Binding of OTULIN to the PUB Domain of HOIP Controls NF-κB Signaling

Molecular Cell

Article

Binding of OTULIN to the PUB Domainof HOIP Controls NF-kB SignalingVeronique Schaeffer,1,4 Masato Akutsu,1,2,4 Michael H. Olma,1,4 Ligia C. Gomes,2 Masato Kawasaki,3 and Ivan Dikic1,2,*1Institute of Biochemistry II, Goethe University Faculty of Medicine, 60590 Frankfurt am Main, Germany2Buchmann Institute for Molecular Life Sciences, Goethe University, 60438 Frankfurt am Main, Germany3Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator ResearchOrganization (KEK), Tsukuba, Ibaraki, 305-0801, Japan4Co-first authors

*Correspondence: [email protected]

http://dx.doi.org/10.1016/j.molcel.2014.03.016

SUMMARY

Linear ubiquitin chains are implicated in the regula-tion of the NF-kB pathway, immunity, and inflamma-tion. They are synthesized by the LUBAC complexcontaining the catalytic subunit HOIL-1-interactingprotein (HOIP) and are disassembled by the linearubiquitin-specific deubiquitinase OTULIN. Little isknown about the regulation of these opposing activ-ities. Here we demonstrate that HOIP and OTULINinteract and act as a bimolecular editing pair for linearubiquitin signals in vivo. The HOIPPUB domain bindsto the PUB interacting motif (PIM) of OTULIN and thechaperone VCP/p97. Structural studies revealed thebasis of high-affinity interaction with the OTULINPIM. The conserved Tyr56 of OTULIN makes criticalcontacts with the HOIP PUB domain, and its phos-phorylation negatively regulates this interaction.Functionally, HOIP binding to OTULIN is required forthe recruitment of OTULIN to the TNF receptor com-plex and to counteract HOIP-dependent activationof the NF-kB pathway.

INTRODUCTION

Ubiquitination is a posttranslational modification involved in

regulating many cellular processes (Grabbe et al., 2011). This

is possible due to a variety of ubiquitin-based signals encoded

by different types of ubiquitin chains and ubiquitin binding pro-

teins that specifically interact with them (Husnjak and Dikic,

2012). Ubiquitin chains include the seven canonical lysine-linked

polymers and the more recently discovered Met1-linked (or

linear) chains (Komander and Rape, 2012; Walczak et al., 2012).

The first biological process regulated by Met1-linked ubiquiti-

nation described was the NF-kB signaling pathway (Gerlach

et al., 2011; Ikeda et al., 2011; Tokunaga et al., 2011). Upon acti-

vation of the TNF receptor (TNFR) by TNFa, several factors are

recruited to the receptor such as the kinase RIP1 and E3 ubiqui-

tin ligases, including TRAFs and IAPs, which are required for the

activation of the downstream pathways (Wertz and Dixit, 2010).

Consequently, the E3 ubiquitin ligase ‘‘linear Ub chain assembly

complex’’ (LUBAC) generates Met1-linked ubiquitin chains on

RIP1 and the IkB kinase (IKK) component NF-kBessential modu-

lator (NEMO). This results in the activation of IKK, followed by the

phosphorylation and degradation of IkB, and subsequent NF-kB

nuclear translocation and transactivation of NF-kB-target genes.

In addition, LUBAC has been shown to be important for the acti-

vation of the pattern recognition receptor NOD2 bymodifying the

receptor component RIP2with linear ubiquitin chains (Damgaard

et al., 2012).

TheE3ubiquitin ligase LUBAC is theonly known ligase capable

of generatingMet1-linked ubiquitin chains. It consists of the three

subunits: HOIP, HOIL-1, and Sharpin (Gerlach et al., 2011; Ikeda

et al., 2011; Tokunaga et al., 2011). HOIP contains two RING

domains separated with an in between RING (IBR)-domain

constituting the active core of the E3 ligase (Stieglitz et al.,

2013; Walczak et al., 2012). Additionally, it contains an unchar-

acterized ‘‘PNGase/UBA or UBX-containing proteins’’ (PUB)

domain at the N terminus, a stretch of zinc finger domains, and

a UBA domain. Recently, ‘‘OTU DUB with linear linkage speci-

ficity’’ (OTULIN, also named GUMBY and FAM105B) was identi-

fied as the deubiquitinase that specifically degradesMet1-linked

ubiquitin chains (Keusekotten et al., 2013; Rivkin et al., 2013). It

contains an OTU-like domain at its C terminus sufficient for its

catalytic activity and anN-terminalmotif proposed to be involved

in binding HOIP (Rivkin et al., 2013). It was shown that OTULIN

inhibits both NF-kB and NOD2 signaling by limiting LUBAC-

mediated Met1 ubiquitination of NEMO and RIPK2. OTULIN

appears also to be essential for murine embryonic development

by counteracting the functions of HOIP in the Wnt signaling

pathway (Rivkin et al., 2013).

The PUBdomainwas identified as a protein-protein interaction

domain consisting of a short, three-stranded b sheet packedonto

an a-helical core domain (Allen et al., 2006; Suzuki et al., 2001).

Three PUB-domain-containing proteins have been described

in humans so far. Beside HOIP, it is also present in the p97 cofac-

tors UBXD1 and ‘‘peptide:N-glycosidase’’ (PNGase). The AAA+

ATPase p97 (Halawani and Latterich, 2006) uses coordinated

ATPhydrolysis to segregate substrate proteins fromprotein com-

plexes or membranes. This functionality is used in a variety of

different cellular processes such as ER-associated degradation

(Stolz et al., 2011). Important for this versatility is the largenumber

of different cofactors linking p97 to its substrates. Most of these

Molecular Cell 54, 349–361, May 8, 2014 ª2014 Elsevier Inc. 349

mailto:[email protected]


http://crossmark.crossref.org/dialog/?doi=10.1016/j.molcel.2014.03.016&domain=pdf

Molecular Cell

PUB Domain-Mediated Binding of HOIP and OTULIN

cofactors attach to the N terminus of p97. However, some

cofactors likeUBXD1or PNGasebinda shortmotif at theextreme

C terminus of p97 using their PUB domains (Madsen et al., 2009).

Here we identified OTULIN and p97 as direct HOIP binding

partners. Crystallographic analysis of the HOIP PUB domain re-

vealed an overall similarity to the PNGase PUB domain, but it

also highlighted unique features of the configuration of the bind-

ing pocket. Additional structural studies identified the tyrosine-

based PUB-interaction motif (PIM) in both OTULIN and p97

to be crucial for binding to HOIP. Isothermal titration calorimetry

(ITC) measurements demonstrated that phosphorylation of

OTULIN on Tyr56 inhibits its interaction with the HOIP PUB

domain. Furthermore, the direct binding of OTULIN and HOIP

revealed the importance of their interaction for the proper action

of the NF-kB signaling in vivo.

RESULTS

OTULIN Binds Directly to HOIPWe investigated the interactome of LUBAC in stable HeLa cell

lines expressing inducible HA/23Strep-tagged LUBAC subunits.

The cellular localization and expression of individual LUBAC

components were verified. In particular, we controlled that exog-

enous expression integrated with endogenous complexes (Fig-

ures S1A–S1E available online). Due to the excessive expression

of HOIL-1 and Sharpin, we excluded them from further mass

spectrometry analysis (Figures S1D and S1E). HOIP-associated

complexes were affinity purified from SILAC-labeled cells and

analyzed using Orbitrap-based mass spectrometry (MS). HOIL-

1 andSharpinwere consistently foundamong the strongest inter-

actors. In addition, we identified the linear ubiquitin-specific DUB

OTULINasan interactionpartner inHOIPMSdata (Figure 1A).We

also confirmed that OTULIN interacts with the LUBAC complex,

as it coprecipitatedHOIPaswell asHOIL-1 (Figure 1B). This inter-

action is independent of OTULIN’s DUB activity, as the catalytic

inactive mutant, C129S, showed similar association with LUBAC

than wild-type OTULIN (Figure 1B). We also showed that endog-

enous OTULIN and HOIP could be coimmunoprecipitated from

HeLa cells (Figure 1C).

To gain additional insight into the mechanism of interaction

between LUBAC and OTULIN, we conducted in vitro interaction

studies using purified recombinant proteins. In glutathione

S-transferase (GST) pull-down assays OTULIN bound to HOIP,

but not to HOIL-1 or Sharpin (Figure 2A). Within HOIP several

functionally distinct domains have already been assigned (Fig-

ure S2A). To test which of these domains binds to OTULIN, frag-

ments of HOIP spanning across the whole protein were tested

for binding to OTULIN in pull-down assays (Figure S2B). OTULIN

bound to the first 179 amino acids (Figures 2B and S2B), which

comprise the proposed PUB domain of HOIP.

Structure of the HOIP PUB DomainWe next performed structural studies on the HOIP PUB domain.

We crystallized the HOIP PUB domain and solved the structure

by single anomalous dispersion phasing (1.7 A resolution). Elec-

tron density iswell defined for all residues fromaa2 to aa 178. The

crystallographic data is summarized in Table 1. The crystal struc-

ture of the HOIP PUB domain consists of nine a helices and three

350 Molecular Cell 54, 349–361, May 8, 2014 ª2014 Elsevier Inc.

antiparallel b strands (Figures 2C, S3A, and S3C). The PNGase

PUB domain (PDB ID 2hpl) shares the closest homology with

theHOIPPUBdomain based on theDALI server (HolmandRose-

nstrom, 2010) with a Z score of 9.8 (root-mean-square [rms] de-

viation in Ca positions of 1.47 A, residues from 12 to 111). Both

PUBdomains share the samecore fold consistingof fiveahelices

and three b strands, but the HOIP PUB domain is slightly bigger

as it contains three additional N-terminal a helices (Figures 2C

and S3A– S3C). To visualize functionally important regions of

the HOIP PUB domain we used the CONSURF algorithm and

thereby the degree of evolutionary conservation of HOIP PUB

domains to color the surface of the domain (Figure 2D) (Ashke-

nazy et al., 2010). The surface representation shows a highly

conserved region containing a pocket formed by the b strand

b1 and the a helices H5, H6, and H7 (Figure 2E). This pocket is

similar to the one in the PNGase PUB domain used for binding

the C-terminal amino acids DLYG of p97 (Zhao et al., 2007) (Fig-

ure 2F). Since this hydrophobic pocket of PNGase mainly inter-

actswith theTyr805of p97,wename itJ–Ypocket. Interestingly,

the corresponding J–Y pocket in HOIP shows structural differ-

ences with the one of PNGase. In the PNGase PUB domain,

one side of the binding pocket is formed by a 310 helix with

Tyr51 as a part of the pocket wall (Figure 2F). Contrary to this,

the HOIP PUB domain shows a unique loop structure due to

the corresponding Trp95 breaking the potential 310 helix forma-

tion and a functional substitution of PNGase Tyr51 by HOIP

Pro92 (Figures 2E and S2C). This allowed the exposure of HOIP

Tyr94 and the formation of a deep groove in proximity to the

pocket unique for the HOIP PUB domain.

Another obvious difference is the orientation of the side chains

of the equivalent amino acids HOIP Lys99 and PNGase Arg55

(Figures 2E and 2F). The guanidine group of PNGase Arg55 inter-

acts with C-terminal carboxylate of p97 and thereby terminates

the path for additional C-terminal amino acids (Figures 2F and

2G). Importantly, the orientation of PNGase Arg55 is independent

of the binding to p97 based on published structures (PDB IDs

2CM0, 2HPJ, and 2HPL). On the contrary, the side chain of the

corresponding residue in theHOIPPUBdomain, Lys99, is flipped

away due to the carboxamido group of the highly conserved

Asn101 and thereby points out of the J–Y pocket, opening a

path for a possible extension of the peptide (Figures 2E, 2H,

S2E, and S2F). This different direction of the HOIP Lys99 and

PNGase Arg55 side chains suggest that HOIP PUB domain may

recognize not onlyC-terminal peptides but also internal peptides.

OTULIN and p97 Interact with the HOIP PUB DomainTo test that OTULIN indeed directly binds to the J–Y pocket

of HOIP, we introduced various point mutations and detected

a decrease in OTULIN binding to the HOIP PUB domain with

both single mutants Y82A and N85E (Figure 2I). The double

mutant Y82A/N85E completely abolished binding of OTULIN

without affecting the fold of the domain, as judged by CD spectra

of wild-type and mutant OTULIN (Figures 2I and S2D). The spec-

ificity of this interaction and its inhibition with the double mutant

Y82A/N85E was confirmed by coimmunoprecipitation experi-

ments using HeLa cells (Figure S2G). Since p97 was detected

in our HOIP mass spectrometric analysis (Figure 1A), and from

a structural point of view the extreme C terminus of p97 could

Figure 1. Identification of LUBAC Interaction Partners

(A) SILAC-basedmass spectrometric analysis of LUBAC interactome. Immunoprecipitation of C-terminally HA/23Strep-tagged (-SSH) HOIP stably expressed in

HeLa cells is shown. Bait-expressing cells were heavy labeled; cells expressing no bait were light labeled. Significantly enriched proteins are indicated.

(B) Immunoprecipitation of tagged Sharpin, OTULIN wild-type, and catalytic inactive mutant.

(C) Immunoprecipitation of endogenous OTULIN with endogenous HOIP. OTULIN was immunoprecipitated, and samples were immunoblotted and stained for

HOIP and OTULIN. See also Figure S1.

Molecular Cell


also directly bind to theHOIPPUBdomain,we tested this directly

using a GST pull-down experiment. p97 bound to the HOIP PUB

domain while the Y82A/N85E double mutant abolished this inter-

action (Figure 2J). Moreover, affinity measurements using ITC

indicated that HOIP binding to OTULIN was stronger than for

p97 (Table 2). The dissociation constant of p97 bound to the

HOIP PUB domain was 47.6 mM affinity, while OTULIN bound

to the HOIP PUB domain with 290 nM affinity, which is more

than 100-fold stronger binding as compared to p97 (Table 2).

Consistently, OTULIN was able to displace p97 from the HOIP

PUB in competition assays while p97 could not outcompete

OTULIN (Figure S2H). In conclusion, both OTULIN and p97

directly bind to HOIP via a conserved J–Y pocket in the PUB

domain, but the affinity of OTULIN for HOIP PUB domain was

much higher than the affinity of p97 for the PUB domain.

Next, we sought to define the region of OTULIN required for

binding to the HOIP PUB domain by using fragments of OTULIN

in GST pull-down assays. The highly conserved stretch between

aa 45 and aa 73 is required to bind to the PUB domain of HOIP

(Figure 3A, lanes 2–7). Interestingly, this region contained the

amino acid sequence EDMYR (aa 53–57), which resembled the

motif DDLYG (aa 802–806) in p97 important for binding to

PNGase PUB domain (Zhao et al., 2007). This posed the possi-

bility for a very similar OTULIN binding mode to the HOIP PUB

domain in comparison to p97 and the PNGase PUB domain.

Structure of the HOIP PUB-OTULIN and HOIP PUB-p97ComplexesTo further investigate the interaction betweenHOIP, OTULIN and

p97, we solved the structure of theHOIP PUBdomain in complex


Figure 2. OTULIN and p97 Bind to a Conserved Pocket on HOIP PUB Domain

(A) Pull-down analysis of immobilized GST-tagged LUBAC components with OTULIN. Ponceau S staining shows GST-HOIP, -HOIL, and -Sharpin.

(B) Pull-down analysis of immobilizedGST-HOIP full-length, PUB-, and NZF-domain constructs with OTULIN. Ponceau-S-stainedmembrane showsGST-tagged

proteins.

(C) Superposition of the ribbonmodel of crystal structures of the HOIP PUB domain (green) and PNGase PUB domain (pink) in complex with p97 peptide (PDB ID

2HPL). p97 peptide (cyan) is shown in ball-and-stick representation.

(D) Surface representation of the HOIP PUB domain, colored by conserved residues of PUB domain.

(E) A close-up view of the conserved pocket of HOIP PUB domain, in the same view as (C). Hydrophobic surface residues are colored in green.

(F) Close-up view of the p97 peptide-binding pocket of the PNGase PUB domain.

(G and H) Surface representation of PUB domain J–Y pocket of PNGase PUB domain and p97 peptide (ball-and-stick representation, cyan) (G) and HOIP PUB

domain (H).

(I) Pull-down analysis of immobilized GST-HOIP PUB wild-type and mutants with OTULIN. Ponceau-S-stained membrane shows GST-tagged constructs.

(J) Pull-down analysis of immobilized GST-HOIP PUB wild-type and mutant with HeLa Lysate, Ponceau S staining as in (I). See also Figures S2and S3.

Molecular Cell


with OTULIN (aa 52–61) and p97 (aa 796–806) peptides by mo-

lecular replacement using the Apo-HOIP PUB domain structure

as a search model. In both structures, the electron density was


well defined except the loops connecting the two b strands b2

and b3 and the two helices H4 and H5. Although there is little

structural difference between the two complex structures in

Table 1. Crystallization Data Collection and Refinement Statistics

HOIP_PUB_Apo (SAD) HOIP_PUB_Apo HOIP_PUB/p97 HOIP_PUB/OTULIN

Data Collection Statistics

Beamline KEK BL17 DIAMOND I03 KEK BL17 KEK BL1A

Wavelength (A) 0.97910 0.97630 1.0000 1.0000

Space group P6122 P41212 P61 P61

Unit cell (A) a, b = 92.78; c = 91.53 a, b = 47.92; c = 152.71 a, b = 48.04; c = 266.62 a, b = 48.16; c = 267.71

Resolution (A) 60.38–3.00 (3.16–3.00) 50.90–1.70 (1.79–1.70) 53.32–2.30 (2.42–2.30) 44.62–2.70 (2.85–2.70)

Observed reflections 399672 (57733) 147391 (16764) 152751 (11289) 98466 (15073)

Unique reflections 5046 (705) 20365 (2713) 15446 (2224) 9674 (1403)

Redundancy 79.2 (81.9) 7.2 (6.2) 9.9 (5.1) 10.2 (10.7)

Completeness (%) 100.0 (100.0) 99.0 (93.6) 99.9 (99.3) 100.0 (100.0)

Rmerge 0.115 (0.578) 0.081 (0.390) 0.090 (0.527) 0.065 (0.728)

<I/sI> 43.4 (12.3) 13.5 (4.2) 14.9 (2.3) 23.8 (4.0)

Phasing Statistics

FOM 0.328

FOM after DM 0.767

Refinement Statistics

Reflections in test set 1020 755 464

Rcryst 18.1 19.9 21.3

Rfree 23.0 24.9 27.0

Number of Groups

Protein residues 177 350 345

Ions and ligand atoms 0 0 0

Water 170 92 55

Wilson B-factor 23.3 42.64 64.71

RMSD from Ideal Geometry

Bond length (A) 0.018 0.010 0.002

Bond angles (�) 1.802 1.192 0.695

Ramachandran Plot Statistics

In favored regions (%) 163 (96.45) 328 (97.04) 308 (93.05)

In allowed regions (%) 6 (3.56) 10 (2.96) 23(6.95)

Outliers (%) 0 (0.00) 0 (0.00) 0 (0.00)

Values in parentheses are for the highest resolution shell.

Molecular Cell


the asymmetric unit (rms deviation of up to 0.62A), the electron

density of one complex pair (HOIP PUB-OTULIN or HOIP PUB-

p97) is more ordered than the other. We used well-ordered com-

plex pair for the Discussion. All three crystal structures of the

HOIP PUB domains (Apo, OTULIN complex, and p97 complex)

superpose well with an rms deviation of up to 0.75A, suggesting

that the association of both peptides does not alter the overall

structure of the PUB domain.

The position of both peptides relative to the HOIP PUB domain

is essentially identical. The electron densities of four residues

of the associated peptides (OTULIN Asp54–Arg57 and p97

Asp803–Gly806) were well defined (Figures 3B, 3C, S4A, and

S4B). The hydrophobic residues OTULIN Met55 and p97

Leu804 fit into the J–Y pocket, and both interact by van der

Waals interactionswithHOIP Ile78/Tyr82 andby hydrogenbonds

with the hydroxyl group of HOIP Tyr82 (Figures 3F and 3G). A few

hydrogenbondsbetweenAsn101ofHOIPPUBdomain andmain

chain oxygen atomsofOTULINor p97peptides also contribute to

the complex formation. The leading residues for the interactions

with theJ–Y pocket are the tyrosines in the interacting peptides.

Tyr56 of OTULIN and Tyr805 of p97 deeply enter theJ–Y pocket

(Figures 3B–3E). Their hydroxyl groups form hydrogen bonds

with the carboxyamide of HOIP Asn85 and interact with the

main chain oxygen of HOIP Tyr82 and the hydroxyl group of

HOIP Tyr124 through a water molecule (Figures 3F and 3G). We

name this interaction motif in both OTULIN and p97 due to the

importance for the interaction with the HOIP PUB domain as

PIM. Importantly, in both complex structures the HOIP Lys99

side chains have the same orientation as in the apo structure,

leaving a path for additional amino acids. In addition, both C-ter-

minal amino acids visible in the complex structures, OTULIN

Arg57 andp97Gly806, sit at the entrance of this potential peptide

route. This shows why the HOIP PUB domain is capable of bind-

ing both C-terminal and internal peptides.


Table 2. Thermodynamic Parameters of the HOIP PUB Domain Interactions with OTULIN

Injectant Cell DH (kcal/mol) DS (cal/mol/K)

�T3DS

(kcal/mol) DG (kcal/mol) Ka (3106 M�1) Kd (mM) N

HOIP PUB 550 mM OTULIN 50 mM �13.1 ± 0.05 �15.1 4.43 �8.67 3.40 ± 0.19 0.29 1.02 ± 0.00

HOIP PUB 550 mM OTULIN Y56A 50 mM n.d.

HOIP PUB 550 mM OTULIN (45–73) 50 mM �11.7 ± 0.16 �10.9 3.20 �8.5 2.26 ± 0.31 0.44 0.99 ± 0.01

HOIP PUB 550 mM OTULIN (52–61) 50 mM �11.7 ± 0.07 �14.8 4.34 �7.36 0.35 ± 0.01 2.90 1.05 ± 0.01

HOIP PUB 550 mM OTULIN (52–61)

Phospho-Y56 50 mM

n.d.

HOIP PUB 1.33 mM P97 50 mM �10.0 ± 0.66 �14.4 4.22 �5.78 2.21 ± 0.15

(3104 M�1)

47.6 1.05 ± 0.06

P97 peptide 2 mM HOIP PUB 150 mM �7.7 ± 0.30 �5.6 1.64 �6.06 3.30 ± 0.31

(3104 M�1)

30.3 0.96 ± 0.03

DH, molar enthalpy of the interaction; DS, molar entropy of the interaction; �T 3 DS, entropy contribution term; DG, molar free (Gibbs) energy of

interaction; N, number of binding sites upon interaction; n.d., no binding detected.

Molecular Cell


Tyr56 Phosphorylation Regulates Interaction betweenHOIP and OTULINThe critical location of the tyrosines led us to test their impact on

the binding to the PUB domain. Upon mutation of OTULIN Tyr56

to phenylalanine, alanine, or glutamate, no interaction with the

PUB domain is detectable in GST pull-down assays (Figure 3H).

The analogous OTULIN Y56Fmutant was not able to coimmuno-

precipitate endogenous HOIP in contrast to wild-type OTULIN

(Figure 3I), suggesting the importance of the hydroxyl group

of Tyr56. To quantify the interaction between the HOIP PUB

domain and OTULIN, we conducted ITC experiments (Table 2;

Figure S3D). Full-length OTULIN bound the PUB domain with a

similar affinity as the OTULIN stretch from aa 45 to aa 73

(KdFL = 294 nM versus Kd45–73 = 440 nM), suggesting that this re-

gion contains the complete interaction surface of OTULIN for the

HOIP PUB domain. As expected, OTULIN Y56A did not show

any measurable affinities. Interestingly, the OTULIN peptide

used for crystallization (aa 52–61) shows a much lower affinity

(Kd52–61 = 2.9 mM) than the longer 28 aa peptide, suggesting

a more extended interaction surface than visible in the cocrystal

structure. It was previously reported that OTULIN Tyr56 is phos-

phorylated in cells (Moritz et al., 2010). Based on the crucial

involvement of the hydroxyl group of Tyr56 in the interaction,

such modification of a phosphate group to the hydroxyl group

of tyrosine could disrupt binding. Indeed, the OTULIN peptide

aa 52–61 bearing a phosphorylation at Tyr56 shows no binding

affinity in ITC-measurements (Table 2; Figure S3D).

Role of the Interaction HOIP-OTULIN in the NF-kBSignaling PathwayTo investigate the functional consequences of the HOIP-

OTULIN interaction, we analyzed the recruitment of OTULIN to

the TNFR upon TNFa-mediated activation. Endogenous HOIP

and OTULIN were recruited to the activated TNFR complex after

2 min of TNFa stimulation. The amount of endogenous OTULIN

at the activated TNFR peaked at 15 min and then decreased

progressively at 30 and 60 min time points (Figure 4A), consis-

tent with the previously described time course of HOIP recruit-

ment to the TNFR (Gerlach et al., 2011). Moreover, ectopically

expressed tagged OTULIN was readily precipitated with the


active TNFR complex (Figure 4B), while the Y56F mutant was

almost not detected at the activated TNFR (Figure 4C). This

suggests that OTULIN is recruited to the activated TNFR

complex, mainly by its direct binding to the HOIP PUB domain.

OTULIN was shown to deubiquitinate several substrates,

including NEMO and RIPK2, critical for the activation of the

NF-kB pathway (Fiil et al., 2013; Keusekotten et al., 2013; Toku-

naga, 2013). In this way OTULIN can act as an integral inhibitor

of HOIP-dependent linear ubiquitination. In accordance, the

activation of the NF-kB signaling upon HOIP/Sharpin overex-

pression was fully inhibited by simultaneous overexpression of

wild-type OTULIN (Figure 5A). However, OTULIN Y56F, which

cannot bind to HOIP, was reduced in its ability to block

NF-kB signaling to a similar extent as the C129S catalytic

mutant of OTULIN. In addition to the effect of OTULIN Y56F

on LUBAC-induced NF-kB signalization, we also investigated

the role of OTULIN-HOIP interaction in the TNFa-induced

pathway. Knockdown of OTULIN induced an increase of phos-

phorylated IkBa (Figure S5A). We showed that wild-type

OTULIN could rescue the effect of OTULIN knockdown on

phospho-IkBa, while the mutant OTULIN Y56F had no effect

on IkBa phosphorylation (Figure S5A). Similarly, both the trans-

location of p65 to the cell nucleus or the activation of a NF-kB

luciferase reporter were not rescued by the reintroduction of

OTULIN Y56F, while wild-type OTULIN could counteract the ef-

fect of the knockdown of endogenous OTULIN (Figures 5B and

S5B). This absence of effect of the OTULIN Y56F mutant on the

TNFa-induced NF-kB signalization was recapitulated at various

time points of TNFa treatment, as shown for p65 nuclear trans-

location (Figure 5C). Moreover, the effect of OTULIN Y56F on

the NF-kB activation was not due to reduced enzymatic activity,

which is similar to wild-type OTULIN as measured by in vitro

deubiquitination assays (Figure 5D).

Our structural and pull-down data indicate that the residues

Tyr82 and Asn85 of HOIP are crucial for its interaction with

OTULIN. Mutations of both residues (Y82A/N85E) abolished

the inhibitory effect of OTULIN on HOIP-dependent activation

of the NF-kB pathway, as assessed by both a luciferase gene

reporter assay and the nuclear localization of p65 (Figures 5E

and 5F). Altogether, these data indicate that the direct binding

Molecular Cell


of OTULIN to HOIP is important for its ability to limit NF-kB

signaling activation.

DISCUSSION

In this study, we showed that the DUB OTULIN interacts with the

E3 ligase LUBAC via the HOIP PUB domain and that this binding

is crucial for their ability to control the NF-kB signaling. TNFa

stimulation promotes the recruitment of the HOIP-OTULIN

complex to the active receptor complex, indicating that they

can act as a bimolecular ubiquitin editing pair (ligase and DUB)

implicated in remodeling of linear ubiquitin chains in the active

receptor complexes. HOIP-mediated linear ubiquitination of

TNFR-associated substrates such as NEMO and RIPK1 is

required for proper NF-kB activation (Gerlach et al., 2011; Ikeda

et al., 2011; Rahighi et al., 2009; Tokunaga et al., 2011). In turn,

these chains can be trimmed down by the linear-specific DUB

OTULIN, which is present in the same complex, leading to the in-

hibition of the entire pathway. Moreover, OTULIN can also act as

an internal regulatory mechanism of HOIP itself by controlling its

autoubiquitination and its ligase activity. Consistent with these

hypotheses, overexpression of OTULIN, unable to bind to

HOIP, was impaired in its ability to inhibit HOIP- or TNFa-induced

NF-kB activation. Similarly, mutation of HOIP residues neces-

sary for OTULIN binding prevented OTULIN to exert its inhibitory

action on the NF-kB pathway. Thus, the activity of HOIP-OTULIN

complex in the activated receptor complexes might be neces-

sary for an efficient and precise regulation of the NF-kBpathway.

This situation is reminiscent of the dual function of A20, which in-

hibits the NF-kB activation by the DUB activity of its N-terminal

domain removing K63 ubiquitin chains while its C-terminal

domain adds K48 chains to RIP1 (Wertz et al., 2004). In this

case both activities lead to efficient inhibition of NF-kB pathway.

It appears that specific pairs of E3 ligases and DUBs are crucial

for spatiotemporal remodeling of distinct ubiquitin chains result-

ing in efficient regulation of the NF-kB pathway.

HOIP PUB Domain Binds Distinct EffectorsAn additional aspect of our study relates to the functional and

structural analysis of the PUB domain of HOIP in interaction

with different effectors such as OTULIN and p97. Both p97

and OTULIN PIMs are oriented in the same way relative to the

PUB domain and bind to theJ–Y pocket. The hydrophobic res-

idue and Tyr of PIM (OTULIN: Met55 and Tyr56; p97: Leu804

and Tyr805) fit into the J–Y pocket and interact with the PUB

domain by van der Waals interactions. Additionally, a few

hydrogen bonds interactions between the hydroxyl group of

Tyr and the main chain oxygen atoms of PIM and Tyr82,

Asn85, Asn102, and Tyr124 stabilize the interaction between

PIM and HOIP PUB domain. Overall, the structure of the HOIP

PUB domain resembles the one of PNGase. However, the

different way of the recognition of the C-terminal carboxylate

of the peptide proves that only HOIP can bind internal and C-ter-

minal motifs. This is different in the PUB domain of PNGase that

has a distinct configuration of the J–Y pocket limiting its PUB

domain to C-terminal PIMs. In short, the position of PNGase

Arg55 binds to C-terminal PIMs and may also block internal

PIMs. Contrary to this, in the HOIP PUB domain the correspond-

ing residue HOIP Lys99 is flipped away, opening a route for not

only C-terminal but also internal PIMs. Although we could not

see the full electron density of OTULIN Arg57 side chain, this

side chain most likely sits between the pit of Tyr94 and Lys99

of HOIP PUB domain in agreement with this notion. Interestingly,

in the direction of this potential peptide route, the PUB domain

harbors an additional highly conserved surface patch potentially

involved in binding amino acids downstream of the PIM of

OTULIN. This idea would fit to the strongly increased affinity of

the longer peptide (aa 45–73) of OTULIN in comparison to the

shorter peptide (aa 52–61) and to the high conservation of

OTULIN between aa 61 and aa 69, a region C-terminal of its

PIM. This data explains why the PUB domain of HOIP can

accommodate both OTULIN PIM with high affinity and P97

PIM with lower affinity.

Interestingly, one of the other proteins identified in our mass

spectrometric analysis of LUBAC was another AAA+ ATPase,

the DNA damage factor Werner helicase interacting protein 1

(WRNIP1). It contains the motif EEHYN (aa 497–501), which

resembles the sequences of OTULIN (EDMYR, aa 53–57) and

p97 (DDLYG, aa 802–806), and we have shown that this motif

binds to the PUB domain of HOIP (Figure S5C). This indicates

that HOIP may exist in complex with different effectors (e.g.,

HOIP-OTULIN, HOIP-p97, and HOIP-WRNIP1). In support of

this hypothesis, we detected lower amount of OTULIN than

HOIP in our cells, and OTULIN coprecipitated only a fraction of

HOIP, leaving a significant amount of HOIP potentially in com-

plex with other partners. The functional significance of these

different complexes is currently unknown. It remains to be

seen if HOIP also recruits p97 beside OTULIN to the activated

TNFR and if it contributes to the regulation of NF-kB pathway.

Indeed, previous work suggested that p97 regulates the NF-kB

pathway (Asai et al., 2002; Custer et al., 2010; Dai et al., 1998;

Li et al., 2014).

In humans, the p97 adaptor UBXD1 was identified as a

third PUB-domain-containing protein along HOIP and PNGase.

Although the structure of PUB domain of UBXD1 has not been

reported, one can expect an overall structure similar to the

PNGase and HOIP PUB domains due to the high sequence

homology. UBXD1 Lys198 andGln200 are equivalent andmaybe

functionally similar to HOIP Lys99 and Asn101 (Figure S2F).

However, HOIP Arg106 is replaced by the shorter amino acid

Gln205 in UBXD1, potentially insufficient to fix the orientation

of Gln200 and thereby Lys198. This would result in closing the

gate of the peptide route and limiting the UBXD1 PUB domain

to the C-terminal PIMs. This is consistent with our biochemical

data, indicating that UBXD1 does not bind to the OTULIN PIM

but is bound to p97 PIM, as previously reported (Kern et al.,

2009; Stapf et al., 2011). Therefore, the unique combination of

amino acids of HOIP Lys99, Asn101, and Arg106 would specif-

ically expand its PUB domain to interact with internal PIMs, as

opposed to the PUB domains of UBXD1 or PNGase.

Regulation of PUB-PIM Interaction by Phosphorylationor UbiquitinationThe direct interaction between the HOIP PUB domain and

OTULIN or p97 could be regulated by various posttranslational

modifications. Based on the structural data showing a tight fit


Figure 3. OTULIN and p97 Bind HOIP via Conserved Tyrosine-Based Motif

(A) Pull-down analysis of immobilized OTULIN wild-type and fragments thereof with MBP-tagged HOIP ZF. Ponceau-S-stained membrane shows GST-tagged

fragments.

(B) A close-up view of the interaction between HOIP PUB domain and OTULIN peptide (Ball-and-stick representation, yellow), in the same view as Figure 2E.

Hydrophobic surface residues are colored by green.

(legend continued on next page)

Molecular Cell



Figure 4. OTULIN Is Recruited at the TNF Receptor via Tyr56

(A–C) TNFR purifications after treatment with HisFlag-TNFa (HF-TNFa) for the indicated times and immunoprecipitation using AntiFlag-resin.

(A) The recruitment of endogenous OTULIN and HOIP to the activated TNFR was detected by immunoblot.

(B and C) Cells expressed transiently HSS-OTULIN wild-type or mutant Y56F prior to the addition of HF-TNFa.

Molecular Cell


of the central tyrosines and the ITC measurements of the

phosphorylated OTULIN-peptide, the phosphorylation of these

tyrosines inhibits the interaction of OTULIN or p97 with HOIP.

Evidence for the phosphorylation of OTULIN Tyr56 in vivo has

been reported (Moritz et al., 2010), but it is still unclear which

enzymes are responsible for placing and removing this phos-

phorylation. Such phosphorylation of OTULIN Tyr56 would regu-

late the general association of OTULIN with LUBAC before their

recruitment to the TNFR complexes. One could also envision

a much more directed regulation at the TNFR. For example,

OTULIN might be specifically phosphorylated after the recruit-

ment to the TNFR as part of an active exclusion mechanism.

This would fit the notion that two independent mechanisms allow

for a stabilization of OTULIN at the TNFR, with the direct recruit-

ment of OTULIN via the HOIP PUB domain only being one way.

(C) Close-up view of the interaction between HOIP PUB domain and p97 peptid

(D and E) Surface representations of HOIP PUB domain J–Y pocket binding to

(F and G) Cartoon representation of HOIP PUB domain (green) binding to p97 s

between HOIP PUB domain the central tyrosines are shown as black dashed lin

(H) Pull-down analysis of immobilizedGST-taggedOTULINwild-type and Tyr56m

(I) Immunoprecipitation of HSS-OTULIN wild-type and Y56F mutant transiently o

For p97, the regulation of the phosphorylation at Tyr805 by c-src

(Li et al., 2008) provides a potential regulatory mechanism for

the interaction of p97 with LUBAC. On the contrary, the non-

receptor-type tyrosine phosphatase PTPL1 has been reported

to directly dephosphorylate Y805 of p97, thereby linking the

dephosphorylation of Y805 to tumorigenic cell transformation

(Abaan et al., 2013). Interestingly, phosphorylation of WRNIP1

on Tyr500, which corresponds to OTULIN Tyr56 and p97

Tyr805, has been reported, suggesting that all three conserved

tyrosine are phosphorylated in vivo and can potentially be nega-

tively regulated in their binding to PUB domains (Shiromizu

et al., 2013).

A second regulatory mechanism may involve ubiquitination of

HOIP. Interestingly, a proteomics study to identify in vivo ubiqui-

tination sites detected HOIP Lys99 to be ubiquitinated (Wagner

e (ball-and-stick representation, cyan), in the same view as Figure 2E.

p97 peptide shown in cyan (D) or to OTULIN peptide shown in yellow (E).

hown in cyan (F) or binding to OTULIN shown in yellow (G). Hydrogen bonds

es.

utantswithMBP-HOIP ZF. Ponceau S staining showsGST-OTULIN constructs.

verexpressed in HeLa cells. See also Figure S4.


Figure 5. OTULIN Recruitment via Tyr56 Is Important for NF-kB Signaling

(A) Luciferase assay for NF-kB activity in HeLa cells transfected with Sharpin, HOIP, OTULIN wild-type, or the indicated point mutants.

(B) Luciferase assay for TNFa-induced NF-kB activity in HeLa cells knockdown for OTULIN (siOTULIN) and expressing OTULIN wild-type (wt) or OTULIN Y56F

mutant.

(C) p65 nuclear translocation in response to TNFa in HeLa cells knockdown for OTULIN (siOTULIN) and expressing OTULIN wt or OTULIN Y56F mutant. Cells

were treated with TNFa for the indicated time. * compared to siOTULIN; # compared to siOTULIN + TNFa + OTULINwt.

(D) DUB activity assessment for GST-OTULIN wt and GST-OTULIN Y56F.

(E) Effect of OTULIN in the luciferase assay for NF-kB activity in HeLa cells expressing sharpin and wt or mutant (Y82A/N85E) HOIP.

(F) Effect of OTULIN on p65 nuclear translocation in cells expressing sharpin and wt or mutant (Y82A/N85E) HOIP. Data are represented asmean ±SEM. See also

Figure S5.

Molecular Cell


et al., 2011). Such ubiquitination may inhibit the interaction with

the PIM of OTULIN but not with the C-terminal PIM peptide of

p97. Although further experiments will be required to address

these questions, ubiquitination could play an important role in

regulating the interaction between HOIP and OTULIN. Further

functional studies of the HOIP PUB domain and the dynamic


regulation of its interactions with other effectors will contribute

to the better understanding of the NF-kB pathway by linear

ubiquitination. We provide here the first demonstration that

HOIP-OTULIN interact through the HOIP PUB domain and act

as a bimolecular editing pair to regulate the NF-kB activation

upon TNF stimulation.

Molecular Cell


EXPERIMENTAL PROCEDURES

Mass Spectrometry/Data Processing

Liquid chromatography-tandemmass spectrometry analyses were performed

on an EasyLC nano-HPLC coupled to an Orbitrap Elite mass spectrometer

(both Thermo Scientific). In gel trypsin digestion of the immunoprecipitates

was performed as described previously (Shevchenko et al., 2006). In brief,

eluates were run on a SDS-PAGE, alkylated with chloroacetamide, overnight

digested with trypsin, and extracted. The desalted peptide mixtures were

injected onto the column in HPLC Solvent A (0.5% acetic acid) and eluted

with a 5%–33% gradient HPLC solvent B (80% acetonitril in 0.5% acetic

acid) running at a constant flow rate of 200 nl/min at 30�C. Full-scan MS

spectra were acquired in a mass range fromm/z 300 to 2,000 with a resolution

of 120.000 without lock mass. The 20 most intense precursor ions were

sequentially CID fragmented in each scan cycle. In all measurements, up to

500 sequenced precursor masses were excluded from further analysis for

90 s. The target values of the mass analyzers were 1 million charges (MS)

and 5,000 charges (MS/MS). The MS data of all SILAC experiments was pro-

cessed using default parameters of the MaxQuant software (1.3.0.5) (Cox and

Mann, 2008). The peak lists were queried against the human UniProt database

(2012_09). Full tryptic specificity was required, and up to two missed cleav-

ages were allowed. Carbamidomethylation of cysteine was set as fixed modi-

fication. Protein N-terminal acetylation, oxidation of methionine, and GlyGly

modification were set as variable modifications. Initial precursor mass toler-

ance was set to 7 ppm and 0.5 Da at the fragment ion level. For protein group

quantitation, a minimum of two quantified peptides were required. False dis-

covery rates were set to 1% at peptide and protein group level. Downstream

analysis of all resulting text files was performed using R (2.14.1).

Cell Culture/Stable Cell Lines/Transient Overexpression/SILAC

HeLa cells were cultured in Dulbecco’smodified Eagle’smedium (DMEM) sup-

plemented with 10% fetal bovine serum, 0.2 mM L-glutamine, and standard

antibiotics. The HeLa FRT/TO cell line for creating stable cell lines using the

Flp-InTM System (Invitrogen) was kindly provided by S. Taylor (University

Manchester) and maintained as described (Tighe et al., 2008). The HSS-/

SSH-tagged HOIP/HOIL-1/Sharpin cell lines were generated as described

(Glatter et al., 2009). For transient overexpression, DNA plasmid transfections

were performed with GeneJuice (Merck) following the manufacturer instruc-

tions. For SILAC-labeling, cells were maintain in custom-made SILAC

DMEM (heavy/light) for 10 days, and incorporation of labeled amino acids to

more the 95% was verified.

DNA Manipulations/Mutagenesis

The OTULIN cDNA (NM_138348.4) was synthesized de novo. The pcDNA5-

HSS and pcDNA5-SSH vectors for N-terminal HA/23Strep and C-terminal

HA/23Strep tagging were obtained from E. Wytler (ETH Zurich). HOIP

(NM_017999.4), HOIL-1 (NM_031229.2), Sharpin (NM_030974.3), or OTULIN

were subcloned into pcDNA5-HSS or -SSH for transient or stable expression

in HeLa cells and into pGEX6P1 for GST-tagged or into pMAX-C2X for

maltose-binding protein (MBP)-tagged bacterial protein purification by sub-

cloning and verified by sequencing. Additional derivates (fragments/point

mutations/siRNA resistance) of the expression plasmids were generated by

site-directed mutagenesis according to standard protocols.

RNAi and Rescue Experiments

The siRNA directed against OTULIN used was published previously

(siOTULIN#2, Fiil et al., 2013). HeLa cells were reverse transfected with control

siRNA or siRNA against OTULIN using Lipofectamine RNAimax. After 24 hr,

cells were transfected with the siRNA-resistant OTULIN construct (wild-type

and Y56F mutant) or empty vector for an additional 36 hr.

Immunoprecipitation

HA purifications were carried out as described (Glatter et al., 2009). Briefly,

expression of the bait protein in stable cells was induced by 1 mg/ml doxycy-

cline for 24 hr before harvesting or transiently overexpressed using GeneJuice

for 16–24 hr. Cells were harvested, washed with ice-cold PBS, and lysed using

26G needles in IP buffer (10 mM Tris [pH 7.5], 100mMKCl, 2 mMMgCl2, 0.5%

NP40, and Complete Protease inhibitors [Roche]) containing 0.5 mM DTT.

Extracts were cleared by centrifugation and incubated with pre-equilibrated

anti-HA-beads (Sigma; 20 ml per 10 cm dish or 30 ml per 15 cm dish) for 1 hr.

For MS analysis, samples were eluted with Laemmli buffer without DTT, and

after elution, DTT was added to 20 mM final concentration. For immunoblot

analysis, samples were eluted with 100 mM glycin (pH 2), and after elution

Laemmli buffer was added and neutralized with 1 M Tris (pH 9.2).

For immunoprecipitation of endogenous OTULIN, cells were lysed in 30 mM

Tris (pH 7.4), 120 mM NaCl, 2 mM EDTA, 2 mM KCl, 1% triton X-100, and

Complete Protease inhibitors (Roche). After clearing, lysates were incubated

overnight at 4�C with anti-OTULIN antibody. Then protein A-agarose (Roche)

was added for an additional 3 hr at 4�C. Beads were then washed, and sam-

ples were eluted with Laemmli buffer.

For TNR-receptor purifications, immunoprecipitations were carried out

similarly to endogenous OTULIN immunoprecipitation with the following

adjustments. Cells were treated with 0.05 ng/ml self-made bacterially purified

HisFlag-tagged TNFa for the time points indicated. Treatments were stopped

by washing with ice-cold PBS prior harvesting. At 0 min time point, TNFa was

added after clearing of the lysate.

Immunoblotting/Antibodies

For immunoblotting, proteins were resolved by SDS-PAGE and transferred to

PVDF amersham hybond-p membrane (GE Healthcare). Blocking and anti-

body incubations were carried out in 5% low-fat milk (Roth) or 5% BSA in

TBS-T (150mMNaCl, 20 mM Tris [pH 8.0], and 0.1% Tween-20) and washings

in TBS-T. Blots were developed using ImmunoCruz (Santa Cruz) or TMA-6

(Lumigen).

The following commercial antibodies were used: anti-HA (Covance

MMS-101P), HOIP (Abcam ab125189 and ab125184), HOIL-1 (Santa Cruz

sc49718), Sharpin (Protein Tech 14626-1-AP), OTULIN (Abcam 151117), p97

(Cell Signaling #2649), MBP (Sigma M-6295), TNFR1 (Santa Cruz sc8436),

RIP1 (BD Transduction 610458), Tubulin (Sigma T9026), Vinculin (Sigma

hVIN-1), p65 (Santa Cruz, sc-372), and phospho-IkBa (Cell Signaling, 9246).

Protein Purification/Pull-Down Experiments

GST or MBP fusion proteins were expressed in Escherichia coli BL21 DE3.

Cells were lysed by sonication in lysis buffer (25 mM Tris and 200 mM NaCl

[pH 8.5]), and the lysate was cleared by centrifugation. The expressed protein

was purified by glutathione-Sepharose 4B or Amylose resin (NEB). For crystal-

lography, proteins were cleaved by PreScission Protease and further purified

by size exclusion chromatography (HiLoad 16/600 Superdex 200 column, GE

Healthcare Life Sciences) in 25 mM Tris and 150 mM NaCl at pH 8.5.

For biochemical assays, OTULIN was cleaved by Precission Protease, MBP-

HOIP ZF was eluted using 10 mM Maltose, and all other fusion proteins

were left on the beads.

Pull-down experiments were conducted by diluting 10 mg proteins on beads

in pull-down buffer (PDB) (150 mM NaCl, 50 mM Tris pH 7.5, 0.1% NP-40,

5 mM DTT) and adding 1 mg solubilized prey protein in PDB plus 0.5 mg/ml

BSA or 100 mg HeLa lysate in IP buffer as used for immunoprecipitations. After

overnight incubation, beads were washed with PDB, eluted with Laemmli-

buffer, and analyzed by SDS-PAGE and immunoblotting.

Crystallization, X-Ray Data Collection, and Structure Determination

Crystals of HOIP PUB domain (1–179) alone or in complex with p97 peptide

(797–806) or with OTULIN peptide (52–61) were obtained using 0.1 M Tris,

20% 2-methyl-2,4-pentanediol, 15% Polyethyleneglycol 3350 (pH 8.5,

pH 6.5, and pH 7.5, respectively) as a reservoir solution by the sitting-drop

vapor diffusion method at 293 K. HOIP PUB domain (1–179) selenomethionine

(SeMet) substituted crystals were obtained using 0.1M Tris, 30%Polyethylene

glycol monomethyl ether 550 (pH 8.5). Data were collected at KEK, photon

factory BL1A, BL17, and Diamond Light Source beam line I03 and processed

with XDS. The crystal structure of HOIP PUB domain was determined by single

anomalous dispersion using SeMet substituted HOIP PUB domain mutant

(L31MR35M) crystals with Phenix AutoSol. The crystal structure of HOIP

PUB domain (1–179) in complex with p97 peptide and OTULIN peptide was

determined by molecular replacement with Molrep using the HOIP PUB

domain structure as a search model. Manual model building and refinement


Molecular Cell


were done with Coot and Phenix. The final statistics of refined models are

shown in Table 2, and the atomic coordinates have been deposited in the

Protein Data Bank (PDB IDs 4P09, 4P0A, and 4P0B). Structures were visual-

ized using PyMOL 1.3 (Schrodinger).

For Consurf-based structures of conserved surface patches, we used

TreeFam 9 to collect all HOIP sequences with complete PUB domains. These,

in total, 45 sequences ranging from Oreochromis niloticus to Homo sapiens

were then submitted as an external multiple sequence alignment file together

with the PUB domain structure data to http://consurf.tau.ac.il (Ashkenazy

et al., 2010). The resulting structure model contained the conservation infor-

mation of each amino acid.

ITC and CD

Calorimetry experiments were performed at 20�C using a VP-ITC microcalo-

rimeter (MicroCal). All samples were prepared in 25 mM Tris, 150 mM NaCl

(pH 8.5). A total of 10 ml of the HOIP PUB domain at concentration of

0.55 mM was titrated into sample cell containing 0.05 mM OTULIN proteins

or peptides. A total of 27 injections were performed with duration of 20 s

and spacing of 400 s. The ITC data were analyzed with the ITC-Origin 7.0

software with a one-site fitting scheme. The heats of dilution control titration

were subtracted from the raw data for model fitting.

CD spectra of the HOIP PUB domain wild-type and mutant were measured

by a Jasco J-820 spectropolarimeter at 25�C using a cuvette with 1 mm path

length.

Luciferase Assay

HeLa cells were transfected with pNFkBx5-Luc (Stratagene) as luciferase

reporter and pUT651 as b-galactosidase reporter using GeneJuice (Merck).

Cells were additionally transfected with HOIP, Sharpin, and OTULIN con-

structs in LUBAC-dependent NF-kB activation experiments. After 48 hr of

transfection, cells were treated with TNFa for 6 hr in TNFa-dependent NF-kB

activation experiments or directly lysed in LUBAC-induced NF-kB activation.

Lysates were subjected to luciferase assays following the manufacturer’s pro-

tocol (Roche). Internal control was measured by b-galactosidase activity using

its substrate (Roche). Three independent experiments were performed using

triplicate samples in each experiment.

P65 Nuclear Translocation

HeLa cells were fixed and stained for p65. The number of cells showing a

nuclear localization of p65 was counted and normalized to the total number

of cells to determine the percentage of translocation.

Statistical Analysis

Results were analyzed using one- or two-way analysis of variance followed

by Tukey’s post hoc tests.

ACCESSION NUMBERS

The atomic coordinates of all three structures have been deposited in the

Protein Data Bank (accession numbers 4P09, 4P0A, and 4P0B).

SUPPLEMENTAL INFORMATION

Supplemental Information includes five figures and can be found with this

article online at http://dx.doi.org/10.1016/j.molcel.2014.03.016.

ACKNOWLEDGMENTS

We are thankful to Fernando Bazan for the bioinformatics analysis of the HOIP

PUB domain; Jan Heering for help with ITC measurements; Alexander Buch-

berger for p97 and UBXD1 constructs; David McEwan, Anja Bremm, and

Jaime Lopez-Mosqueda for critical reading of the manuscript; Masuda Sader

for help with protein purifications; and all members of the Dikic lab for discus-

sions and constructive comments. Crystallographic data were collected at

KEK, photon factory, beamline BL1A and BL17 and DIAMOND light source,

beamline I03. The work was supported by the Cluster of Excellence ‘‘Macro-


molecular Complexes’’ of the Goethe University Frankfurt (EXC115), the

LOEWE funded OSF network, the LOEWE Gene and Cell Therapy Center,

and the European Research Council/ERC grant agreement n� [250241-

LineUb] to I.D. The research leading to these results received also funding

from the European Union Seventh Framework Programme (FP7/2007-2013)

PRIMES project under grant agreement number FP7-HEALTH-2010-278568

and from the FSP SPP1365.

Received: October 16, 2013

Revised: January 24, 2014

Accepted: February 28, 2014

Published: April 10, 2014

REFERENCES

Abaan, O.D., Hendriks, W., Uren, A., Toretsky, J.A., and Erkizan, H.V. (2013).

Valosin containing protein (VCP/p97) is a novel substrate for the protein tyro-

sine phosphatase PTPL1. Exp. Cell Res. 319, 1–11.

Allen, M.D., Buchberger, A., and Bycroft, M. (2006). The PUB domain functions

as a p97 binding module in human peptide N-glycanase. J. Biol. Chem. 281,

25502–25508.

Asai, T., Tomita, Y., Nakatsuka, S., Hoshida, Y., Myoui, A., Yoshikawa, H., and

Aozasa, K. (2002). VCP (p97) regulates NFkappaB signaling pathway, which is

important for metastasis of osteosarcoma cell line. Jpn. J. Cancer Res. 93,

296–304.

Ashkenazy, H., Erez, E., Martz, E., Pupko, T., and Ben-Tal, N. (2010). ConSurf

2010: calculating evolutionary conservation in sequence and structure of pro-

teins and nucleic acids. Nucleic Acids Res. 38 (Web Server issue), W529–

W533.

Cox, J., and Mann, M. (2008). MaxQuant enables high peptide identification

rates, individualized p.p.b.-range mass accuracies and proteome-wide pro-

tein quantification. Nat. Biotechnol. 26, 1367–1372.

Custer, S.K., Neumann, M., Lu, H., Wright, A.C., and Taylor, J.P. (2010).

Transgenic mice expressing mutant forms VCP/p97 recapitulate the full spec-

trum of IBMPFD including degeneration in muscle, brain and bone. Hum. Mol.

Genet. 19, 1741–1755.

Dai, R.M., Chen, E., Longo, D.L., Gorbea, C.M., and Li, C.C. (1998).

Involvement of valosin-containing protein, an ATPase Co-purified with

IkappaBalpha and 26 S proteasome, in ubiquitin-proteasome-mediated

degradation of IkappaBalpha. J. Biol. Chem. 273, 3562–3573.

Damgaard, R.B., Nachbur, U., Yabal, M., Wong, W.W., Fiil, B.K., Kastirr, M.,

Rieser, E., Rickard, J.A., Bankovacki, A., Peschel, C., et al. (2012). The ubiqui-

tin ligase XIAP recruits LUBAC for NOD2 signaling in inflammation and innate

immunity. Mol. Cell 46, 746–758.

Fiil, B.K., Damgaard, R.B., Wagner, S.A., Keusekotten, K., Fritsch, M., Bekker-

Jensen, S., Mailand, N., Choudhary, C., Komander, D., and Gyrd-Hansen, M.

(2013). OTULIN restricts Met1-linked ubiquitination to control innate immune

signaling. Mol. Cell 50, 818–830.

Gerlach, B., Cordier, S.M., Schmukle, A.C., Emmerich, C.H., Rieser, E., Haas,

T.L., Webb, A.I., Rickard, J.A., Anderton, H., Wong, W.W., et al. (2011). Linear

ubiquitination prevents inflammation and regulates immune signalling. Nature

471, 591–596.

Glatter, T., Wepf, A., Aebersold, R., and Gstaiger, M. (2009). An integrated

workflow for charting the human interaction proteome: insights into the

PP2A system. Mol. Syst. Biol. 5, 237.

Grabbe, C., Husnjak, K., and Dikic, I. (2011). The spatial and temporal organi-

zation of ubiquitin networks. Nat. Rev. Mol. Cell Biol. 12, 295–307.

Halawani, D., and Latterich, M. (2006). p97: The cell’s molecular purgatory?

Mol. Cell 22, 713–717.

Holm, L., and Rosenstrom, P. (2010). Dali server: conservation mapping in 3D.

Nucleic Acids Res. 38 (Web Server issue), W545–W549.

Husnjak, K., and Dikic, I. (2012). Ubiquitin-binding proteins: decoders of ubiq-

uitin-mediated cellular functions. Annu. Rev. Biochem. 81, 291–322.

http://consurf.tau.ac.il


Molecular Cell


Ikeda, F., Deribe, Y.L., Skanland, S.S., Stieglitz, B., Grabbe, C., Franz-

Wachtel, M., van Wijk, S.J., Goswami, P., Nagy, V., Terzic, J., et al. (2011).

SHARPIN forms a linear ubiquitin ligase complex regulating NF-kB activity

and apoptosis. Nature 471, 637–641.

Kern, M., Fernandez-Saiz, V., Schafer, Z., and Buchberger, A. (2009). UBXD1

binds p97 through two independent binding sites. Biochem. Biophys. Res.

Commun. 380, 303–307.

Keusekotten, K., Elliott, P.R., Glockner, L., Fiil, B.K., Damgaard, R.B., Kulathu,

Y., Wauer, T., Hospenthal, M.K., Gyrd-Hansen, M., Krappmann, D., et al.

(2013). OTULIN antagonizes LUBAC signaling by specifically hydrolyzing

Met1-linked polyubiquitin. Cell 153, 1312–1326.

Komander, D., and Rape, M. (2012). The ubiquitin code. Annu. Rev. Biochem.

81, 203–229.

Li, G., Zhao, G., Schindelin, H., and Lennarz, W.J. (2008). Tyrosine phosphor-

ylation of ATPase p97 regulates its activity during ERAD. Biochem. Biophys.

Res. Commun. 375, 247–251.

Li, J.M., Wu, H., Zhang, W., Blackburn, M.R., and Jin, J. (2014). The p97-

UFD1L-NPL4 protein complex mediates cytokine-induced IkBa proteolysis.

Mol. Cell. Biol. 34, 335–347.

Madsen, L., Seeger, M., Semple, C.A., and Hartmann-Petersen, R. (2009).

New ATPase regulators—p97 goes to the PUB. Int. J. Biochem. Cell Biol.

41, 2380–2388.

Moritz, A., Li, Y., Guo, A., Villen, J., Wang, Y., MacNeill, J., Kornhauser, J.,

Sprott, K., Zhou, J., Possemato, A., et al. (2010). Akt-RSK-S6 kinase signaling

networks activated by oncogenic receptor tyrosine kinases. Sci. Signal. 3,

ra64.

Rahighi, S., Ikeda, F., Kawasaki, M., Akutsu, M., Suzuki, N., Kato, R., Kensche,

T., Uejima, T., Bloor, S., Komander, D., et al. (2009). Specific recognition of

linear ubiquitin chains by NEMO is important for NF-kappaB activation. Cell

136, 1098–1109.

Rivkin, E., Almeida, S.M., Ceccarelli, D.F., Juang, Y.C., MacLean, T.A.,

Srikumar, T., Huang, H., Dunham, W.H., Fukumura, R., Xie, G., et al. (2013).

The linear ubiquitin-specific deubiquitinase gumby regulates angiogenesis.

Nature 498, 318–324.

Shevchenko, A., Tomas, H., Havlis, J., Olsen, J.V., and Mann, M. (2006). In-gel

digestion for mass spectrometric characterization of proteins and proteomes.

Nat. Protoc. 1, 2856–2860.

Shiromizu, T., Adachi, J., Watanabe, S., Murakami, T., Kuga, T., Muraoka, S.,

and Tomonaga, T. (2013). Identification of missing proteins in the neXtProt

database and unregistered phosphopeptides in the PhosphoSitePlus

database as part of the Chromosome-centric Human Proteome Project.

J. Proteome Res. 12, 2414–2421.

Stapf, C., Cartwright, E., Bycroft, M., Hofmann, K., and Buchberger, A. (2011).

The general definition of the p97/valosin-containing protein (VCP)-interacting

motif (VIM) delineates a new family of p97 cofactors. J. Biol. Chem. 286,

38670–38678.

Stieglitz, B., Rana, R.R., Koliopoulos, M.G., Morris-Davies, A.C., Schaeffer, V.,

Christodoulou, E., Howell, S., Brown, N.R., Dikic, I., and Rittinger, K. (2013).

Structural basis for ligase-specific conjugation of linear ubiquitin chains by

HOIP. Nature 503, 422–426.

Stolz, A., Hilt, W., Buchberger, A., and Wolf, D.H. (2011). Cdc48: a power

machine in protein degradation. Trends Biochem. Sci. 36, 515–523.

Suzuki, T., Park, H., Till, E.A., and Lennarz, W.J. (2001). The PUB domain: a

putative protein-protein interaction domain implicated in the ubiquitin-protea-

some pathway. Biochem. Biophys. Res. Commun. 287, 1083–1087.

Tighe, A., Staples, O., and Taylor, S. (2008). Mps1 kinase activity restrains

anaphase during an unperturbed mitosis and targets Mad2 to kinetochores.

J. Cell Biol. 181, 893–901.

Tokunaga, F. (2013). Linear ubiquitination-mediated NF-kB regulation and its

related disorders. J. Biochem. 154, 313–323.

Tokunaga, F., Nakagawa, T., Nakahara, M., Saeki, Y., Taniguchi, M., Sakata,

S., Tanaka, K., Nakano, H., and Iwai, K. (2011). SHARPIN is a component of

the NF-kB-activating linear ubiquitin chain assembly complex. Nature 471,

633–636.

Wagner, S.A., Beli, P., Weinert, B.T., Nielsen, M.L., Cox, J., Mann, M., and

Choudhary, C. (2011). A proteome-wide, quantitative survey of in vivo ubiqui-

tylation sites reveals widespread regulatory roles. Mol. Cell Proteomics 10,

http://dx.doi.org/10.1074/mcp.M111.013284.

Walczak, H., Iwai, K., and Dikic, I. (2012). Generation and physiological roles of

linear ubiquitin chains. BMC Biol. 10, 23.

Wertz, I.E., and Dixit, V.M. (2010). Signaling to NF-kappaB: regulation by ubiq-

uitination. Cold Spring Harb. Perspect. Biol. 2, a003350.

Wertz, I.E., O’Rourke, K.M., Zhou, H., Eby, M., Aravind, L., Seshagiri, S., Wu,

P., Wiesmann, C., Baker, R., Boone, D.L., et al. (2004). De-ubiquitination and

ubiquitin ligase domains of A20 downregulate NF-kappaB signalling. Nature

430, 694–699.

Zhao, G., Zhou, X., Wang, L., Li, G., Schindelin, H., and Lennarz, W.J. (2007).

Studies on peptide:N-glycanase-p97 interaction suggest that p97 phosphory-

lation modulates endoplasmic reticulum-associated degradation. Proc. Natl.

Acad. Sci. USA 104, 8785–8790.


http://dx.doi.org/10.1074/mcp.M111.013284

Binding of OTULIN to the PUB Domain of HOIP Controls NF-κB Signaling

Documents

Transcript of Binding of OTULIN to the PUB Domain of HOIP Controls NF-κB Signaling