Frank J Ivins

Mark G Montgomery

Susan J M Smith

Aylin C Morris-Davies

Ian A Taylor

Katrin Rittinger
email: katrin.rittinger@nimr.mrc.ac.uk

NEMO OLIGOMERISATION AND ITS UBIQUITIN-BINDING PROPERTIES

Keywords: coiled-coil, ubiquitin, protein-protein interaction, AUC, MALS, ITC, IKK complex NF-B, nuclear factor-kappa B, NEMO, NF-B essential modulator, CC, coiled-coil, LZ, leucine zipper, ZF, zinc finger, ITC, isothermal titration calorimetry, AUC, analytical ultracentrifugation, MALS, multi-angle laser light scattering

The IKK complex is a key regulatory component of NF-B activation and is responsible for mediating the degradation of IB, thereby allowing nuclear translocation of NF-B and transcription of target genes. NEMO, the regulatory subunit of the IKK complex plays a pivotal role in this process by integrating upstream signals, in particular the recognition of poly-ubiquitin chains, and relaying these to the activation of IKK and IKK, the catalytic subunits of the IKK complex. The oligomeric state of NEMO is controversial and the mechanism by which it regulates activation of the IKK complex is poorly understood. Using a combination of hydrodynamic techniques we now show that apo-NEMO is a highly elongated, dimeric protein that is in weak equilibrium with a tetrameric assembly. Interaction with peptides derived from IKK disrupts formation of the tetrameric NEMO complex indicating that interaction with IKK and IKK and tetramerisation are mutually exclusive. Furthermore, we show that NEMO binds to linear di-ubiquitin with a stoichiometry of one molecule of di-ubiquitin per NEMO dimer. This stoichiometry is preserved in a construct comprising the second coiled-coil region and the leucine zipper and in one that essentially spans the full-length protein. However, our data show that at high di-ubiquitin concentrations a second, weaker binding site becomes apparent, implying that two different NEMO/di-ubiquitin complexes are formed during the IKK activation process. We propose that the role of these two complexes is to provide a threshold for activation thereby ensuring sufficient specificity during NF-B signaling.

Introduction

NF-B transcription factors are key regulators of immune and inflammatory responses (reviewed in [START_REF] Hayden | Shared principles in NF-kappaB signaling[END_REF][START_REF] Ghosh | Missing pieces in the NF-kappaB puzzle[END_REF]). In unstimulated cells they are found in complex with inhibitor B proteins (IBs), an interaction that prevents their translocation to the nucleus and retains NF-Bs in the cytosol. Stimulus-dependent phosphorylation of IBs induces their Lys48-linked ubiquitination and subsequent degradation by the 26S proteasome, thereby liberating NF-B and allowing nuclear translocation. A key regulator of this process is the multi-subunit IKK complex (sometimes referred to as the signalosome), which is composed of two kinases, IKK and IKK and a noncatalytic regulatory subunit called IKK or NEMO (for NF-B essential modulator) [START_REF] Rothwarf | IKK-gamma is an essential regulatory subunit of the IkappaB kinase complex[END_REF][START_REF] Yamaoka | Complementation cloning of NEMO, a component of the IkappaB kinase complex essential for NF-kappaB activation[END_REF][START_REF] Scheidereit | IkappaB kinase complexes: gateways to NF-kappaB activation and transcription[END_REF]. The apparent molecular mass of the resting IKK complex has been shown to be in the 700-900 kDa range, suggesting that it may contain multiple copies of its constituents [START_REF] Didonato | A cytokine-responsive IkappaB kinase that activates the transcription factor NF-kappaB[END_REF][START_REF] Mercurio | IKK-1 and IKK-2: cytokine-activated IkappaB kinases essential for NF-kappaB activation[END_REF][START_REF] Regnier | Identification and characterization of an IkappaB kinase[END_REF]. Nonetheless, its precise composition is controversial and it may be that its size varies in a stimulus-dependent manner [START_REF] Poyet | Activation of the Ikappa B kinases by RIP via IKKgamma /NEMO-mediated oligomerization[END_REF]. NEMO is the regulatory hub of the IKK complex and integrates multiple signals including phosphorylation, ubiquitination and interaction with ubiquitin chains [START_REF] Sebban | Posttranslational modifications of NEMO and its partners in NF-kappaB signaling[END_REF]. In addition to being the target of multiple post-translational modifications, NEMO also acts as an assembly platform that facilitates incorporation of a variety of proteins into the IKK signalosome such as the viral proteins v-FLIP and Tax, which can act as potent activators of NF-B [START_REF] Sun | Activation of NF-kappaB by HTLV-I and implications for cell transformation[END_REF]12].

NEMO is a ~50 kDa protein predicted to consist of two coiled-coil (CC1 and CC2) regions, a leucine zipper (LZ) and a zinc finger (ZF)(Figure1). In spite of extensive studies aimed at defining the assembly of NEMO, its oligomeric state still remains a matter of some controversy. The apo form of the full-length protein has been described to exist in a monomeric, dimeric, trimeric or tetrameric state [START_REF] Agou | NEMO trimerizes through its coiled-coil C-terminal domain[END_REF][START_REF] Fontan | NEMO oligomerization in the dynamic assembly of the IkappaB kinase core complex[END_REF][START_REF] Tegethoff | Tetrameric oligomerization of IkappaB kinase gamma (IKKgamma) is obligatory for IKK complex activity and NF-kappaB activation[END_REF]. A number of studies have suggested that the second coiled-coil region, together with the leucine zipper, constitutes the minimal oligomerisation domain (from now on referred to as CoZi) [START_REF] Tegethoff | Tetrameric oligomerization of IkappaB kinase gamma (IKKgamma) is obligatory for IKK complex activity and NF-kappaB activation[END_REF]16]. However, like the full-length protein, the oligomeric state of this region is not clear and has, for example, been described to exist as a trimer that can associate to "pseudo-hexamers" upon appropriate stimulation [16]. In contrast, two recent crystal structures of this region have shown it to form a parallel, dimeric coiled-coil in accordance with other studies that reported a dimeric assembly [START_REF] Tegethoff | Tetrameric oligomerization of IkappaB kinase gamma (IKKgamma) is obligatory for IKK complex activity and NF-kappaB activation[END_REF][START_REF] Lo | Structural Basis for Recognition of Diubiquitins by NEMO[END_REF][START_REF] Rahighi | Specific recognition of linear ubiquitin chains by NEMO is important for NF-kappaB activation[END_REF][START_REF] Bloor | Signal processing by its coil zipper domain activates IKK gamma[END_REF]. The oligomeric state of the N-terminal coiled-coil region is similarly unresolved and a range of oligomeric states have been reported [START_REF] Drew | Inhibitor kappaB kinase beta binding by inhibitor kappaB kinase gamma[END_REF][START_REF] Lo | High-affinity interaction between IKKbeta and NEMO[END_REF][START_REF] Marienfeld | Dimerization of the I kappa B kinase-binding domain of NEMO is required for tumor necrosis factor alpha-induced NF-kappa B activity[END_REF].

A key step towards a better understanding of the regulatory role of NEMO in NF-B activation was the observation that NEMO interacts specifically with polyubiquitin chains [START_REF] Wu | Sensing of Lys 63-linked polyubiquitination by NEMO is a key event in NF-kappaB activation [corrected][END_REF][START_REF] Ea | Activation of IKK by TNFalpha requires site-specific ubiquitination of RIP1 and polyubiquitin binding by NEMO[END_REF]. These experiments initially utilized linear ("genetically linked", i.e. G76 to M1 linked) poly-ubiquitin chains in a yeast two-hybrid screen. However, K63-linked ubiquitin chains attached to RIP1 (receptor interaction protein kinase) are believed to be the physiological targets [START_REF] Wu | Sensing of Lys 63-linked polyubiquitination by NEMO is a key event in NF-kappaB activation [corrected][END_REF]. This interaction is suggested to recruit the IKK complex to receptor complexes and thereby promote the subsequent activation of the kinase activity of IKK and IKK. The binding site for ubiquitin has been mapped to a region of NEMO that has homology with ABIN and optineurin and has been named the NUB or UBAN motif [START_REF] Wu | Sensing of Lys 63-linked polyubiquitination by NEMO is a key event in NF-kappaB activation [corrected][END_REF][START_REF] Ea | Activation of IKK by TNFalpha requires site-specific ubiquitination of RIP1 and polyubiquitin binding by NEMO[END_REF][START_REF] Wagner | Ubiquitin binding mediates the NF-kappaB inhibitory potential of ABIN proteins[END_REF]. This conserved binding region is located within the CoZi domain. Although a number of studies have shown that specific recognition of K63-linked ubiquitin chains is important for NF-B activation [START_REF] Wu | Sensing of Lys 63-linked polyubiquitination by NEMO is a key event in NF-kappaB activation [corrected][END_REF][START_REF] Ea | Activation of IKK by TNFalpha requires site-specific ubiquitination of RIP1 and polyubiquitin binding by NEMO[END_REF], it has recently been reported that linear di-ubiquitin binds with a 100-fold higher affinity to the CoZi domain than K63-linked di-ubiquitin [START_REF] Lo | Structural Basis for Recognition of Diubiquitins by NEMO[END_REF][START_REF] Rahighi | Specific recognition of linear ubiquitin chains by NEMO is important for NF-kappaB activation[END_REF][START_REF] Komander | Molecular discrimination of structurally equivalent Lys 63-linked and linear polyubiquitin chains[END_REF]. This observation is particularly interesting in light of the recent report that NEMO itself becomes modified with linear ubiquitin chains that are attached by the ligase complex LUBAC [27].

In this study we have applied a combination of complementary hydrodynamic methods to define the oligomeric state of NEMO and resolve the current controversy surrounding its self-association. We show that apo-NEMO forms a tight dimer that is in a relatively weak equilibrium with a tetrameric assembly. Importantly, binding of IKK-derived peptides prevented formation of the tetramer, indicating that the IKKbinding region in the N-terminal portion of NEMO is responsible for the formation of higher molecular mass species and that IKK-binding and tetramerisation are mutually exclusive. Furthermore, we show that the CoZi domain and a nearly full-length NEMO construct bind di-ubiquitin with similar affinities but slightly different energetics. These data suggest that no major conformational changes take place upon interaction with ubiquitin chains but that minor rearrangements may be induced in the N-terminal coiled-coil region. Surprisingly, only one di-ubiquitin chain binds to the symmetrical NEMO dimer. However, a second binding site becomes apparent at high ubiquitin concentrations, implying that two different NEMO/poly-ubiquitin complexes are formed during the activation process. This observation may have implications for the in vivo function of NEMO and suggests that association with protein assemblies that provide a high density of poly-ubiquitin chains might induce clustering of NEMO which in turn will lead to activation of the IKK complex. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Experimental Plasmids

The cDNA for full-length mouse and human NEMO and the expression plasmid for diubiquitin were kindly provided by Dr. F. Randow (MRC-LMB, Cambridge, UK). NEMO 251-355 (from now on referred to as CoZi), NEMO (1-355, C54S, K285N referred to as NEMO 355 ) and human NEMO 218-363 were cloned into the pGEX-6P1 expression vector. The C54S mutation was introduced to prevent disulfide bond formation, K285N has been described in [START_REF] Bloor | Signal processing by its coil zipper domain activates IKK gamma[END_REF]. Bovine mono-Ub was purchased from Sigma.

Protein Purification

CoZi purification. BL21(DE3) cells containing the plasmid were grown in Luria Broth (LB) 37°C to an OD 600 of ~0.8. Protein expression was induced by the addition of 0.5mM IPTG and cells were incubated for a further 3 hours. Cell pellets were resuspended in Buffer A (50mM Tris pH8, 500mM NaCl and 0.5mM TCEP), lysed by sonication and the crude lysate was applied to a pre-equilibrated glutathione sepharose column (GE Healthcare) at 1ml/min. Bound protein was eluted with Buffer A containing 20mM reduced glutathione. The GST affinity tag was removed by the addition of 3C protease and incubation at 4°C overnight. The sample was concentrated in 10kDa MWCO Vivaspin concentrators (Sartorius) and applied to a Superdex 75 column (GE Healthcare) pre-equilibrated with Buffer B (50mM Tris pH8, 100mM NaCl and 0.5mM TCEP). Fractions containing CoZi were pooled and loaded onto another GSH column to remove contaminating GST and 3C proteins. CoZi was concentrated to 20 mg/ml. NEMO 355 purification . Expression was carried out in Rosetta2(DE3)pLysS cells at 18˚C for 16hr. Cell pellets were resuspended in Buffer GST (20 mM Tris pH8, 500 mM NaCl, 10% glycerol, 0.25 mM TCEP) and lysed by sonication. Crude lysate was applied to a column of glutathione sepharose resin followed by extensive washing with Buffer GST . The GST affinity tag was removed on-column by overnight incubation with 1.5 mg 3C protease. GST-free NEMO 355 was eluted in Buffer Q (20 mM Tris pH 8, 50 mM NaCl, 10% glycerol, 0.25 mM TCEP) and applied to a Source Q ion exchange column (GE Healthcare). The sample was eluted with a 1M NaCl 20 column volume gradient in Buffer Q , and concentrated to 5ml on a 50kDa MWCO Vivaspin concentrator. The sample was then applied to a Superdex 200 sizeexclusion column pre-equilibrated in Buffer GST . The relevant fractions were concentrated to 13.7mg/ml. Linear di-Ubiquitin. Expression was carried out in BL21(DE3) cells for 3 hours at 37°C. Purification was carried out as described previously for CoZi except the affinity tag was removed by incubation with TEV protease instead of 3C protease. Pure protein was concentrated using 5kDa MWCO Vivaspin columns to 13.3mg/ml. Protein concentrations were determined by UV spectrometry using calculated 280 nm molar extinction coefficients. Concentrations were confirmed by analysis of the differential refractive index eluate peaks from the SEC-MALS experiments described below. Specifically, integration of an RI peak yields total mass that, in conjunction with known loading volume, allows determination of the loading concentration independent of UV absorbance. Concentration values from both methods were in good agreement. Furthermore, expected formula molecular weights were confirmed for each protein using mass spectrometry. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Isothermal Titration Calorimetry

All samples were extensively dialyzed into fresh buffer (50mM Tris pH8, 0.5mM TCEP and 100mM NaCl (CoZi) or 150 mM NaCl (NEMO 355 ). Protein concentration was determined by UV absorbance and confirmed by the integration of refractive index peaks from SEC-MALS (see below). ITC measurements were carried out at 10°C, 18°C and 25° C on a VP-ITC or ITC200 MicroCalorimeter (MicroCal Inc). In most cases, VP-ITC titrations were carried out with 30 consecutive 10 μl injections of ubiquitin into the cell containing NEMO on the VP-ITC or alternatively with 19 consecutive 2 μl injections on the ITC200 (all at varying concentrations). ITC data were analyzed with Origin7 (MicroCal) supplied by the manufacturer. The heat capacity change was determined from the slope of a linear regression fit of H plotted against the temperature of the titration.

Multi-Angle Light Scattering (MALS)

Molecular mass and molecular mass distributions were determined using on line multi-angle laser light scattering coupled with size exclusion chromatography (SEC-MALS). Samples were applied in a volume of 100 µl to a Superdex 200 10/300 GL column equilibrated in 20 mM Tris-HCl, 150 mM NaCl 0.25 mM TCEP, pH 8.0 at a flow rate of 0.5ml/min. The column was mounted on a Jasco HPLC controlled by the Chrompass software package. The scattered light intensity of the column eluate was recorded at sixteen angles using a DAWN-HELEOS laser photometer (Wyatt Technology Corp., Santa Barbara, CA). The Protein concentration of the eluent was determined from the refractive index change (dn/dc = 0.186) using an OPTILAB-rEX differential refractometer equipped with a Peltier temperature-regulated flow cell, maintained at 25°C (Wyatt Technology Corp., Santa Barbara, CA). The wavelength of the laser in the DAWNHELEOS and the light source in the OPTILABrEX was 658nm. The weight-averaged molecular mass of material contained in chromatographic peaks was determined using the ASTRA software version 5.1 (Wyatt Technology Corp., Santa Barbara, CA). Briefly, at 1-second intervals throughout the elution of peaks the scattered light intensities together with the corresponding protein concentration were used to construct Debye plots (KC/R θ  vs sin 2 (θ/2)). The weight-averaged molecular mass was then calculated at each point in the chromatogram from the intercept of an individual plot. An overall average molecular mass and polydispersity term for each species was calculated by combining and averaging the data from the individual measurements.

Sedimentation Velocity Analytical Ultracentrifugation

Sedimentation velocity experiments were performed in a Beckman Optima Xli analytical ultracentrifuge, using conventional charcoal filled Epon double-sector quartz cells or aluminium double-sector sapphire cells in an An-50 Ti rotor. The rotor speed was 40,000 rpm or 50,000 rpm, respectively, and the temperature was maintained at 293 K. Prior to centrifugation, protein samples were dialyzed exhaustively against the buffer blank, 10 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.25 mM TCEP. The protein concentration was varied from 0.37 mg/ml to 1.76 mg/ml. Interference images were collected every 180 seconds during the sedimentation run. The data recorded from moving boundaries were analyzed by the program SEDFIT in terms of both discrete species and continuous distribution function of sedimentation coefficient C(S).

Results NEMO 355 exists in a dimer/tetramer equilibrium

In order to resolve the discrepancies concerning the exact multimeric state of NEMO, the solution molecular mass of a construct containing all of the predicted coiled-coil regions was examined by size exclusion chromatography coupled to multiangle laser light scattering (SEC-MALS). This construct is missing only the C-terminal zinc finger (ZF), which has been shown to be monomeric [28] and will be referred to from now on as NEMO 355 (Figure 1). SEC-MALS allows the determination of an absolute molecular mass during analytical gel filtration, independent of the shape of the molecule under investigation, and hence is well suited to determine the oligomeric state of proteins. An initial MALS experiment reports a weight-averaged molecular mass (M W ) of 85.7 kDa for an average peak concentration of 1.4 μM, a value in close agreement with the formula molecular mass of dimeric NEMO 355 (83.8 kDa). However, at increased concentrations elevated weight-averaged M W values are apparent, a phenomenon that was unaffected when the NaCl concentration was increased to 500 mM (data not shown). We therefore further analyzed the concentration-dependent M W of NEMO 355 with a sequence of SEC-MALS analyses ranging from 1.4 to 16.9 μM (average peak concentration), as shown in Figure 2. These experiments reported corresponding weight-averaged M W values of 85.7 -113.8 kDa and clearly demonstrate that NEMO 355 is subject to concentrationdependent self-association based upon a minimum assembling unit equivalent to a dimer. The assembly of dimers into tetramers is the simplest model explaining these data.

In order to further characterize the size of the multimers involved in selfassociation, we investigated NEMO 355 using sedimentation velocity analytical ultracentrifugation (AUC) at three concentrations: 8.8 μM, 17.6 μM, and 42 μM (monomer concentration). C(S) distribution analyses of the data clearly showed the presence of two major species centered at S 20,w values of approximately 3 and 4 (Figure 2). Consistent with the MALS data above, the relative peak proportions varied in a concentration-dependent manner. At low concentration, the predominant peak was centered on the lower S 20,w value, while the reverse was true at higher concentration. At intermediate concentration (17.6 μM), the peaks were nearly indistinguishable. As the peaks were fully separated in the C(S) function for the low concentration experiment, we were able to analyze these data as discrete species. Data fit well to a two-component discrete species model with S 20,w values of 3.00 and 4.23 and D t,20,w values of 3.48 and 2.30, respectively (Figure 2 and Table 1). The respective weight-averaged molecular masses calculated from these values are 77.6 and 164.9 kDa. These values are in good agreement with the molecular mass expected from NEMO 355 dimers (83.8 kDa) and tetramers (167.6 kDa). Using the relative peak proportions from the C(S) function for the low concentration experiment (8.8 μM), an apparent dissociation constant (K d ) of 25.6 μM (dimer-tetramer equilibrium) was obtained. Notably, a fit of the SEC-MALS concentration-dependent data to a two-state self-association model resulted in an apparent K d of 27.5 μM (data not shown).

In order to help identify the region responsible for NEMO tetramerisation, isolated NEMO CoZi domain was also subjected to sedimentation velocity analysis at 46, 93 and 139 µM monomer concentration (data not shown). The data from this experiment fit well to a discrete species model showing a single significant component and weight-averaged molecular mass with a range of 25.0-27.8 kDa, consistent with the formula molecular mass of dimeric CoZi domain (25.9 kDa) (see Table 3). These data do not indicate the presence of the tetramers evident in the NEMO 355 construct, suggesting that the N-terminal region of NEMO is responsible for higher order self-association.

NEMO adopts a highly elongated conformation

Importantly, the discrete species analysis of the AUC data also allowed us to calculate the frictional ratio (f/f 0 ) for CoZi and the NEMO 355 dimer and tetramer. The frictional ratio is a measure of the degree of asymmetry in a molecule with respect to an anhydrous sphere of the same mass. For CoZi a f/f0 of 1.7 was measured, which deviates considerably from the value (1.0) characteristic of a spherical protein molecule and points towards a highly elongated structure. Remarkably, the frictional ratios for NEMO 355 dimers and tetramers were significantly higher, 2.3 and 2.6, respectively, demonstrating that both are unusually elongated and asymmetric molecules in solution (Table 1).

IKK binding abolishes NEMO 355 tetramerisation

The most highly recognized function of NEMO within the NF-B signaling pathway is in the recruitment and regulation of IKKs responsible for the phosphorylation and inactivation of IB. In order to further investigate the nature of the NEMO/IKK interaction, we analyzed a complex of NEMO 355 and a 41-mer peptide comprising the IKK NEMO binding domain (IKK NBD ; 4.7 kDa) by SEC-MALS analysis. The resulting weight-averaged molecular mass of the complex (92 kDa) was in close agreement with the formula molecular mass expected of dimeric NEMO 355 bound to two IKK NBD peptides (93.3 kDa) (Figure 3). This result agrees with a recently published heterotetrameric crystal structure comprising a short, dimeric NEMO N-terminal fragment bound to two IKK peptides [29]. Interestingly, the weightaveraged molecular mass reported by SEC-MALS remained constant over a wide NEMO 355 concentration range, demonstrating that the interaction is tight in accordance with previous studies [START_REF] Drew | Inhibitor kappaB kinase beta binding by inhibitor kappaB kinase gamma[END_REF]29]. Moreover, it shows that binding of IKK NBD completely abrogates the concentration-dependent tetramerisation of NEMO 355 . These results indicate that the IKK binding and tetramerisation regions are overlapping. Importantly, these data demonstrate that the oligomeric state of NEMO is modulated by binding of specific ligands.

The CoZi/di-ubiquitin interaction

Most ubiquitin binding domains characterized so far interact with mono-ubiquitin, often with relatively low affinities [START_REF] Hurley | Ubiquitin-binding domains[END_REF][START_REF] Hicke | Ubiquitin-binding domains[END_REF]. NEMO also binds very weakly to monoubiquitin [START_REF] Bloor | Signal processing by its coil zipper domain activates IKK gamma[END_REF] and surprisingly, only weakly to K63-linked di-ubiquitin, while the interaction with linear di-ubiquitin is around 100-fold stronger [START_REF] Lo | Structural Basis for Recognition of Diubiquitins by NEMO[END_REF][START_REF] Rahighi | Specific recognition of linear ubiquitin chains by NEMO is important for NF-kappaB activation[END_REF][START_REF] Komander | Molecular discrimination of structurally equivalent Lys 63-linked and linear polyubiquitin chains[END_REF] . In order to better understand the thermodynamics of the interaction with linear ubiquitin chains we used isothermal titration calorimetry (ITC) to follow complex formation. No binding was detected between the CoZi domain and mono-ubiquitin at three different temperatures at concentrations of 50 µM CoZi in the cell and 500 µM ubiquitin in the syringe, indicating that any interaction must be very weak (data not shown). Titration of CoZi with increasing concentrations of di-ubiquitin at 18 o C resulted in heats of interaction that were too small for quantitative analysis. However, titrations at 25 o C and 10 o C produced sufficient signal to be able to fit the binding isotherm, resulting in dissociation constants of 1.4 and 2.6 µM, respectively (see Table 2 and Figure 4A). Surprisingly, the stoichiometry of complex formation was ~0.5 in terms of polypeptide chains, demonstrating that one CoZi dimer interacts with one molecule of di-ubiquitin. Given the pseudo-symmetrical structure of dimeric CoZi [START_REF] Lo | Structural Basis for Recognition of Diubiquitins by NEMO[END_REF][START_REF] Rahighi | Specific recognition of linear ubiquitin chains by NEMO is important for NF-kappaB activation[END_REF] one might expect that equivalent binding sites on each NEMO chain exist, and hence that two molecules of di-ubiquitin would bind, one either side (CoZi dimer /2xUb2). In contrast, these binding studies suggest that the di-ubiquitin molecule interacts with NEMO in an asymmetric fashion. To test if this binding mode is preserved in the context of full length NEMO, we titrated NEMO 355 with di-ubiquitin. As observed with the isolated CoZi domain, the enthalpy of complex formation was strongly temperaturedependent and endothermic at lower temperatures but exothermic at room temperature and above. Notably, the binding stoichiometry and strength of complex formation are similar to that found for the CoZi domain with K d s of 0.95 µM and 3.45 µM at 25 o C and 10 o C, respectively (Table 2 and Figure 4 B). However, there is some difference in the heat capacity change of complex formation which is -587cal . mol -1. K -1 for CoZi and -360 cal . mol -1. K -1 for the NEMO 355 di-ubiquitin interaction (Figure 4C).

Analysis of NEMO/ubiquitin interaction by analytical ultracentrifugation

To further investigate the composition of the CoZi/di-ubiquitin complex and confirm the stoichiometry suggested by our ITC experiments, we characterized the CoZi/di-ubiquitin interaction by sedimentation velocity AUC which allows direct observation of the complex(es) under investigation. Multiple experiments were performed in which molar ratios of CoZi dimer and di-ubiquitin were varied (Ub2);

(1:1, 1:2, 1:4, and 1:8 (CoZi dimer :Ub2)). Molar concentrations and the resulting hydrodynamic data from these experiments are detailed in Table 3. In each of the three experiments where di-ubiquitin was present in stoichiometric excess, two discrete species were observed. The smallest component in each experiment clearly represents free di-ubiquitin, as the S, D t , and M W values closely correspond to the values obtained from analysis of di-ubiquitin alone (Table 3). In the 1:1 experiment, the largest and most abundant component has a mass of 45.3 kDa, close to the predicted molecular mass of a complex consisting of dimeric CoZi bound to one diubiquitin chain (CoZi dimer /Ub2 = 43.5 kDa). As expected, increasing excess of diubiquitin resulted in increasing proportions of free di-ubiquitin. Unexpectedly, however, the apparent molecular mass of the complex component also increased with increasing excess of di-ubiquitin (Figure 5 and Table 3). At the highest ratio (1:8 CoZi dimer :Ub2), the apparent mass increased to 53.3 kDa. This trend suggests formation of a complex containing a second di-ubiquitin bound to dimeric CoZi. We used the weight-averaged molecular mass (53.3 kDa) for the 1:8 experiment to determine the proportions of (CoZi dimer /Ub2 and CoZi dimer /(Ub2) 2 from which we can estimate a 100-fold weaker binding constant for the second di-ubiquitin binding site.

Discussion

NEMO is the key regulatory component of the IKK complex that senses upstream stimulatory events and relays them to the activation of IKK and IKK. A large proportion of the protein is predicted to form coiled-coils, including the regions that are involved in ubiquitin recognition and interaction with the catalytic IKK subunits. The molecular mechanism by which NEMO induces activation of IKK and IKK remains largely unknown, although various models have been suggested. One model assumes the CoZi domain to undergo stimulus-dependent conformational changes that in turn might induce activation of the kinases [START_REF] Bloor | Signal processing by its coil zipper domain activates IKK gamma[END_REF]. Alternatively, NEMO might act as a carrier or platform that is responsible for bringing IKK and IKK in close proximity to activators such as TAK1 (TGF-activated kinase-1) to promote phosphorylation and activation [START_REF] Wu | Sensing of Lys 63-linked polyubiquitination by NEMO is a key event in NF-kappaB activation [corrected][END_REF][START_REF] Ea | Activation of IKK by TNFalpha requires site-specific ubiquitination of RIP1 and polyubiquitin binding by NEMO[END_REF]. On the other hand, the interaction with ubiquitin chains may induce clustering of NEMO, which could lead to IKK activation, possibly by inducing trans-phosphorylation. In this study we have characterized the oligomeric state of NEMO and its interaction with di-ubiquitin in order to provide a molecular basis for a better understanding of its role in NF-B activation.

The oligomeric state of NEMO

The oligomeric state of NEMO has been controversial and a number of different models have been suggested for the quaternary structure of the full-length protein as well as various fragments. Using a combination of MALS and analytical ultracentrifugation, we show that NEMO forms a very tight dimer that exists in a relatively weak equilibrium (~27 µM) with tetramers. Importantly, our extensive analysis shows that the protein preparations used in our study are homogeneous and monodisperse and that no aggregates are present that may interfere with the measurements. Our results partially agree with a previous study that described human NEMO to predominantly form tetramers [START_REF] Tegethoff | Tetrameric oligomerization of IkappaB kinase gamma (IKKgamma) is obligatory for IKK complex activity and NF-kappaB activation[END_REF] but contradict a number of others that described NEMO to form monomers and trimers [START_REF] Agou | NEMO trimerizes through its coiled-coil C-terminal domain[END_REF][START_REF] Fontan | NEMO oligomerization in the dynamic assembly of the IkappaB kinase core complex[END_REF]16]. The most likely explanation for this discrepancy is the presence of detergent in the work by Agou and colleagues, which was, apparently, required to prevent the formation of aggregates, suggesting that the protein used in those studies may not have been in its native conformation. This interpretation is supported by the presence of DnaK in the protein preparations used for some of these studies. Furthermore, we believe that the varying and highly contradicting oligomeric states determined for different N-terminal constructs of NEMO [START_REF] Drew | Inhibitor kappaB kinase beta binding by inhibitor kappaB kinase gamma[END_REF][START_REF] Lo | High-affinity interaction between IKKbeta and NEMO[END_REF] are due to an inherent instability and heterogeneity of shorter NEMO fragments, which may have compromised earlier work. This interpretation is supported by Rushe and colleagues who reported a similar lack of homogeneity for shorter N-terminal fragments of NEMO [29].

In contrast to NEMO 355 , the CoZi domain has no tendency to tetramerise over a range of concentrations. Furthermore, complex formation with an IKK-derived peptide completely abolished the ability of dimeric NEMO 355 to form higher oligomers, showing that the region surrounding the IKK binding site is responsible for tetramerisation. These data are in contrast to previous cross-linking studies, which suggested that NEMO binds the IKKs in its tetrameric form and that tetramerisation is required for kinase activity [START_REF] Tegethoff | Tetrameric oligomerization of IkappaB kinase gamma (IKKgamma) is obligatory for IKK complex activity and NF-kappaB activation[END_REF]. It now seems likely that cross-linking has overestimated the oligomeric state of NEMO due to non-specific association. Nevertheless, the model that oligomerisation of NEMO might be part of the IKK activation mechanism is intriguing and is fully compatible with the notion that binding to poly-ubiquitin chains induces clustering of NEMO as described below. Importantly, the observation that interaction with IKK abolishes tetramerisation clearly illustrates that the oligomeric state of NEMO in its ligand complexes might differ from that of the apo-protein.

Similar to full-length NEMO, the state of the minimal oligomerisation domain, CoZi, has been controversial. Two recent crystal structures of the apo form of this domain show it to form a parallel coiled-coil that is around 120Ǻ in length [START_REF] Lo | Structural Basis for Recognition of Diubiquitins by NEMO[END_REF][START_REF] Rahighi | Specific recognition of linear ubiquitin chains by NEMO is important for NF-kappaB activation[END_REF]. This is in accordance with the data reported here, yet contradicts a number of other studies that reported trimeric and hexameric assemblies [START_REF] Agou | NEMO trimerizes through its coiled-coil C-terminal domain[END_REF]16]. A CoZi construct comprising amino acids 250-339, has been reported to exist in a monomer-dimer equilibrium with a K d of 30 µM [START_REF] Bloor | Signal processing by its coil zipper domain activates IKK gamma[END_REF]. The fragment used in our study is slightly extended at the C-terminus and includes amino acids 340-355, which are not predicted to be part of the LZ. Interestingly, the inclusion of this region significantly strengthens dimerisation, suggesting that this C-terminal extension, which is absent in the crystallised fragments, is part of the coiled-coil.

Analytical ultracentrifugation studies of NEMO 355 show that it adopts an extremely elongated conformation, as illustrated by the frictional ratios for NEMO 355 dimers and tetramers (2.3 and 2.6, respectively). These values are significantly higher than would be expected for a globular protein and are in fact similar to the value measured for fibrinogen (2.3), a rod-like protein that has been shown by electron microscopy to have an average axial ratio (length divided by diameter) of 7.5 [START_REF] Hall | The fibrinogen molecule: its size, shape, and mode of polymerization[END_REF][33][START_REF] Smith | Handbook of Biochemistry-Selected Data for Molecular Biology[END_REF]. The recently reported structures of NEMO 44-111 bound to IKK-derived peptides [29] and NEMO 150-272 bound to vFLIP [35] show the respective, ligandbound fragments of NEMO to form an elongated dimeric coiled-coil. In combination with our AUC studies, these observations indicate that binding of target proteins will not induce gross conformational changes, but can alter oligomerisation. Instead, apo-NEMO forms a highly elongated coiled-coil that is poised to accommodate the binding of different proteins, apparently without major structural rearrangements. Nevertheless, it is tempting to speculate that binding to ligands may induce slight local changes that are propagated along the coiled-coil, ultimately inducing activation of IKK and IKK.

The NEMO/poly-ubiquitin interaction

The interaction of NEMO with poly-ubiquitin chains is important for the activation of the IKK complex in response to TNF stimulation [START_REF] Wu | Sensing of Lys 63-linked polyubiquitination by NEMO is a key event in NF-kappaB activation [corrected][END_REF][START_REF] Ea | Activation of IKK by TNFalpha requires site-specific ubiquitination of RIP1 and polyubiquitin binding by NEMO[END_REF]. Binding occurs via the UBAN motif within the CoZi domain. The interaction of mono-ubiquitin with CoZi is extremely weak and we could not detect any binding under the conditions of our ITC experiments although high sensitivity LUMIER assays showed weak binding can occur [START_REF] Bloor | Signal processing by its coil zipper domain activates IKK gamma[END_REF]. In contrast, linear di-ubiquitin binds with an affinity in the low micromolar range, clearly indicating that the interaction with each individual ubiquitin is additive. Unexpectedly, binding of NEMO to K63-linked di-ubiquitin is significantly weaker than to linear chains [START_REF] Lo | Structural Basis for Recognition of Diubiquitins by NEMO[END_REF][START_REF] Rahighi | Specific recognition of linear ubiquitin chains by NEMO is important for NF-kappaB activation[END_REF][START_REF] Komander | Molecular discrimination of structurally equivalent Lys 63-linked and linear polyubiquitin chains[END_REF] , suggesting that linear poly-ubiquitin chains may be the true physiological targets [START_REF] Rahighi | Specific recognition of linear ubiquitin chains by NEMO is important for NF-kappaB activation[END_REF].

The stoichiometry of the CoZi/di-ubiquitin interaction determined in our ITC experiments indicated that one NEMO dimer interacts with one molecule of diubiquitin. This stoichiometry is in accordance with an independent study by Lo and colleagues that also detected a 2:1 complex in solution [START_REF] Lo | Structural Basis for Recognition of Diubiquitins by NEMO[END_REF]. Based on NMR chemical shift perturbations and mutagenesis studies these authors suggested a model for the complex, in which di-ubiquitin clasps around dimeric NEMO roughly perpendicular to the long axis of the NEMO coiled-coil. However, this stoichiometry and the derived model strongly contradict the recently published CoZi/di-ubiquitin structure, which instead shows two molecules of di-ubiquitin to bind to a NEMO dimer in an orientation that is parallel to the coiled-coil axis [START_REF] Rahighi | Specific recognition of linear ubiquitin chains by NEMO is important for NF-kappaB activation[END_REF]. Moreover, the authors of this study present NF-B activation assays with NEMO mutants defective in ubiquitin binding, which support the notion that binding of two di-ubiquitin molecules to NEMO is physiologically important. A simple explanation for the apparent discrepancy between these studies could be the partial dissociation of CoZi in the solution studies. However, this explanation seems highly unlikely, as our AUC sedimentation analysis of CoZi at concentrations used in the ITC experiments shows that CoZi exists entirely in its dimeric form.

Interestingly, in solution NEMO 355 shows similar binding properties towards diubiquitin in terms of stoichiometry and apparent affinity like the CoZi domain, while the thermodynamic characteristics of complex formation show small but distinctive differences. This observation implies that binding of ubiquitin does not induce any large conformational changes in the region N-terminal to the CoZi domain since this would likely be reflected in the affinity of the interaction. Nevertheless it indicates that some communication exists between the CoZi domain and the N-terminal part of NEMO.

To gain further insight into the CoZi/di-ubiquitin interaction and understand the apparently conflicting binding stoichiometries, we analysed complexes at different NEMO/di-ubiquitin ratios by analytical ultracentrifugation. Sedimentation velocity experiments at equimolar concentrations of CoZi dimers and di-ubiquitin showed a complex of the molecular mass expected from the stoichiometry observed in our ITC studies. Intriguingly however, increases in the relative ratio of di-ubiquitin over CoZi led to an increase in the apparent molecular mass of the complex, suggesting that at higher concentrations of di-ubiquitin a second binding site in NEMO becomes occupied. Affinity for this second site is significantly weaker, and thus it is possible that binding of the first molecule of di-ubiquitin may occlude binding of a second. However, high ubiquitin concentrations might induce slight structural rearrangements in NEMO, which in turn allow the second binding site to become accessible. Alternatively, apo-NEMO might exist in an equilibrium between two states, of which only one may be capable of binding two di-ubiquitin chains. This state might be unfavourable and hence would only be able to be occupied at high ubiquitin concentrations. The crystallization conditions might have selected for this state over the other. It is tempting to speculate that, in vivo, a multi-protein complex containing ubiquitinated proteins such as RIP1 may provide the high ubiquitin density required for occupation of the second site. Population of the second site might be the trigger to initiate IKK activation, possibly through clustering of NEMO. In fact, this two-site binding event might constitute a "threshold for activation" to prevent non-specific activation of NF-B, which can have detrimental consequences for the host. Further studies are now required to better characterize the in vivo role of these two binding sites and understand the molecular events leading to their formation.

Table 1 Hydrodynamic parameters of the 8.8 μM NEMO 355 sedimentation velocity experiment (discrete species model).

Formula molecular weight (M r ); partial specific volume (V bar ); weight-averaged molecular mass (M w ) reported by experiment; sedimentation coefficient (S 20,w ); translational diffusion coefficient (D t,20,w ); frictional coefficient ratio (f/f 0 ); dissociation constant (K d ) calculated from known loading concentration and relative peak proportions for a dimer-tetramer equilibrium; average root mean square deviation of the fit (RMSD). Parameters with only one value are independent of the particular species involved. A c c e p t e d M a n u s c r i p t

Parameters

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Table 2

Thermodynamic parameters for the NEMO/di-ubiquitin interaction.

All titrations were carried out between 3-4 times and the estimated error is the standard deviation between measurements. A c c e p t e d M a n u s c r i p t

Component in cell

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Biochemical

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Biochemical

  Journal Immediate Publication. Published on 07 May 2009 as manuscript BJ20090427 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090427

Biochemical

  Journal Immediate Publication. Published on 07 May 2009 as manuscript BJ20090427 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090427 A c c e p t e d M a n u s c r i p t

  Biochemical Journal Immediate Publication. Published on 07 May 2009 as manuscript BJ20090427 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090427A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

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Figure 1 .

 1 Figure 1. A. Schematic representation of the domain structure of NEMO and the constructs used in this study. B. Coomassie stained SDS gel showing the purity of the protein preparations used. The faint bands on the NEMO 355 and CoZi lanes at twice the molecular mass represent incompletely denatured dimers.

Figure 2 .

 2 Figure 2. NEMO 355 is dimeric and exists in a concentration-dependent equilibrium with a higher order species. A. SEC-MALS measurements of NEMO 355 (dimeric formula M r 83.8 kDa) loaded at concentrations ranging from 0.5 to 8.0 mg/ml. Refractive index-derived weight concentrations (mg/ml) of the peaks are shown to the right. For clarity, derived weight-averaged molecular mass (kDa) of the peaks are shown separately to the left along with a legend detailing the peak weight-averaged M w for each experiment. B. NEMO 355 exists in a dimer/tetramer equilibrium. Sedimentation velocity experiments of NEMO 355 at three concentrations (8.8, 17.6, and 42 μM) shown as best fit C(s) functions.

Figure 3 .

 3 Figure 3. IKK C-terminal peptide tightly binds NEMO 355 and abrogates tetramerisation. SEC-MALS measurements of high concentration NEMO 355 (dimeric formula M 83.8 kDa) bound to excess 41-mer C-terminal IKK peptide (4.7 kDa). Refractive index-derived weight concentrations (mg/ml, left axis) of the peaks are shown for the entire run. In corresponding line types, derived apparent molecular mass (kDa, right axis) are shown for the center of each peak. Shown for comparison is a measurement of a comparable concentration of NEMO 355 alone demonstrating an increase in weight-averaged M w caused by tetramerisation. Apparent molecular mass for the binding experiments (~92kDa) agree with the formula molecular mass of dimeric NEMO 355 bound to two IKK peptides (93.3 kDa).

Figure 4 .Figure 5 .

 45 Figure 4. ITC titrations of A. 49.5 µM NEMO 355 titrated with 453 µM di-ubiquitin, and B. 112 µM CoZi titrated with 693 µM di-ubiquitin, both at 10 o C. C. Enthalpies of complex formation are plotted as a function of temperature. The change in heat capacity is the slope of a linear regression fit Figure 5. CoZi and di-ubiquitin interact to form CoZi dimer /Ub2 and at higher ubiquitin ratios CoZi dimer /(Ub2) 2 . Best fit C(s) functions for sedimentation velocity analysis of CoZi/Ub2 interaction(s).

Figure 1 AB

 1 Figure 1

Figure2 A B

 A Figure2

  Figure 3

  Figure 4

Biochemical

  Figure 5

  Biochemical Journal Immediate Publication. Published on 07 May 2009 as manuscript BJ20090427 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090427

  Biochemical Journal Immediate Publication. Published on 07 May 2009 as manuscript BJ20090427 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090427

		Dimer Tetramer
	M r (kDa) V bar	83.8	167.6 0.728
	M w (kDa)	77.6	164.9
	S 20,w	3.00	4.23
	D t,20,w	3.5	2.3
	f/f 0 Relative Proportions	2.3 0.87	2.6 0.13
	K d (μM) RMSD		25.6 0.057

  Biochemical Journal Immediate Publication. Published on 07 May 2009 as manuscript BJ20090427 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090427

		T	N	K d (M)	H (kcal/mol)
	mouse CoZi	10 o C	0.53 ± 0.06	2.6 ± 0.4	4.8 ± 0.6
		25 o C	0.62 ± 0.07	1.4 ± 1.0	-4.0 ± 0.6
	mouse Nemo 355	10 o C	0.44 ± 0.05	3.45 ± 0.4	4.1 ± 0.7
		25 o C	0.52 ± 0.04	0.95 ± 0.2	-1.3 ± 0.5

© 2009 The Authors Journal compilation © 2009 Portland Press Limited

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

Acknowledgements

We thank Steve Smerdon (NIMR), Felix Randow (MRC-LMB) and Ben Stieglitz (NIMR) for helpful discussions and critical reading of the manuscript. This work was supported by the Medical Research Council, UK.

Table 3 Hydrodynamic parameters of the CoZi/Ub2 interaction(s).

The first three columns denote the composition, reduced ratio, and molar ratios, respectively at which the sedimentation velocity experiments were carried out. All parameters are derived from discrete species modeling. Formula molecular mass: CoZi dimer 25.9 kDa; Ub2 17.5 kDa; CoZi dimer /Ub2 43.5 kDa; CoZi dimer /(Ub2) 2 61.0 kDa. Partial specific volumes (V bar ): CoZi 0.737; Ub2 0.746; and averaged V bar was used for CoZi/Ub2 complexes. Weight-averaged molecular mass (M w ) reported by experiment; sedimentation coefficient (S 20,w ); translational diffusion coefficient (D t,20,w ); frictional ratio (f/f 0 ); average root mean square deviation of the fit (RMSD). Data are organized into sections pertaining either to free (unbound) or complex species and the nature of the free species is noted as CoZi dimer , Ub2, or Mixture (a weight-averaged mixture of unbound species). A c c e p t e d M a n u s c r i p t