Methods
Yeast two-hybrid screening
The ubiquitin (ΔGG) coding sequence was cloned into pYTH9 vector creating a fusion protein with GAL4-
DNA-binding domain. The vector was introduced into Saccharomyces cerevisiae Y190 strain using standard lithium acetate/polyethylene glycol transformation. cDNA libraries were introduced similarily and cells were plated on synthetic dropout medium lacking leucine, tryptophan and histidine (BD Biosciences) that was supplemented with 25 mM 3-amino-1,2,4-triazole. Colonies that grew on the selection medium were assayed for
β-galactosidase activity. Plasmid DNA was extracted by lycticase digestion followed by spin column purification, amplified and sequenced.
Cell Lines and reagents
HEK 293T and HeLa S3 cells were grown in D-MEM supplemented with 10% fetal bovine serum, 2 mM Lglutamine, 0.4 mM sodium pyruvate and antibiotics. Recombinant human TNF was produced in Escherichia coli and purified to at least 99% homogeneity. TNF had a specific biological activity of 4.7x10
7 IU/mg of purified protein, as determined with the international standard code 87/650 (National Institute for Biological Standards and Control, Potters Bar, UK).
Plasmids and antibodies
The plasmid pconaLUC (LMBP 3248) expressing the luciferase (Luc) gene under the control of a minimal NF-

B-responsive chicken conalbumin promoter, was a gift from Dr. A. Israël (Institut Pasteur, Paris, France). The plasmid pUT651, encoding

-galactosidase, was supplied by Eurogentec (Seraing, Belgium). The ABIN and
MAD expression constructs in pCAGGS vector have been described in (Heyninck et al.
, 2003; Van Huffel et al.
,
2001; Wullaert et al.
, 2007). All mutations were introduced by PCR mutagenesis. GST-NEMO has been described before (Wu et al.
, 2006). HA-ubiquitin was a gift from Dr. K. Burns (University of Lausanne,
Epalinges, Switzerland), pCD-FLAG-NEMO was made by M. Kreike (LMBP 5496), pCal-c-A7M8K expressing
API2/MALT1 was given by Dr. M. Baens (VIB - Katholieke Universiteit Leuven, Leuven, Belgium). The plasmids that have been assigned a LMBP accession number are available from the BCCM/LMBP Plasmid collection, Department of Molecular Biology, Ghent University, Belgium
( http://bccm.belspo.be/about/lmbp.htm
). pGEX 4T1 vector was used for expression of all other GST fused proteins. Monoclonal anti-E tag horseradish peroxidase-linked antibody was obtained from GE Healthcare
(Diegem, Belgium), monoclonal anti-FLAG and monoclonal anti-FLAG horseradish peroxidase-linked
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antibodies from Sigma-Aldrich (St. Louis, MO, USA), monoclonal anti-HA antibody from Eurogentec (Seraing,
Belgium) and polyclonal anti-hMALT1 antibody (H-300) from Santa Cruz Biotechnology (Santa Cruz, CA,
USA). Anti-mouse and anti-rabbit horseradish peroxidase-linked antibodies were obtained from GE Healthcare
(Diegem, Belgium).
Immunoprecipitation and western blotting
HEK 293T cells (1.5×10 6 ) were plated on 10 cm dishes and transfected by calcium phosphate precipitation with
5 μg of total DNA. Twenty-four hours after transfection cells were lysed in 200 μl 0.5 % SDS (v/v) dissociation buffer (1% Triton-X 100, 0.5% deoxycholate, 120 mM NaCl, 50 mM HEPES pH 7.5) supplemented with protease and phosphatase inhibitors, and boiled to dissociate any non-covalently bound proteins. Lysates were diluted 1:5 in dissociation buffer without SDS and cleared, followed by anti-FLAG immunoprecipitation and immobilization on protein G sepharose beads (Pierce Chemicals, Rockford, IL, USA). Beads were washed four times with 0.1% SDS dissociation buffer. Precipitated proteins were resolved by SDS-PAGE and analyzed by western blotting using ECL detection (PerkinElmer Life and Analytical Sciences, Waltham, MA, USA).
GST-pull down assays
GST-proteins were expressed in Escherichia coli BL21 strain and purified from bacterial cell lysates. For binding assays, HEK 293T cells were transfected with the indicated constructs and lysed for 10 min on ice in lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton-X-100, 0.5 mM DTT, 10

M
ZnCl
2
) supplemented with 10 mM NEM, protease- and phosphatase inhibitors. Cell lysates were collected, centrifuged for 15 min (13,000× g ) to remove the insoluble fraction and incubated with GST-protein or GST alone coupled to glutathione sepharose 4B (GE Healthcare, Diegem, Belgium) for 3 h at +4 °C. After incubation, the sepharose matrix was washed three times with lysis buffer. Bound proteins were resolved by SDS-PAGE and analyzed by immunoblotting using the indicated antibodies.
NF-κB reporter gene assay
NF-

B dependent luciferase reporter assays were performed as previously described (De Valck et al.
, 1997).
IL-8 determination
IL-8 levels in cell supernatants were determined via specific enzyme-linked immunosorbent assay (BD
Biosciences) according to manufacturer’s instructions.
Protein expression and purification for NMR studies
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The coding sequence of human ubiquitin was cloned into the pET-30 Xa/LIC (Novagen), containing N-terminal
6×His- and S-tag, and into the pQE-9 (QIAGEN), containing a N-terminal 6×His-tag and an additional
Tryptophan residue for the accurate calculation of the concentration. The cloned proteins were expressed in 15 N-, or 13 C, 15 N-enriched M9 media following standard protocols and isolated using Ni-NTA chromatography. Further purification and equilibration with NMR buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.5) were achieved with size-exclusion chromatography. The GST- or NusA-6×His- fused ABIN-1 constructs were expressed in LB media and isolated with corresponding affinity chromatography. The intact fused and/or protease cleaved ABIN-
1 polypeptides were subjected to size-exclusion chromatography in NMR buffer.
NMR spectra acquisition
The NMR spectra were collected at 298 K on Bruker Avance700, Avance900 and DRX600 spectrometers equipped with cryogenic probes with Z-gradient capability. Proton chemical shifts were referenced relative to internal DSS. The 15 N and 13 C chemical shifts were referenced indirectly using the consensus Ξ ratios (Wishart et al.
, 1995). For the reassignement of the ubiquitin amide backbone resonances, HNCACB spectrum of 1.1 mM
13 C, 15 N-labelled ubiquitin was collected.
NMR titration experiments
For the titration experiments, series of the 15 N, 1 H-TROSY spectra of the pure 15 N-labelled ubiquitin at 0.2 mM concentration were collected in presence of various concentrations (0 – 0.4 mM) of non-labelled (fused or protease cleaved) ABIN-1
459-545 or it’s mutants. Chemical shift differences (Δσ) were calculated for each individual amide group using the formula:

=[

HN
2 +(

N
/6.5) 2 ] 1/2
The dissociation constants, Kd , were calculated by a least squares fit to the titration data under the assumptions of a fast exchange regime and a “one binding site” mode of the protein interaction. The equations for the Kd calculations have been described (Kang et al.
, 2003; Kim et al.
, 2001). Kd and Δδ max
=Δδ bound
-Δδ free
were used as minimization parameters.
References
De Valck D, Heyninck K, Van Criekinge W, Vandenabeele P, Fiers W, Beyaert R (1997). A20 inhibits NFkappaB activation independently of binding to 14-3-3 proteins. Biochem Biophys Res Commun 238: 590-4.
Heyninck K, Kreike MM, Beyaert R (2003). Structure-function analysis of the A20-binding inhibitor of NFkappa B activation, ABIN-1. FEBS Lett 536: 135-40.
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Kang RS, Daniels CM, Francis SA, Shih SC, Salerno WJ, Hicke L et al (2003). Solution structure of a CUEubiquitin complex reveals a conserved mode of ubiquitin binding. Cell 113: 621-30.
Kim S, Cullis DN, Feig LA, Baleja JD (2001). Solution structure of the Reps1 EH domain and characterization of its binding to NPF target sequences. Biochemistry 40: 6776-85.
Van Huffel S, Delaei F, Heyninck K, De Valck D, Beyaert R (2001). Identification of a novel A20-binding inhibitor of nuclear factor-kappa B activation termed ABIN-2. J Biol Chem 276: 30216-23.
Wishart DS, Bigam CG, Yao J, Abildgaard F, Dyson HJ, Oldfield E et al (1995). 1H, 13C and 15N chemical shift referencing in biomolecular NMR. J Biomol NMR 6: 135-40.
Wu CJ, Conze DB, Li T, Srinivasula SM, Ashwell JD (2006). NEMO is a sensor of Lys 63-linked polyubiquitination and functions in NF-kappaB activation. Nat Cell Biol 8: 398-406.
Wullaert A, Verstrepen L, Van Huffel S, Adib-Conquy M, Cornelis S, Kreike M et al (2007). LIND/ABIN-3 is a novel lipopolysaccharide-inducible inhibitor of NF-kappaB activation. J Biol Chem 282: 81-90.
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Supplementary figure legends:
Supplementary Figure 1A. The Ubiquitin – UBAN interaction is observed with different constructs.
Interaction of 15 N-labelled 6×His-Trp-Ubiquitin with uncleaved NusA-6×His-ABIN-1
459-545
(left panel), interaction of 15 N-labelled 6×His-S-Tag-Ubiquitin with uncleaved GST-ABIN-1
465-525
. The black contours shows unassigned resonanses from the S-Tag region (right panel).
Supplementary Figure 1B.
Results of the best fitting analysis for 10 Ub residues showing most prominent Δσ upon titration are presented as individual plots. Each plot contains experimentally observed Δσ values (open circles) and values calculated according to “monomolecular binding in fast exchange” assumption (line).
Additionally, residue and Kd value obtained during individual fitting are given in each plane. The concentration of Ub was calculeted from UV-spectra; the concentration for ABIN-1
459-545 was estimated by numbers of methods for each step of titration, the accuracy of ABIN-1
459-545
concentration corresponds to ~50%.
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