Evidence for M1-Linked Polyubiquitin-Mediated Conformational Change in NEMO
nstein, Guozhou Xu1, Ven
kataraman Kabaleeswaran and Hao Wu
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, and Program in Cellular and MolecularMedicine, Boston Children's Hospital, Boston, MA 02115, United States
Correspondence to Hao Wu: [email protected]://dx.doi.org/10.1016/j.jmb.2017.10.026Edited by M Yaniv
Abstract
The NF-κB essential modulator (NEMO) is the scaffolding subunit of the inhibitor of κB kinase (IKK)holocomplex and is required for the activation of the catalytic IKK subunits, IKKα and IKKβ, during thecanonical inflammatory response. Although structures of shorter constructs of NEMO have been solved,efforts to elucidate the full-length structure of NEMO have proved difficult due to its apparent highconformational plasticity. To better characterize the gross dimensions of full-length NEMO, we employedin-line size exclusion chromatography–small-angle X-ray scattering. We show that NEMO adopts a morecompact conformation (Dmax = 320 Å) than predicted for a fully extended coiled-coil structure (N500 Å). Inaddition, wemap a region of NEMO (residues 112–150) in its coiled-coil 1 domain that impedes the binding oflinear (M1-linked) di-ubiquitin to its coiled-coil 2–leucine zipper ubiquitin binding domain. This ubiquitinbinding inhibition can be overcome by a longer chain of linear, but not K63-linked polyubiquitin. Collectively,these observations suggest that NEMO may be auto-inhibited in the resting state by intramolecularinteractions and that during signaling, NEMO may be allosterically activated by binding to long M1-linkedpolyubiquitin chains.
Cellular responses to various pathogenic andpro-inflammatory signaling molecules, includingviral and bacterial nucleic acids, lipopolysaccharide,interleukin-1β, and tumor necrosis factor alpha(TNFα), depend on the activation of a family ofdimeric transcription factors known as NF-κB [1].During basal conditions, NF-κBs are sequestered inthe cytosol bound to inhibitor molecules called IκBs,with IκBα being the most ubiquitous. Upon binding ofextracellular ligands to their cognate receptors,including members of the Toll-like receptor/interleu-kin-1 receptor and TNF receptor superfamilies, acentral effector kinase complex, called the inhibitorof κB kinase (IKK), is activated [1,2]. Once activated,the catalytic subunits of the IKK complex phosphor-ylate two N-terminal serines of IκBα leading to itsK48-linked polyubiquitination and subsequent pro-teolytic degradation by the 26S proteasome. Thisfrees NF-κB to translocate into the nucleus to inducetranscription of pro-inflammatory and/or anti-apopto-tic gene suites [1,2].
er Ltd. All rights reserved.
The IKK holocomplex is composed of the catalyticsubunits, IKKβ (or IKK2) and/or IKKα (or IKK1), andthe helical scaffolding protein NF-κB essentialmodulator (NEMO) that is the central regulatorysubunit [2,3]. Genetic ablation of the 48-kDa NEMOin mice results in embryonic lethality by day 13 ofdevelopment and a complete unresponsiveness to avariety of canonical NF-κB inducers in mouseembryonic fibroblast cells [4–6]. Although lackingcatalytic activity of its own, NEMO articulatesupstream signals to the activation of the IKK catalyticsubunits. It does this mainly through its ability to bindnon-degradative polyubiquitin chains (mainly M1-and K63-linked) synthesized during signaling [7–10].NEMO has two distinct ubiquitin binding domains.One is composed of the coiled-coil 2 and leucinezipper domains, together called the UBAN domain(also known as the NOA, NUB, or CoZi domain) andthe other is composed of the C-terminal zinc fingermotif (Fig. 1a) [11–15]. Other domains in NEMOinclude the helix–loop–helix (HLX) 1, coiled-coil 1
Fig. 1. Size characterization of IKKβ, NEMO, and the IKKβ/NEMO complex. (a) Domain architecture of human NEMOshowing regions of protein–protein interactions. CC2, coiled-coil 2; LZ, leucine zipper; ZF, zinc finger. (b) A linear model offull-length NEMO with ~500 Å in length constructed from known structures of NEMO and its complexes. (c) SEC-MALS ofHis-IKKβ, showing a predominant dimer. (d) SEC-MALS of NEMO-His, showing a predominant dimer. (e) SEC-MALS ofthe IKKβ/NEMO complex, showing a megadalton higher-order oligomer. (f) Coomassie Blue-stained, 10% SDS-PAGE gelshowing the Superose 6 10/300GL elution profile of IKKβ, NEMO, and the IKKβ/NEMO complex. All NEMO constructswere sub-cloned into either pET28a or pET26b vectors between NdeI and XhoI restriction sites for expression in E. coliBL21-CodonPlus® (DE3) RIPL cells (Agilent Technologies). IKKβ was sub-cloned into pFastBacHTb transfer vector forbacmid generation and expression in High Five™ insect cells using the Bac-to-Bac® baculoviral expression system(Thermo Fisher). All proteins were purified by nickel affinity chromatography followed by SEC. MALS measurements wereperformed with an inline three-angle light scattering detector (mini-DAWN TRISTAR) coupled to a refractive index detector(Optilab DSP) (Wyatt Technology). Data were analyzed using ASTRA VI software.
3794 Conformational Change in NEMO
(CC1), and HLX2 domains (Fig. 1a). While a part ofHLX1 and CC1 interacts with IKKα or IKKβ [16],HLX2 binds the FLICE inhibitory protein (vFLIP) fromKarposi sarcoma-associated herpesvirus and Taxfrom the human T-lymphocyte virus to result inNEMO activation during viral infection [17,18] (Fig.1a). Mutations in various domains of NEMO havebeen implicated in cases of anhidrotic ectodermaldysplasia with immune deficiency and incontinentiapigmenti [19–21].Structural studies show that many regions of
NEMO exhibit coiled-coil structures, including thoseresponsible for binding to IKKα or IKKβ [16], vFLIP[17], and polyubiquitin [11–13], at least in thepartner-bound forms (Fig. 1b). However, it is unclearif full-length NEMO is indeed an extended, long
dimeric coiled-coil with a predicted length of ~500 Å(Fig. 1b). Previous studies have also reported thatNEMO can exist as trimer or tetramer depending onthe signaling condition, protein concentration, andthe NEMO construct under study [22–24]. A recentanalysis further suggests that as much as half ofNEMO may be intrinsically disordered in its restingstate, suggesting that partner binding may induceconformational changes in NEMO [25].In this study, we aimed to characterize conforma-
tional or oligomeric changes introduced by thebinding of NEMO interacting proteins includingIKKβ, vFLIP, and polyubiquitin in the context offull-length NEMO. We show that both full-lengthIKKβ and full-length NEMO are primarily dimericindividually, but form a megadalton complex when
3795Conformational Change in NEMO
together. For some NEMO constructs, a minortetramer peak can also be detected in addition tothe dimer peak in size exclusion chromatography(SEC), rationalizing previous observations of trimer-ization as a mixture of dimers and tetramers, and oftetramerization as dimers of dimers. We report byinline SEC–small-angle X-ray scattering (SEC–SAXS) that NEMO does not adopt a fully extendedcoiled-coil conformation (predicted to be N500 Å),but instead exhibits a comma-shaped form with aDmax of ~320 Å. We also identify a region (residues112–150) of NEMO in the CC1 domain that hindersM1-linked di-ubiquitin binding to the UBAN domain.Longer-chain M1-linked polyubiquitin but not withK63-linked polyubiquitin can overcome this inhibi-tion, suggesting polyubiquitin-induced NEMO con-formational changes during signaling.
Characterization of NEMO and the IKKholocomplex
Initial biophysical characterization of the IKKholocomplex from TNFα stimulated HeLa cellsshowed that it purifies as a 700- to 900-kDa complexwith IκBα phosphorylating activity [4,26,27]. Subse-quent studies revealed that this complex composedcanonically of IKKβ, IKKα, and NEMO can bereconstituted in vitro [28,29]. Several crystal struc-tures of nearly full-length IKKβ and a recent IKKαcryo-EM structure showed that these catalyticsubunits exist primarily as dimers [30–32]. However,the oligomerization state of NEMO is controversial,and full-length NEMO has been observed to elutefrom SEC at a position that corresponds to a~600-kDa globular protein [28].To obtain a shape-independent assessment of the
oligomerization state of IKKβ and full-length NEMO,we coupled multi-angle light scattering (MALS) andrefractive index detection to SEC (Fig. 1c, d). BothIKKβ and NEMO exist primarily as dimers with ameasured molecular mass of 186 kDa (4% error)and 113 kDa (3% error), respectively, in comparisonwith the calculated monomeric molecular weight of87 and 48 kDa. In contrast, the NEMO/IKKβ com-plex exhibits a measured molecular mass of~2000 kDa (Fig. 1e), suggesting higher-order olig-omerization of the IKK holocomplex. The earlierelution volume of NEMO relative to IKKβ (Fig. 1c, d,f) despite its smaller molecular mass also suggestedthat NEMO is an especially elongated molecule.
Auto-inhibition in full-length NEMOshown by M1-linked di-ubiquitin binding
Short constructs of NEMO that encompass theUBAN domain have been shown to bind with lowmicromolar affinity to M1-linked polyubiquitin chains
[11]. Indeed, M1-linked di-ubiquitin co-eluted withUBAN (192–350) and UBAN–zinc finger (246–419)by SEC (Fig. 2a, b). Surprisingly, this interaction wascompletely abrogated in the context of full-lengthNEMO (Fig. 2c), and this inhibition could not berescued with the addition of other NEMO interactingproteins like IKKβ or MBP–vFLIP (Fig. 2d, e). Wewondered if a specific region of NEMO is responsiblefor the interference of M1-di-ubiquitin interaction.Because the C-terminal portion of NEMO binds wellto M1-di-ubiquitin, we truncated NEMO from theN-terminus. While NEMO (112–350) did not interactwith M1-di-ubiquitin (Fig. 2f), NEMO (150–350) didinteract with M1-di-ubiquitin (Fig. 2g), suggestingthat the region between 112 and 150 in the CC1domain of NEMO may be important for thisinhibition.As controls, we conducted SEC-MALS to measure
the molecular masses of the NEMO (112–350) (Fig.3a) and NEMO (150–350) constructs (Fig. 3b) sinceit is possible that higher-order oligomerizationbeyond the putative dimer could compete forubiquitin binding sites. For NEMO (112–350), themeasured molecular mass is 72.8 kDa (6% error),consistent with primarily a dimer of the calculatedmonomer molecular mass of 31.1 kDa (Fig. 3a). ForNEMO (150–350), the major peak has a measuredmolecular mass of 50.1 kDa (3% error), which is alsoin agreement with a dimer of the calculatedmonomer molecular mass of 26.5 kDa (Fig. 3b).However, for this construct, there is also a minorpeak with the measured molecular mass of 95.6 kDa(3% error) (Fig. 3b), which indicated a tetramer andsuggested potential higher-order oligomerization.Interestingly, it appears that only the dimeric regionof the construct near 14 ml in elution volumeinteracted with M1-di-ubiquitin (Fig. 2g).
M1-linked tetra-ubiquitin overcomes theinhibition in full-length NEMO
Lack of binding to M1-di-ubiquitin by full-lengthNEMO indicates that in the resting state, NEMOexists in an auto-inhibited conformation throughinteractions within the dimer, either intramolecularor intermolecular. Because NEMO activation re-quires longer M1-linked polyubiquitin [25,34], wetested M1-tetra-ubiquitin and K63-tetra-ubiquitin forbinding to full-length NEMO. While M1-tetra-ubiqui-tin robustly interacted with NEMO, K63-tetra-ubiqui-tin did not (Fig. 2h). These data indicate that longM1-linked polyubiquitin is able to overcome NEMOauto-inhibition. Thus, our experiments revealedevidence for allosteric control of NEMO conforma-tion including auto-inhibition in the resting state andM1-linked polyubiquitin-mediated conformationalchange that results in ubiquitin binding and NEMOactivation.
(a)
)d()c(
(f)
(h)
(b)
(e)
(g)
NEMO-His (FL)
His-M1-Di-Ub
(250 M)
His-IKKβ (FL)
His-M1-Di-Ub
His-IKKβ (FL) +
NEMO-His (FL) +
His-M1-Di-Ub
7.0
ml
8.0
ml
10
.0 m
l
11
.0 m
l
13
.0 m
l
16
.0 m
l
9.0
ml
12
.0 m
l
14
.0 m
l
15
.0 m
l
17
.0 m
l
18
.0 m
l
19.0
ml
20
.0 m
l
Superose 6 10/300GL
S / GSuperdex 200 10/300GL
7.0
ml
8.0
ml
10
.0 m
l
11
.0 m
l
13
.0 m
l
16
.0 m
l
9.0
ml
12
.0 m
l
14
.0 m
l
15
.0 m
l
17
.0 m
l
18
.0 m
l
19
.0 m
l
20
.0 m
lNEMO-His (FL)
His-M1-Di-Ub
(100 M)
NEMO-His (FL)
+ His-M1-Di-Ub
His-MBP-vFLIP
+ NEMO-His (FL)
+His-M1-Di-Ub
Superdex 200 10/300GL
His-M1-Di-Ub
(30 M)
His-MBP-vFLIP
NEMO-His (FL)
NEMO-His (FL)
His-MBP-vFLIP
His-M1-Di-Ub
Complex
7.0
ml
8.0
ml
10
.0 m
l
11.0
ml
13
.0 m
l
16
.0 m
l
9.0
ml
12
.0 m
l
14
.0 m
l
15
.0 m
l
17
.0 m
l
18
.0 m
l
19
.0 m
l
20
.0 m
l
His-NEMO (192-350)
His-M1-Di-Ub
(250 M)
His-NEMO (192-350)
His-M1-Di-Ub
His-NEMO (192-350)
+ His-M1-Di-Ub
Superdex 200 10/300GL
Complex
His-NEMO (246-419)
7.4
ml
7.9
ml
8.9
ml
9.4
ml
10
.4 m
l
11.9
ml
8.4
ml
9.9
ml
10
.9 m
l
11.4
ml
12
.4 m
l
12
.9 m
l
His-M1-Di-Ub
(250 M)
His-NEMO (246-419)
+ His-M1-Di-Ub
His-NEMO (246-419)
His-M1-Di-Ub
13
.4 m
l
13
.9 m
l
Superdex 75 10/300GL
)
His-NEMO (150-350)
His-M1-Di-Ub
(100 M)
complex
His-NEMO (150-350)
+ His-M1-Di-Ub
His-NEMO (150-350)
His-M1-Di-Ub
7.0
ml
8.0
ml
10.0
ml
11
.0 m
l
13.0
ml
16.0
ml
9.0
ml
12.0
ml
14.0
ml
15.0
ml
17.0
ml
18.0
ml
Superdex 200 10/300GL
)
Superdex 200 10/300GL
His-NEMO (112-350)
His-M1-Di-Ub
(100 M)
His-NEMO (112-350)
+ His-M1-Di-Ub
His-NEMO (112-350)
His-M1-Di-Ub
7.0
ml
8.0
ml
10.0
ml
11
.0 m
l
13.0
ml
16.0
ml
9.0
ml
12.0
ml
14.0
ml
15.0
ml
17.0
ml
18.0
ml
7.5
ml
8.5
ml
10.5
ml
11
.5 m
l
13.5
ml
16.5
ml
19.5
ml
9.5
ml
12.5
ml
14.5
ml
15.5
ml
17.5
ml
18.5
ml
Superdex 200 10/300GL
His-K63-Tetra-Ub
(60 M)
NEMO-His (FL)
His-M1-Tetra-Ub
(60 M)
NEMO-His (FL) NEMO-His (FL) +
His-M1-Tetra-Ub
NEMO-His (FL) +
His-K63-Tetra-Ub
Complex
Fig. 2. The CC1 domain (aa 112–150) of NEMO inhibits linear di-ubiquitin binding. (a–h) SDS-PAGE of SEC fractions ofvarious NEMO constructs mixed with M1-di-ubiquitin and/or IKKβ, MBP–vFLIP, or tetra-ubiquitin chains. Complexformation is indicated as well as the SEC column used in each panel. Purified NEMO constructs were incubated withpurified IKKβ, MBP–vFLIP, and/or ubiquitin proteins in a roughly 1:1 or 1:2 molar ratio for 30 min at room temperature priorto loading onto a Superdex 75, Superdex 200, or Superose 6 column pre-equilibrated with buffer containing 20 mM Tris atpH 8.0, 150 mM NaCl, and 1 mM DTT run at 4°C. The synthesis of K63-linked tetra-ubiquitin was accomplished asdescribed in Ref. [33].
3796 Conformational Change in NEMO
NEMO adopts a more compact confor-mation than predicted for a fully ex-tended coil–coil structure
To further understand the conformational control inNEMO, we measured the gross shape and dimen-sions of full-length NEMO using an inline SEC–SAXS experimental setup at the NSLS X9 beamline.The inline SEC was aimed to eliminate largeaggregates in the SAXS measurements. SAXSwas measured in real time as protein eluted off aSuperose 6 10/300GL column, as shown by thechromatograph and SDS-PAGE gel of peak fractionsof full-length NEMO in complex with MBP–vFLIPfusion protein (Fig. 4a). MBP–vFLIP was added to
provide a large globular domain for the purpose ofstructure orientation since NEMO itself is notpredicted to have any globular domain. Importantly,NEMO in complex with MBP–vFLIP did not appre-ciably change the SEC elution profile in comparisonto NEMO alone, suggesting that vFLIP binding doesnot result in any large-scale conformational changes(Fig. 2e). SAXS data were collected every 35 s asprotein eluted off the column and the raw scatteringcurves are shown (Fig. 4b).The particle size is relatively homogenous under
the elution peak with Guinier approximations of theradii of gyration (Rg) ranging from 88.5 to 98.6 Å(Fig. 4b). The pairwise distribution function p(r),determined from the central peak scattering curvedata, showed an Rg of 101.2 Å that is similar to
(a) (b)
0
0.2
0.4
0.6
0.8
1
1.2
0
20
40
60
80
100
120
140
10 12 14 16 18
Re
fra
ctive
In
de
x
Mo
lecu
lar M
ass (
kD
a)
Elution Volume (ml)
50.1 kDa
(3%)
(NEMO)2
95.6 kDa (3%)
(NEMO)4
His-NEMO (150-350): calculated monomer 26.5 kDaIn
pu
t
7.5
ml
8.5
ml
9.5
ml
10.5
ml
11.5
ml
12.5
ml
13.5
ml
14
.5 m
15.5
ml
Superdex 200 10/300GL
16.5
ml
His-NEMO
(112-350)
Superdex 200 10/300GL
7.5
ml
8.5
ml
9.5
ml
10.5
ml
11.5
ml
12.5
ml
15.5
ml
16.5
ml
Inp
ut
13.5
ml
14.5
ml
17.5
ml
18.5
ml
19.5
ml
His-NEMO
(150-350)
0
0.2
0.4
0.6
0.8
1
1.2
0
20
40
60
80
100
120
9 11 13 15 17 19
Re
fra
ctive
In
de
x
72.8 kDa (6%)
(NEMO)2
His-NEMO (112-350): calculated monomer 31.1 kDa
Elution Volume (ml)
Mo
lecu
lar M
ass (
kD
a)
Fig. 3. Size characterization of NEMO constructs. (a) SEC-MALS of NEMO (aa 112–350) along with the associatedeluted fractions run on a 10% SDS-PAGE gel, showing that it behaves as a dimer with a measured molecular mass of72,820 Da. (b) SEC-MALS of NEMO (aa 150–350), showing that it exists primarily as a dimer (molecular mass of50,090 Da) in equilibrium with a small amount of a tetramer species (molecular mass of 95,550 Da). SEC-MALS wasconducted as described in Fig. 1.
3797Conformational Change in NEMO
those estimated from Guinier approximations (Fig.4b, c) and a maximum intramolecular distance(Dmax) of 320 Å (Fig. 4c). This maximal dimensionis considerably shorter than the predicted Dmax of500 Å for fully extended NEMO (Fig. 1b). Ab initiomodeling of the electron density envelope using theprogram DAMMIF generated 10 independentmodels with an average normalized spatial discrep-ancy value of 1.112 ± 0.098 (Fig. 4d). These modelswere averaged and filtered in DAMAVER to give thefinal refined model (Fig. 4d), which agreed well withthe experimental scattering curve (Fig. 4e). Thecomma-shaped envelope has one end that isglobular, presumably due to the presence of MBP–vFLIP, a middle section that is kinked, and the otherend that is tapered. Although it is difficult to dock thevarious NEMO subdomain crystal structures into theenvelope due to the low resolution, it is obvious thatNEMO does not adopt a fully extended conformationin solution.
Discussion
Our SAXS data and M1-linked di-ubiquitin bindingdata together provided a number of importantinsights into conformational regulation of NEMO.First, full-length NEMO assumes a more compactconformation with auto-inhibition in the resting-statedimer. While either the N-terminal or the C-terminalregion may fold back to create the comma shape of
the NEMO/MBP–vFLIP complex (Fig. 4d), given theapparent allosteric control in the CC1 domain (Fig.2f–g), we propose that an N-terminal region (res-idues 112–150) including the CC1 domain folds backonto the vicinity of the HLX2 NEMO region where theMBP–vFLIP fusion protein binds (Fig. 4d). However,our data do not exclude the possibility that theC-terminal end of NEMO may also fold back to exertauto-inhibition. Our detailed biochemical character-izations are consistent with a previous study in whichNEMO was shown to be a dimeric protein that is inweak equilibrium with a tetrameric assembly [39].They further rationalize that previously concludedtrimerization of NEMO may represent mixture ofdimers and tetramers.Second, there are a number of established NEMO
mutations in the proposed allosteric regulatoryregion of NEMO that are associated with thehuman disease incontinentia pigmenti [20,21]. Twosuch point mutations are D113N and R123W. Sincethis region of NEMO has not been shown to interactwith any other binding partners, a likely explanationof the disease phenotype may be related to the roleof the region in NEMO allosteric regulation. Intrigu-ingly, normal lipopolysaccharide-induced NF-κBactivation was observed in NEMO−/− cells recon-stituted with these mutants [20], suggesting that thisallosteric regulation may be influenced by overex-pression in the reconstitution. Furthermore, the basalNF-κB activity may need to be assessed to revealthe functional phenotypes of these mutants in
-200.00
-150.00
-100.00
-50.00
0.00
50.00
100.00
0 5 10 15 20
mA
U
Volume (ml)
NEMO-His (FL) + His-MBP-vFLIP
His-MBP-vFLIP
(1-178)
NEMO-His (FL)
Inp
ut
9.5
ml
9.7
5 m
l
10.0
ml
10.2
5 m
l
10.5
ml
10.7
5 m
l
11.0
ml
11.2
5 m
l
11.5
ml
11.7
5 m
l
12.0
ml
12.2
5 m
l
12.5
ml
Superose 6 10/300GL
ab initio models
NSD= 1.112 ± 0.098Averaged and
refined model
320 Å
(a) (b)
(c) (d)
Log (
I)
9.86
10.04
10.22
10.39
10.56
10.74
10.91
11.09
11.27
11.44
11.62
Rg
(Å)
Volume
(ml)
98.6
93.6
92.5
92.1
91.3
89.7
88.8
88.3
87.4
89.5
88.5
s (Å-1)
0.05 0.1 0.15 0.2 0.25
2
1
0
-1
-2
P(r)
r (Å)
Dmax
: 320 Å
Rg: 101.2 Å
Volume: 10.56 ml0.15
0.1
0.05
0
0 100 200 300
(e)
s (Å-1)
2
1.5
0
0.5
-0.5
1
Log (
I)0.05 0.1 0.15 0.2 0.25 0.3
Experimental
Theoretical
Fig. 4. SAXS analysis of the NEMO/MBP–vFLIP complex. (a) Inline Superose 6 10/300GL SEC of the NEMO/MBP–vFLIP complex before SAXS analysis (top). The MBP–vFLIP fusion construct was cloned into the pET28a vector for E. coliexpression, and the expressed protein was purified by amylose resin followed by SEC. Peak fractions of the NEMO/MBP–vFLIP complex were run on a 10% SDS-PAGE gel (bottom). (b) Raw scattering curves of peak fractions collected every35 s (flow rate, 0.3 ml/min). The incident light wavelength was 0.92 Å and the sample to detector distance was 1.5 m.Radial averaging and buffer subtraction were accomplished using pyXS (Brookhaven, NSLS). The radii of gyration (Rg) inangstroms were estimated from Guinier plots using PRIMUS [35] near I(0) for each scattering curve. (c) The pairwisedistribution plot, P(r), for the complex eluted at 10.56 ml. The estimated maximum intra-molecular particle distance, Dmax,is 320 Å. Both calculation of P(r) and determination of Dmax were performed using GNOM [36]. (d) Ten ab initio beadmodels of NEMO/MBP–vFLIP generated by DAMMIF [37] along with the averaged and refined envelope generated byDAMAVER [38]. (e) Superimposed experimental scattering curve at 10.56 ml and the calculated scattering curve from theaveraged and refined model.
3798 Conformational Change in NEMO
auto-inhibition. Finally, the higher binding affinity ofM1-linked tetra-ubiquitin to NEMO can also over-come NEMO auto-inhibition. Therefore, M1-linkedpolyubiquitin may act as a switch to allostericallyalter NEMO conformational and activate NEMO fordownstream signal transduction in a physiologicalcontext.
Acknowledgments
We thank Dr. Lin Yang at the X9 beamline ofNational Synchrotron Light Source for assistancewith data collection. This research is funded by
National Institutes of Health grant 5R37 AI050872awarded to Dr. Hao Wu. Arthur V. Hauenstein issupported by a National Institutes of Health T32institutional training grant at Boston Children'sHospital.
Received 1 July 2017;Received in revised form 23 October 2017;
Accepted 24 October 2017Available online 27 October 2017
[1] Q. Zhang, M.J. Lenardo, D. Baltimore, 30 Years of NF-kappaB: a blossoming of relevance to human pathobiology,Cell 168 (2017) 37–57.
[2] F. Liu, Y. Xia, A.S. Parker, I.M. Verma, IKK biology, Immunol.Rev. 246 (2012) 239–253.
[3] M. Hinz, C. Scheidereit, The IkappaB kinase complex in NF-kappaB regulation and beyond, EMBO Rep. 15 (2014)46–61.
[4] D.M. Rothwarf, E. Zandi, G. Natoli, M. Karin, IKK-gamma isan essential regulatory subunit of the IkappaB kinasecomplex, Nature 395 (1998) 297–300.
[5] D. Rudolph, W.C. Yeh, A. Wakeham, B. Rudolph, D.Nallainathan, J. Potter, et al., Severe liver degenerationand lack of NF-kappaB activation in NEMO/IKKgamma-deficient mice, Genes Dev. 14 (2000) 854–862.
[6] S. Yamaoka, G. Courtois, C. Bessia, S.T. Whiteside, R. Weil,F. Agou, et al., Complementation cloning of NEMO, acomponent of the IkappaB kinase complex essential forNF-kappaB activation, Cell 93 (1998) 1231–1240.
[7] Z.J. Chen, L. Parent, T. Maniatis, Site-specific phosphoryla-tion of IkappaBalpha by a novel ubiquitination-dependentprotein kinase activity, Cell 84 (1996) 853–862.
[8] L. Deng, C. Wang, E. Spencer, L. Yang, A. Braun, J. You,et al., Activation of the IkappaB kinase complex by TRAF6requires a dimeric ubiquitin-conjugating enzyme complexand a unique polyubiquitin chain, Cell 103 (2000) 351–361.
[9] T.T. Huang, S.M. Wuerzberger-Davis, Wu ZH, S. Miyamoto,Sequential modification of NEMO/IKKgamma by SUMO-1and ubiquitin mediates NF-kappaB activation by genotoxicstress, Cell 115 (2003) 565–576.
[10] C.K. Ea, L. Deng, Z.P. Xia, G. Pineda, Z.J. Chen, Activationof IKK by TNFalpha requires site-specific ubiquitination ofRIP1 and polyubiquitin binding by NEMO, Mol. Cell 22 (2006)245–257.
[11] Y.C. Lo, S.C. Lin, C.C. Rospigliosi, D.B. Conze, C. Wu, J.D.Ashwell, et al., Structural basis for recognition of diubiquitinsby NEMO, Mol. Cell 33 (2009) 602–615.
[12] S. Rahighi, F. Ikeda, M. Kawasaki, M. Akutsu, N. Suzuki, R.Kato, et al., Specific recognition of linear ubiquitin chains byNEMO is important for NF-kappaB activation, Cell 136 (2009)1098–1109.
[13] A. Yoshikawa, Y. Sato, M. Yamashita, H. Mimura, A.Yamagata, S. Fukai, Crystal structure of the NEMOubiquitin-binding domain in complex with Lys 63-linked di-ubiquitin, FEBS Lett. 583 (2009) 3317–3322.
[14] O. Grubisha, M. Kaminska, S. Duquerroy, E. Fontan, F.Cordier, A. Haouz, et al., DARPin-assisted crystallography ofthe CC2–LZ domain of NEMO reveals a coupling betweendimerization and ubiquitin binding, J. Mol. Biol. 395 (2010)89–104.
[15] F. Cordier, O. Grubisha, F. Traincard, M. Veron, M.Delepierre, F. Agou, The zinc finger of NEMO is a functionalubiquitin-binding domain, J. Biol. Chem. 284 (2009)2902–2907.
[16] M. Rushe, L. Silvian, S. Bixler, L.L. Chen, A. Cheung, S.Bowes, et al., Structure of a NEMO/IKK-associating domainreveals architecture of the interaction site, Structure 16(2008) 798–808.
[17] C. Bagneris, A.V. Ageichik, N. Cronin, B. Wallace, M. Collins,C. Boshoff, et al., Crystal structure of a vFlip–IKKgammacomplex: insights into viral activation of the IKK signalosome,Mol. Cell 30 (2008) 620–631.
[18] G.J. Huang, Z.Q. Zhang, D.Y. Jin, Stimulation of IKK-gammaoligomerization by the human T-cell leukemia virus oncopro-tein Tax, FEBS Lett. 531 (2002) 494–498.
[19] E. Vinolo, H. Sebban, A. Chaffotte, A. Israel, G. Courtois, M.Veron, et al., A point mutation in NEMO associated withanhidrotic ectodermal dysplasia with immunodeficiencypathology results in destabilization of the oligomer andreduces lipopolysaccharide- and tumor necrosis factor-mediated NF-kappa B activation, J. Biol. Chem. 281 (2006)6334–6348.
[20] F. Fusco, T. Bardaro, G. Fimiani, V. Mercadante, M.G. Miano,G. Falco, et al., Molecular analysis of the genetic defect in alarge cohort of IP patients and identification of novel NEMOmutations interfering with NF-kappaB activation, Hum. Mol.Genet. 13 (2004) 1763–1773.
[21] F. Fusco, A. Pescatore, E. Bal, A. Ghoul, M. Paciolla, M.B.Lioi, et al., Alterations of the IKBKG locus and diseases: anupdate and a report of 13 novel mutations, Hum. Mutat. 29(2008) 595–604.
[22] F. Agou, F. Ye, S. Goffinont, G. Courtois, S. Yamaoka,A. Israel, et al., NEMO trimerizes through its coiled-coilC-terminal domain, J. Biol. Chem. 277 (2002)17464–17475.
[23] S. Tegethoff, J. Behlke, C. Scheidereit, Tetrameric oligomer-ization of IkappaB kinase gamma (IKKgamma) is obligatoryfor IKK complex activity and NF-kappaB activation, Mol. Cell.Biol. 23 (2003) 2029–2041.
[24] E. Fontan, F. Traincard, S.G. Levy, S. Yamaoka, M. Veron, F.Agou, NEMO oligomerization in the dynamic assembly of theIkappaB kinase core complex, FEBS J. 274 (2007)2540–2551.
[25] D.A. Catici, J.E. Horne, G.E. Cooper, C.R. Pudney,Polyubiquitin drives the molecular interactions of the NF-kappaB essential modulator (NEMO) by allosteric regulation,J. Biol. Chem. 290 (2015) 14130–14139.
[26] J.A. DiDonato, M. Hayakawa, D.M. Rothwarf, E. Zandi, M.Karin, A cytokine-responsive IkappaB kinase that activatesthe transcription factor NF-kappaB, Nature 388 (1997)548–554.
[27] E. Zandi, D.M. Rothwarf, M. Delhase, M. Hayakawa, M.Karin, The IkappaB kinase complex (IKK) contains twokinase subunits, IKKalpha and IKKbeta, necessary forIkappaB phosphorylation and NF-kappaB activation, Cell91 (1997) 243–252.
[28] X.H. Li, X. Fang, R.B. Gaynor, Role of IKKgamma/nemo inassembly of the Ikappa B kinase complex, J. Biol. Chem. 276(2001) 4494–4500.
[29] B.S. Miller, E. Zandi, Complete reconstitution of humanIkappaB kinase (IKK) complex in yeast. Assessment of itsstoichiometry and the role of IKKgamma on the complexactivity in the absence of stimulation, J. Biol. Chem. 276(2001) 36320–36326.
[30] G. Xu, Y.C. Lo, Q. Li, G. Napolitano, X. Wu, X. Jiang, et al.,Crystal structure of inhibitor of kappaB kinase beta, Nature472 (2011) 325–330.
[31] S. Polley, D.B. Huang, A.V. Hauenstein, A.J. Fusco, X.Zhong, D. Vu, et al., A structural basis for IkappaB kinase 2activation via oligomerization-dependent trans auto-phos-phorylation, PLoS Biol. 11 (2013), e1001581. .
[32] S. Polley, D.O. Passos, D.B. Huang, M.C. Mulero, A.Mazumder, T. Biswas, et al., Structural basis for theactivation of IKK1/alpha, Cell Rep. 17 (2016) 1907–1914.
[33] K.C. Dong, E. Helgason, C. Yu, L. Phu, D.P. Arnott, I.Bosanac, et al., Preparation of distinct ubiquitin chainreagents of high purity and yield, Structure 19 (2011)1053–1063.
[34] T. Kensche, F. Tokunaga, F. Ikeda, E. Goto, K. Iwai, I. Dikic,Analysis of nuclear factor-kappaB (NF-kappaB) essential
modulator (NEMO) binding to linear and lysine-linkedubiquitin chains and its role in the activation of NF-kappaB,J. Biol. Chem. 287 (2012) 23626–23634.
[35] P.V. Konarev, V.V. Volkov, A.V. Sokolova, M.H.J. Koch, D.I.Svergun, PRIMUS: a Windows PC-based system for small-angle scattering data analysis, J. Appl. Crystallogr. 36 (2003)1277–1282.
[36] D.I. Svergun, Determination of the regularization parameterin indirect-transform methods using perceptual criteria, J.Appl. Crystallogr. 25 (1992) 495–503.
[37] D. Franke, D.I. Svergun, DAMMIF a program for rapid ab-initio shape determination in small-angle scattering, J. Appl.Crystallogr. 42 (2009) 342–346.
[38] V.V. Volkov, D.I. Svergun, Uniqueness of ab initio shapedetermination in small-angle scattering, J. Appl. Crystallogr.36 (2003) 860–864.
[39] F.J. Ivins, M.G. Montgomery, S.J. Smith, A.C. Morris-Davies,I.A. Taylor, K. Rittinger, NEMO oligomerisation and itsubiquitin-binding properties, Biochem. J. 421 (2009)243–251.
