linked tetra ubiquitin to GST-NEMO 250-339 (wt and mutants) were analyzed by densitometry. Three independent GST-pull down assays were performed and blots were analyzed using Scion Image for Windows (Scion Cooperation, USA). (B) NEMO knock out MEF cells were reconstituted with NEMO mutants (F305A or E313A) as indicated.
Total cell lysates of TNFα -treated cells were analyzed for activation of NFκ B by immunoblotting with phosphor-specific I κ B α and I κ B α antibodies. As a control, cell lysates were also subjected to immunoblotting with antibodies against NEMO and tubulin.
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Figure S5.
Models of different ubiquitin-chains recognitions by NEMO-UBAN
(A) Model of Lys63-linked tetraubiquitin bound to NEMO-UBAN as a hetero-trimeric complex in which one NEMO dimer and one tetraubiquitin are present. The binding between them is made possible by the canonical Ile44 interactions of the first and the fourth ubiquitin moieties with the distal binding surface on the NEMO-UBAN coiled-coil. (B) Interface between distal and proximal ubiquitin moieties of linear diubiquitin bound to the
NEMO-UBAN motif. In complex with NEMO, residues from Ub distal with Ub proximal
(Pro37 to Gln40) interact
(Glu92 to Glu94) in the linear diubiquitin molecules. These residues form, together with the C-terminal tail of Ub distal
(Arg72 to Gly76), a structural core that sustains the two moieties in a semi-fixed orientation.
The contacting residues from Ub
Ub proximal distal
(orange) and
(gold) are shown in stick model. Final 2 F o – F c electron density map is contoured at
0.5
σ .
(C) Model of Lys63-linked diubiquitin (orange) bound unfavorably to the
NEMO-UBAN in a manner similar to NEMO ● linear diubiquitin structure (green). The model was made with a slight modification of the position of Met1 of Ub proximal
by about 2 Å away from the NEMO-UBAN. Lys63-linkage moves towards NEMO dimer and carbonyl oxygen of
Gly75 and pushes N ε atom of Gln2 away. This is, however, unfavorable for the interaction with
Arg309 of NEMO, which would in turn repel N ε of Gln2. If the N ε of Gln2 moves in the opposite direction, then it would now be repelled by the N ζ of Lys63 of Ub proximal hydrogen bond network formed by Gln2/Glu16 of Ub proximal
. Thus the
and Arg309/Arg312 of NEMO could be affected by the linkage formation between Gly76 and Lys63. In addition, the shift of the nitrogen atom of Met1 by ~2 Å further away from NEMO might cause a rotation of
Ub proximal
. This would affect the interaction between the two ubiquitin moieties of the linear diubiquitin and might be detrimental for binding of Lys63 linked diubiquitin to the NEMO
UBAN motif.
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Figure S6. A structural model of full length NEMO and IKK complex formation and a schematic model of NEMO-ubiquitin interaction in NFκ B signaling
(A) NEMO is modeled as a long alpha helical protein based on the reported structures of
NEMO fragments. Two chains of NEMO are coloured in magenta, and their binding partners are drawn in cyan (IKK α ), yellow (IKK β ) and green (Lys 63-linked polyubiquitin chains). (A) HLX1 domain: Modified from Rushe M., et al., Structure 16, 798-808 (2008), (B) HLX2 domain: Modified from Bagneris C., et al., Mol. Cell 30, 620-631, (2008), (C) CoZi domain: Current work, (D) ZF domain:
Cordier F., et al., J. Mol. Biol . 377, 1419-1432 (2008). (B) TNFα activates NFκ B signaling pathway via
TNFR-I, by recruitment of RIP and TRAFs. This complex formation induces Lys63-linked ubiquitination of RIP and TRAFs with Ubc13 and IAPs, hence mediates TAK1-TAB2 complex recruitment and TAK1 activation. TAK1 was shown to be an upstream regulator of both MAPK and
IKK pathways, while Ubc13 is more important for MAPK signaling. At present, it remains unclear how
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TAK1 can impact on NEMO-dependent activation of IKK and NFκ B. LUBAC, an E3 ligase building linear ubiquitin chains, mediates ubiquitination of NEMO, which can be recognized by the UBAN motifs of neighboring NEMO molecules in IKK complexes. Both of these steps, linear ubiquitination and binding to linear chains by NEMO, are critical for NFκ B, but not MAPK, activation in response to
TNFα stimulation.
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