Molecular Cell
Article
Ubiquitin Binding to A20 ZnF4 Is Required
for Modulation of NF-kB Signaling
Ivan Bosanac,1 Ingrid E. Wertz,2 Borlan Pan,1 Christine Yu,1 Saritha Kusam,2 Cynthia Lam,2 Lilian Phu,3 Qui Phung,3
Brigitte Maurer,1 David Arnott,3 Donald S. Kirkpatrick,3 Vishva M. Dixit,4,* and Sarah G. Hymowitz1,*
1Department of Structural Biology
2Department of Protein Engineering
3Department of Protein Chemistry
4Department of Physiological Chemistry
Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA
*Correspondence: dixit@gene.com (V.M.D.), hymowitz@gene.com (S.G.H.)
DOI 10.1016/j.molcel.2010.10.009
SUMMARY
Inactivating mutations in the ubiquitin (Ub) editing
protein A20 promote persistent nuclear factor (NF)-
kB signaling and are genetically linked to inflamma-
tory diseases and hematologic cancers. A20 tightly
regulates NF-kB signaling by acting as an Ub editor,
removing K63-linked Ub chains and mediating addi-
tion of Ub chains that target substrates for degrada-
tion. However, a precise molecular understanding of
how A20 modulates this pathway remains elusive.
Here, using structural analysis, domain mapping,
and functional assays, we show that A20 zinc finger 4
(ZnF4) does not directly interact with E2 enzymes but
instead can bind mono-Ub and K63-linked poly-Ub.
Mutations to the A20 ZnF4 Ub-binding surface result
in decreased A20-mediated ubiquitination and
impaired regulation of NF-kB signaling. Collectively,
our studies illuminate the mechanistically distinct
but biologically interdependent activities of the A20
ZnF and ovarian tumor (OTU) domains that are
inherent to the Ub editing process and, ultimately,
to regulation of NF-kB signaling.
INTRODUCTION
Covalent attachment of ubiquitin (Ub) to protein substrates
requires the sequential action of three classes of enzymes: Ub-
activating enzymes (E1), Ub-conjugating enzymes (E2), and Ub
ligases (E3) (Pickart, 2001). E3 ligases containing RING, HECT,
or U box domains directly interact with E2 enzymes and recruit
protein substrates for Ub modification. E2 and E3 enzymes
cooperate to assemble poly-Ub chains on substrates by linking
the C-terminal glycine of one Ub to the 3-amino group of one
of the seven lysine (K) residues of another Ub, or alternately,
the amino terminus (Iwai and Tokunaga, 2009). Addition of
mono-Ub or K63-linked poly-Ub chains is generally associated
with substrate activation or relocalization, whereas attachment
of K48 or K11 linkages promotes proteasomal degradation (Gar-
nett et al., 2009; Williamson et al., 2009; Xu et al., 2009).
548 Molecular Cell 40, 548–557, November 24, 2010 ª2010 Elsevier Inc.
A20, a bifunctional Ub editing protein, is required for proper
termination of cytokine-induced NF-kB signaling pathways.
A20 knockout mice display persistent NF-kB activation and
cytokine production, resulting in early lethality due to severe mul-
tiorgan inflammation (Lee et al., 2000). In humans, defects in A20
are genetically linked to autoimmune disorders and hematologic
malignancies (Compagno et al., 2009; Graham et al., 2008;
Honma et al., 2009; Kato et al., 2009; Musone et al., 2008; Novak
et al., 2009; Plenge et al., 2007; Schmitz et al., 2009; Thomson
et al., 2007). A20 removes K63-linked Ub chains from substrates,
such as receptor-interacting protein (RIP1), and facilitates addi-
tion of K48-linked Ub chains, thereby targeting RIP1 for protea-
somal degradation (Coornaert et al., 2009; Hymowitz and Wertz,
2010).
The N-terminal region of A20, containing an ovarian tumor
(OTU) domain, functions as a deubiquitinating (DUB) enzyme
that hydrolyzes poly-Ub chains (Evans et al., 2004; Komander
and Barford, 2008; Lin et al., 2008; Wertz et al., 2004).
Conversely, the seven zinc fingers (ZnFs) forming the C-terminal
portion of A20 are required for the assembly of poly-Ub chains
(Opipari et al., 1990; Shembade et al., 2008, 2010; Wertz et al.,
2004). The A20-like ZnF in Rabex-5 has also been shown to
have E3 ligase activity (Lee et al., 2006; Mattera et al., 2006).
The minimal portion of A20 required for this activity was mapped
to the ZnF3-4 region, with ZnF4 being essential (Wertz et al.,
2004). A20 ZnF4 has recently been shown to be important for
TAXBP1 binding, which is necessary for Ubc13 and UbcH5C
degradation during A20-dependent NF-kB signaling inhibition
(Shembade et al., 2010). The C-terminal ZnF region of A20 also
mediates interactions with additional proteins such as the A20-
binding inhibitor of NF-kB (ABIN) family members (Van Huffel
et al., 2001) and TAX1 binding protein-1 (TAXBP-1) (De Valck
et al., 1999). Consistent with the functional importance of both
the N-terminal OTU and C-terminal ZnF domains, genetic alter-
ations throughout A20 have been associated with the develop-
ment of B cell lymphomas (Kato et al., 2009).
To better elucidate the functional importance of ZnF4 in A20
activities, we examined the molecular basis of A20 interaction
with accessory components of the Ub editing complex including
Ub, E2, and substrate, by using a combination of biophysical,
structural, and functional techniques. These studies reveal that
A20 ZnF4 selectively binds K63-linked Ub chains and that muta-
tions of residues in this interface result in impaired Ub editing and
Molecular Cell
Ubiquitin Binding to A20 ZnF4

decreased modulation of NF-kB signaling. In contrast, we Table 1. Data Collection and Refinement Statistics
demonstrate that both depolymerization of poly-Ub chains and
A20 ZnF4-Ub A20 ZnF4-Ub-UbcH5A
interactions with substrate and E2 are not dependent on A20
Data Collection
ZnF4.
Space group P21 P321
RESULTS Cell Dimensions
a, b, c (Å) 42.9, 170.2, 66.2 102.6, 102.6, 112.7
A20 ZnF4 Has Three Ub-Binding Sites a, b, g ( ) 90, 90.1, 90 90, 90, 120
Because of generally low affinities between Ub and its binding Resolution (Å) 50–2.5 25–3.4
partners, nuclear magnetic resonance (NMR) spectroscopy was Rsym 8.0 (40.9) 11.5 (75.0)
used to assess the association between recombinant A20
I/sI 15.3 (3.5) 16.8 (3.3)
ZnF3-4 and Ub and to demonstrate the formation of a stable
Completeness (%) 96.2 (98.2) 98.2 (99.8)
A20 ZnF-Ub complex in solution (Figures S1A–S1D, available on-
Redundancy 3.9 (3.9) 6.1 (6.0)
line). Backbone assignments of A20 ZnF3-4 enabled us to map
the Ub interaction area to ZnF4, because only resonances Refinement
belonging to ZnF4 were perturbed upon addition of Ub (Figures Resolution (Å) 43–2.5 25–3.4
S1C and S2). Complementary studies using labeled Ub confirmed Number of reflections 31,511 9,612
the existence of this interaction and revealed that A20 ZnF4 Rwork/Rfree 20.4 / 22.6 28.0 / 31.9
contacts Ub in the vicinity of residue D58 (Figures S1A and S1B). Number of Atoms
To better understand the association of A20 ZnF4 with Ub, we
Protein 6,602 3,984
determined the crystal structure of the A20 ZnF4-Ub complex to
Ligand/ion 8 2
a resolution of 2.5 Å (Table 1, Figure 1, and Figures S1E–S1K).
Despite participating in very different biological processes, the Water 127
core A20 ZnF4 structure resembles that of the ZnF from the B-factors
Rabex-5, a regulator of endosomal trafficking (Lee et al., 2006; Protein 55.5 86.5
Penengo et al., 2006) (Figures S1E and S1F). The crystal structure Ligand/ion 54.3 73.6
of A20 ZnF4 is consistent with the unpublished NMR structure Water 41.6
(Protein Data Bank [PDB] ID code 2EQE), with a root-mean- Rms deviations
square deviation (rmsd) of 0.8 Å across 99 main chain atoms,
Bond lengths (Å) 0.009 0.009
although careful comparison shows that both backbone and
Bond angles ( ) 1.054 1.013
side chain conformations differ in detail. Despite the one-to-one
X-ray diffraction data were collected on one crystal for each structure.
stoichiometry between A20 ZnF4 and Ub in the crystallographic
Values in parentheses are for the highest-resolution shell.
asymmetric unit, the crystal packing unexpectedly revealed
that A20 ZnF4 interacts with three separate Ub molecules (Ub1,
Ub2, and Ub3), each via a unique interface (Figures 1A and 1B). 94
The first Ub-binding site (site I) involves a cryptic inverted Ub- TEK96 sequence in the cell-cycle regulatory protein Securin
interacting motif (cIUIM) formed by the C-terminal a-helix of A20 (Jin et al., 2008). This motif has been implicated in the formation
ZnF4, which contacts the I44 hydrophobic patch of Ub1 of K11-linked poly-Ub chains leading to degradation of modified
centered at residues L8, I44, and V70 (Figures 1C and Fig- proteins (Jin et al., 2008). The A20 ZnF4-TEK box interface is
ure S1G). The binding mode of this interaction shows significant relatively small, burying 500 Å2 of solvent accessible surface
differences from what has previously been observed for UIM and area. Of this area, 270 Å2 is contributed by seven Ub residues,
IUIM motifs (Figures S1H–S1K). In particular, the position of the and 230 Å2 is contributed by 12 A20 ZnF4 residues. NMR spec-
invariant alanine in the consensus sequence defining the UIM- troscopy confirms that all three interfaces are formed in solution,
IUIM motif is occupied by the bulkier L626 from the a-helix of with the tightest binding observed at site II, with Kd of approxi-
A20 ZnF4. As a consequence, the A20 ZnF4 a-helix is tilted mately 40 mM (Figure 2A and Figure S1D). The binding of
30 compared to a typical a-helix orientation in UIM-IUIM-Ub mono-Ub to the A20 ZnF4 cIUIM (site I) is relatively weak and
interactions. was detected only when concentrations of Ub exceeded
The second A20 ZnF4-Ub contact (site II) is mediated by the 500 mM. Interactions with site III were observed at intermediate
50 s loop of Ub2 (residues E51–K63) and the N-terminal portion concentrations of Ub. The weak association measured between
of the A20 ZnF4 preceding the a-helix (Figure 1D). Comparison of A20 ZnF4 and mono-Ub is consistent with the affinities of other
the A20 ZnF4-Ub2 and Rabex-5 ZnF-Ub structures (Lee et al., Ub-binding domains (UBDs) for mono-Ub, which vary signifi-
2006; Penengo et al., 2006) reveals that this interaction is cantly with Kd values in the range of 1 to1,000 mM (Harper and
observed in both complexes (Figures S1E and S1F), while sites Schulman, 2006; Hicke et al., 2005; Hurley et al., 2006).
I and III are not present in the Rabex-5 ZnF-Ub complex.
The third A20 ZnF4-Ub interaction engages a Ub-binding A20 ZnF4 Selectively Recognizes K63-Linked
surface, which encompasses the TEK-box motif on Ub3 Poly-Ub Chains
(site III, Figures 1E and 1F). The TEK-box motif consists of a short Intriguingly, the A20 ZnF4-Ub complex suggests that A20 ZnF4
region of similarity around the 9TGK11 sequence in Ub and the selectively interacts with K63-linked poly-Ub, because the K63

Molecular Cell 40, 548–557, November 24, 2010 ª2010 Elsevier Inc. 549
 Molecular Cell
Ubiquitin Binding to A20 ZnF4

A B Figure 1. The A20 ZnF4-Ub Complex

Ub1 C
Reveals Three Ub-Binding Sites Including
K63 C a TEK-Box-Mediated Interaction
Site I
R74
* K63
Ub1 I44
(A) Three contacts between A20 ZnF4 and Ub are
Site II observed in the crystal lattice. A20 ZnF4 is colored
I44
C ZnF4 in blue with the Zn2+ cation shown as a gray
50 s Ub2
C sphere, and Zn2+-coordinating Cys (C607, C612,
L73
N C624, and C627) in red. The three Ubs are colored
ZnF4 TEK
K63
orange, yellow, and pink with I44 shown as red
C
Site III spheres. The positions of K63 and the last residue
I44
* Ub3 visible in the crystal structure of 76 amino acid Ub
are highlighted. The position of D58 in the 50 s loop
D
(residues E51–K63) is indicated with an asterisk.
N Ub2 ZnF4 Ub2 (B) Cartoon illustrating A20 ZnF4 interaction with
I44 K63 L626 R54 G53
Ub1, Ub2, and Ub3. Colors are as in (A). The region
T55
T625 E51 of Ub contacting A20 ZnF4 is labeled. The poten-
R74
* C624
Y59
D58
S57
tial for K63 linkage is indicated by dashed lines
Ub3 T604
Y614 between the C-terminus of both Ub2 and
N60 Q62
G603
F615 Ub3, and the K63 side chains of Ub1 and Ub2,
respectively.
(C) The A20 ZnF4 a-helix interaction with the I44
C E F ZnF4
ZnF4 hydrophobic patch on Ub (site I). The key contact-
C
ing residues are shown as sticks with oxygen and
N634
T604 K606 C607 nitrogen atoms colored red and blue, respectively.
C
C L71 K606 N Black dashed lines represent hydrogen bonding
R42 S605 G622
E633 K11 T9
S605 R608
typically observed in a-helical interactions with Ub.
K621
V70 Q49
C (D) Surface representation of the open-book view
I629
L8 of the protein interface between the A20 ZnF4
I44 K11 region preceding the a-helix and the Ub 50 s loop
I44 E34 (site II). Atoms within a distance of 3.5 Å, 4.0 Å,
L626 H68
G10
and 4.5 Å from the binding partner molecule are
colored red, orange and yellow, respectively.
ZnF4 Ub1 Ub3 T9 C
N (E) Ribbon representation of the A20 ZnF4 interac-
Ub3 tion with the Ub TEK-box (site III).
(F) Surface representation in the open-book view of
the protein interface shown in (E). Surface is
colored as in (D).
See Figure S1 for supporting material.

residues of Ub1 and Ub2 are near the C-termini of Ub2 and Ub3,
respectively (Figures 1A and 1B). We tested this hypothesis by
examining the interaction of A20 ZnF4 with mono-Ub, as well
as various forms of tri-Ub chains, in solution using size exclusion
chromatography and NMR spectroscopy (Figure 2 and Fig-
ure S2). Addition of a very high concentration of mono-Ub
(2 mM) to A20 ZnF4 resulted in perturbations of resonances
from all three sites, which is consistent with the observation of
three binding sites in the crystal structure. Addition of K63-linked
tri-Ub to A20 ZnF4 at concentrations far lower than those tested
with mono-Ub led to resonance perturbations at all three sites,
indicating that K63-linked tri-Ub interacts with all three sites (Fig-
ure 2D). The increase in affinity is K63-linkage-specific, because
NMR spectroscopy analysis using K48-linked, K11-linked, and
linear tri-Ub showed no increase in affinity (Figures S2B–S2E),
implying that only one Ub from these tri-Ubs can bind at
a time. K63-linked tri-Ub association is A20 ZnF4 specific,
because resonances belonging to A20 ZnF3 remained
unchanged upon addition of K63-linked tri-Ub to A20 ZnF3-4
(Figure S2B). Size exclusion chromatography also showed an
association between A20 ZnF4 and K63-linked tri-Ub, as the
two proteins coeluted together (Figure 2C). In contrast, size
exclusion chromatography showed no coelution of linear,
K11-, and K48-linked tri-Ub with A20 ZnF4, indicating lower-
affinity interactions. Although the conformation of linear and
K63-linked poly-Ub is similar, our NMR and chromatography
data reveal that A20 ZnF4 distinguishes between the two link-
ages and selectively binds to K63-linked tri-Ub with higher
affinity.
Mutations in the A20 ZnF4 Ub-Binding Sites Impair
A20 Function
In order to determine the importance of the enhanced affinity for
K63-linked poly-Ub, we mutated residues in the Ub-binding
sites. In vitro ubiquitination analysis using either wild-type A20
or A20 ZnF4 point mutants targeting each of the three Ub-
binding sites (site I: I629A, I629R; sites I and II: L626A, L626R;
site II: Y614A, F615A; site III: S605R, R608E, K606E, G622R)
showed that this surface is necessary for A20-mediated ubiqui-
tination. Full-length A20 carrying a single point mutation Y614A,
F615A, or L626R, or the double mutations Y614A, L626R or
Y614A, F615A failed to generate significant amounts of poly-
Ub chains when compared to wild-type A20 (Figure 3A). The
severity of the effect was comparable to mutating the cysteines
coordinating the Zn2+ ion, which has been shown to markedly
attenuate A20 activity (Figure S3A; Wertz et al., 2004). Mutations

550 Molecular Cell 40, 548–557, November 24, 2010 ª2010 Elsevier Inc.
Molecular Cell
Ubiquitin Binding to A20 ZnF4

A Site I Site II B
I629
G622
E633 E633 N634
E633 E630
N634 Y631 T617 105
I629 I629
# # G616
N620 L626 R632 #
N620 L626 G599

L626 T604 110

C627 T617
C624
G611
G616 Y614 T625
K621

T602

N (ppm)
Y614 T604 115
F615 T597
F615 S605 I629
N634
N620
R596 E633 E630
E619 Y631
F628 R601 D600
120

15
V613 #
L626 R632 # #

F623
Q593
C624 C627 A595
A594 C612 125
ZnF4 site Y614 K606
K606 C607
R608

N634 [Ub] I II III K609 C607 K635

F615
R608 60 µM - ++ + 130
G616 A610
150 µM - +++ ++
T617 K621 ZnF4 apo
500 µM ++ +++ +++
ZnF4 + mono-Ub
10 9 8 7 6
Site III 1
H (ppm)

C D
40 K63 apo I629
ZnF4 apo G622
N634
Absorptoin 280nM (mAU)

ZnF4 + K63 E633 E630

30 ZnF4 + Linear Y631 T617 105
ZnF4 + K11 # G616
ZnF4 + K48 L626 R632 # #
G599
20
T604 110
K621
G611
10 T625

N (ppm)
T602
115
T597
0 F615 S605 I629 N620
N634
E633 E630
R596
E619 Y631
10 11 12 13 14 15 F628 R601 D600 120

15
V613 #
# #
Volume (ml) L626 R632
Q593 F623
C624 C627 A595
kDa MS ZnF4 + K63-linked tri-Ub MS ZnF4 + Linear tri-Ub A594
C612 125
Y614 K606
64 R608
K635
50 K609 C607

36
130
A610
22
ZnF4 apo
16
ZnF4 + K63 tri-Ub
6 10 9 8 7 6
4 1
H (ppm)

Figure 2. The A20 ZnF4 Preferentially Interacts with K63-Linked Ub Chains

(A) The N-terminal segment of A20 ZnF4 binds Ub (site II) with higher affinity than the C-terminal a-helix portion (site I). Residues of A20 ZnF4 that changed in
intensity or position as 100 mM 15N-A20 ZnF4 was titrated with increasing concentrations of Ub are labeled in violet (60 mM), blue (150 mM), and green
(>500 mM). The three Ub-binding sites defined in the cocrystal structure are colored orange (site I), yellow (site II), and pink (site III). The inset table summarizes
the magnitude of occupancy of each binding site at various concentrations of Ub. Degree of site occupancy at the indicated concentrations was determined by
chemical shift perturbation of the resonances in an HSQC spectrum and was defined as no binding detected (-), low (+), moderate (++), and fully saturated (+++).
(B) Binding of Ub to all three sites on A20 ZnF4 is detected at very high concentrations of mono-Ub. 15N,1H-HSQC spectrum of 100 mM A20 ZnF4 is shown alone
(blue) and in the presence of 2 mM unlabeled mono-Ub (red). The insert shows an enlarged part of the spectrum corresponding to residues of the A20 ZnF4
a-helix. Broadening of these residues, especially L626 and I629 (shown by an arrow), is indicative of very weak Ub binding to site I. Additional peaks probably
representing Arg side-chains are denoted by #.
(C) A20 ZnF4 preferentially binds to tri-Ub with K63-linkage. Size exclusion chromatography analysis of ZnF4 interaction with various forms of tri-Ub (linear, K63-,
K48-, and K11-linked) is shown. Elution fractions analyzed by Coomassie-stained SDS-PAGE are indicated by the straight black line with sample load (designated
as S) in the lane next to the marker (designated as M). The coelution of A20 ZnF4 with K63-linked tri-Ub and a lack of coelution in the presence of linear tri-Ub
indicate a higher-affinity interaction between ZnF4 and K63-linked tri-Ub than with linear tri-Ub.
(D) A20 ZnF4 binds K63-linked Ub chains with higher affinity than mono-Ub. 15N,1H-HSQC spectrum of 100 mM A20 ZnF4 is shown alone (blue) and in the pres-
ence of 120 mM of unlabeled K63-linked tri-Ub (red). The inset shows an enlarged part of the spectrum corresponding to residues of the A20 ZnF4 a-helix. Broad-
ening of these residues, especially L626 and I629 (highlighted with an arrow), is indicative of Ub binding to site I.
See Figure S2 for supporting material.

Molecular Cell 40, 548–557, November 24, 2010 ª2010 Elsevier Inc. 551
 Molecular Cell
Ubiquitin Binding to A20 ZnF4

A C 0 0
wild type Y614A L626R F615A
Y614A Y614A A2 20 A2 20
L626R F615A M no A no A

A20 construct: – 250

148
250

250- 148
98
WB: biotin 148- 64 98
50 64
36 50
98- 36

160

Total linkage (fmol)

K63
K48
WB: FLAG 98- 120
K11

80

40

0
Linkage type
B
120 120
cyclin D2 c-FLIP
100 100
Relative expression (%)

Relative expression (%)

0 YA 0
A2 /- + A2

+Y R
A
AF
L
80 80

0 WT
A2 /- +
A2 /-

-/-
-

-
0

0
60 60

A2
40 40
WB: A20
20 20

0 WB: actin
0
-/-

-/-

0
R

A
A2

A2
AF
AL

AF
AL
0

0
A2

A2
T

T
+Y

+Y

+Y

+Y
+W

+W
-/-

-/-

-/-

-/-
-/-

-/-
0

0
A2

A2

A2

A2
0

0
A2

A2

D
A20-OTU A20-OTU A20-FL A20-FL
K48-linked K63-linked K11-linked Linear K48-linked K63-linked K11-linked Linear
kDa 0 0.5 1 2 5 21 0 0.5 1 2 5 21 kDa 0 0.5 1 2 5 21 0 0.5 1 2 5 21 hrs kDa 0 0.5 1 2 5 21 0 0.5 1 2 5 21 kDa 0 0.5 1 2 5 21 0 0.5 1 2 5 21 hrs
98 98 98 98
64 64 64 64
50 50 50 50
36 4Ub 36 36 4Ub
36
3Ub 3Ub
22 22 22 22
2Ub 2Ub
16 16 16
16

1Ub 1Ub
6 6
6 6

Figure 3. The Ub-Binding Site on A20 ZnF4 Is Required for Ub Chain Polymerization and for Modulation of NF-kB Signaling
(A) Mutations to the Ub-binding sites in A20 ZnF4 impair A20-dependent ubiquitination. Full-length A20 protein wild-type (WT), the single point mutants
Y614A or L626R or F615A, and the double mutants Y614A, L626R and Y614A, F615A were expressed in 293T cells and assayed with UbcH7 for the
ability to auto-ubiquitinate A20. Only A20 with an intact Ub-binding site II on A20 ZnF4 was able to promote effective auto-ubiquitination. Mutations
to this site result in reduced ubiquitination comparable to mutations of the Zn2+ ion coordinating cysteines (Figure S4A). The A20 double
mutant Y614A, F615A is as deficient in activity as control assays lacking A20, indicating the critical importance of Ub-binding site II on ZnF4 for A20
function.
(B) Complementation studies to monitor NF-kB response. A20 null MEFs were transfected with vector, wild-type, Y614A, L626R (YALR) or the double
mutant Y614A, F615A (YAFA) A20, and the relative expression of NF-kB responsive genes cyclin D2 and c-FLIP was monitored by real-time polymerase
chain reaction (PCR) after activation of cells with 100 ng/mL human TNF-a for 45 min. Cyclin D2 and c-FLIP mRNA levels were normalized against b-actin
expression. The mean of triplicate experiments is shown ± SD. Western blots quantitating A20 levels are shown at right. Similar amounts of wild-type or
mutant A20 are present, indicating that the mutations do not affect expression or stability of A20. Error bars in (B) and (C) are the standard deviation of
triplicate measurements.
(C) Recombinant A20 and UbcH5A catalyzed ubiquitination generates K11, K48, and K63 linkages. Coomassie blue-stained gel of Ub chains is generated
by in vitro ubiquitination reaction using A20 purified from 293T cells compared to a control reaction without A20. The red box marks the gel region that
was excised and subjected to in-gel trypsin digestion followed by Ub-absolute quantification (Blankenship et al., 2009; Kirkpatrick et al., 2006); the red
arrowhead points to the band corresponding to the full-length A20 protein. The corresponding western-blot analysis of Ub incorporation into poly-Ub
chains is shown. A higher amount of K11 linkage was detected than the K48 and K63 linkages.

552 Molecular Cell 40, 548–557, November 24, 2010 ª2010 Elsevier Inc.
Molecular Cell
Ubiquitin Binding to A20 ZnF4
targeting sites I and III had a less pronounced effect (summarized
in Figure S3B), which is consistent with these sites contributing
less to mono-Ub binding but still being important for chain
recognition. NMR analysis of recombinant A20 ZnF4 carrying
some of these mutations is consistent with proper folding of
the domain, thereby indicating that the impairment in ubiquitina-
tion is not due to a general folding defect (data not shown).
To evaluate whether these mutations affect the ability of A20 to
regulate NF-kB signaling, we introduced the A20 ZnF4 double
mutants (Y614A, L626R referred to as YALR, or Y614A, F615A
referred to as YAFA) into A20 / murine embryonic fibroblast
(MEF) cells. Cells without functional A20 have increased expres-
sion of NF-kB reporter genes cyclin D2 and c-FLIP in response to
stimulation with TNF-a (Honma et al., 2009). Transfection with
DNA encoding wild-type A20 reduced relative expression of
these genes by 50%. However, transfection with A20
harboring mutations in the Ub-binding sites (YALR or YAFA)
was unable to attenuate NF-kB signaling (Figure 3B). Because
expression of wild-type and A20 mutants was comparable, the
mutations are unlikely to have adversely affected the stability
of A20 (Figure 3B, right panel). While these assays do not differ-
entiate between impaired Ub ligase activity and the inability to
bind K63-linked poly-Ub, collectively these results indicate that
the capacity of A20 ZnF4 to attenuate NF-kB signaling is depen-
dent on the Ub-binding surface of A20 ZnF4. Quantitative mass
spectrometry examination of the in vitro A20-mediated ubiquiti-
nation unexpectedly revealed the generation of both K48- and
K11-linkages in an A20-dependent manner (Figure 3C). These
data suggest that linkages associated with substrate downregu-
lation via proteosome-mediated degradation are the predomi-
nant result of A20-mediated ubiquitination.
The preference of A20 ZnF4 for K63-linkages may serve to
localize A20 to the proximity of substrates and/or it may facilitate
depolymerization of K63-linked poly-Ub chains to mono-Ub for
use in the resynthesis of a poly-Ub degradation signal. To further
explore the importance of the A20 ZnF domain in the deubiquiti-
nase step of Ub editing, we assessed the ability of full-length or
OTU-only A20 constructs to disassemble linear, K63-, K48-, and
K11-linked poly-Ub chains. These studies show that in vitro,
both full-length and OTU-only constructs are able to disas-
semble K63-, K48-, and K11-linked poly-Ub chains but not linear
poly-Ub chains (Figure 3D). Librated mono-Ub would then be
available for incorporation into new chains (Figures S3C and
S3D). However, while the entire A20 ZnF region, including
ZnF4, is dispensable for deubiquitinase activity, both the A20
OTU and A20 ZnF4 domains are required for the Ub editing
process because targeted mutations to either domain result in
stabilization of the A20 substrate RIP1 in a cellular context (Wertz
et al., 2004). Moreover, because the physiological role of A20 is
to facilitate downregulation of NF-kB activity by removal of
substrate from signaling complexes, additional interactions
may be present in vivo that modulate which Ub-linkages are
subject to depolymerization by A20 DUB activity.
(D) A20 OTU does not depolymerize linear poly-Ub. Depolymerization time course for tetra-Ub with linear, K48, K63, and K11 linkages in the presence of
full-length WT A20 (right) or a construct containing only the A20 OTU domain (left) is shown. K48-, K63-, and K11-linked poly-Ub chains are broken
down.
See Figure S3 for supporting material.
Molecular Cell 40, 548–557, November 24, 2010 ª2010 Elsevier Inc. 553
UbcH5A and RIP Interact with Regions of A20 Distinct
from ZnF4
Full-length A20 shows clear specificity for the E2 UbcH5A and
UbcH7 over other tested E2s, including the closely related
UbcH5B and UbcH5C (Wertz et al., 2004). To determine whether
or not ZnF4 contributes to this specificity, we examined the inter-
action between UbcH5A and A20 ZnF3-4 by NMR spectroscopy.
Surprisingly, the 15N,1H-HSQC spectrum of UbcH5A did not
change upon addition of excess A20 ZnF3-4, indicating that
the proteins do not interact appreciably under these conditions
(Figure S4). In contrast, the 15N,1H-HSQC spectra of Ub changed
upon addition of a slight excess of UbcH5A (Figure 4A). Chemical
shift mapping showed that the I44 hydrophobic patch on Ub was
responsible for UbcH5A binding (Figure 4B). Further changes in
the 15N,1H-HSQC spectra of Ub were observed upon addition of
A20 ZnF3-4, thereby suggesting that a trimeric complex was
formed (Figure 4C). The A20 ZnF3-4 interaction area on Ub
resides away from the UbcH5A contacting surface and was
mapped to the 50 s loop (Figure 4D). Therefore, in solution Ub
mediates the interaction between A20 ZnF3-4 and UbcH5A by
acting as a molecular bridge.
To further characterize this interaction, we determined the
crystal structure of a noncovalent ternary complex of A20
ZnF4, Ub, and UbcH5A to a 3.4 Å resolution (Table 1). Consistent
with the NMR data, this structure revealed minimal direct interac-
tion between A20 ZnF4 and UbcH5A (Figure 4E). In this ternary
complex, the 50 s loop of Ub associates with the N-terminal
region of A20 ZnF4 and recapitulates the interaction seen at
site II of the A20 ZnF4-Ub complex. Simultaneously, the ‘‘back-
side’’ of UbcH5A binds the I44 hydrophobic patch on Ub, as was
observed in the NMR structure of the UbcH5C-Ub complex
(Brzovic et al., 2006) and in the recent crystal structure of the
UbcH5BUb covalent complex (Sakata et al., 2010). Although
the backside interaction between UbcH5B/C and Ub has been
shown to be important for the processivity of several E3 ligases
(Brzovic et al., 2006; Capili and Lima, 2007; Duda et al., 2007; Ed-
dins et al., 2006; Knipscheer et al., 2007; Sakata et al., 2010), its
relevance for A20 ligase activity is not clear. A model of the
charged thioester-linked UbcH5AUb and A20 ZnF4 ternary
complex based on charged Ubc13Ub (Eddins et al., 2006)
also indicates no direct contact between A20 ZnF4 and UbcH5A
(Figure 4F). Thus, the segment of A20 responsible for E2 speci-
ficity probably lies outside ZnF4.
In order to map the molecular determinants for E2 specificity
within A20, we monitored the auto-ubiquitination of full-length
A20 as well as truncated versions of the ZnF region (ZnF1-7,
ZnF1-4, ZnF3-4, and ZnF4-7) in the concomitant presence of
UbcH5 homologs (Figure 5A). Only segments of A20 containing
ZnF5-7 retained specificity for UbcH5A. In contrast, fragments
containing ZnF3-4 and ZnF1-4 lacked E2 specificity and worked
equally well with UbcH5A, UbcH5B, and UbcH5C. As a control,
we also monitored the ability of these fragments to facilitate
ubiquitination in conjunction with UbcH7. All fragments promote
 Molecular Cell
Ubiquitin Binding to A20 ZnF4

A B E
C
105

L73 Ub
110
T9
K11 L71 Q40
ZnF4

N (ppm)
115
V70
K6 R74 I44
L69
120
S22

15
I44 R42 90o
Q49
125 G47
A46 L50

130
F46 K48
Ub apo UbcH5A C85
Ub + UbcH5A
10 9 8 7 6
1
H (ppm)

C D F
C
105

L73
110 T9 Ub
K11 L71 Q40
N (ppm)

115
V70 R74
K6
L69 R42
120
15

I44 Q49
D52
125 A46 G47 L50 E51
F46 K48 ZnF4 UbcH5A
Y59 R54
130 T55
Ub apo S57 D58
Ub + UbcH5A + ZnF3-4 Q62
10 9 8 7 6
1
H (ppm)

Figure 4. A20 ZnF3-4 Is Not Sufficient for E2 Specificity

(A) Overlay of the 15N,1H-HSQC spectrum of 100 mM Ub alone (blue) and in the presence of excess unlabeled UbcH5A (red). The differences between these two
spectra indicate that these two proteins interact under these conditions.
(B) Surface of Ub with residues that exhibited the most significant chemical shift perturbations upon addition of UbcH5A shown in green. The residues whose
intensity was less than 50% of the reference are colored and labeled. These residues correspond to surfaces of Ub that have been shown to interact with UbcH5C
by NMR (Brzovic et al., 2006).
(C) Surface of Ub as in (B) after addition of both UbcH5A and A20 ZnF3-4. Additional changes to the Ub spectra in comparison to (A) indicate that both UbcH5A
and A20 ZnF3-4 are simultaneously interacting with Ub.
(D) Surface of Ub with residues that exhibited the most significant chemical shift perturbations after addition of UbcH5A and A20 ZnF3-4 colored green and blue,
respectively. The residues whose intensity was less than 50% of the reference are colored and labeled. Different sets of residues shift upon addition of UbcH5A
(green) and A20 ZnF3-4 (blue), suggesting that both proteins can bind simultaneously to Ub.
(E) Crystal structure of the noncovalent trimeric complex between A20 ZnF4 (blue), Ub (yellow), and UbcH5A (green). No significant contact between A20 ZnF4
and UbcH5A is observed. Ub serves as a bridge by interacting with A20 ZnF4 via the 50 s loop and with UbcH5A through the I44 hydrophobic patch. The critical
residues for the backside interaction are shown in red. At right, a 90 rotation of the complex better illustrates the minimal interaction between ZnF4 and UbcH5A.
(F) Ub mediates interaction between A20 ZnF4 and UbcH5A. This is a model of a possible trimeric complex between A20 ZnF4 (cyan), Ub (yellow), and UbcH5A
(green) with a covalent link between the C-terminal tail of Ub and catalytically active cysteine of UbcH5A. The model was generated by superimposing the
UbcH5A structure onto a structure of Ubc13 covalently linked to Ub via a thioester bond (PDB code 2GMI) (Eddins et al., 2006). The contact between A20
ZnF4 and Ub is based on site II interaction in the A20 ZnF4-Ub crystal structure involving the Ub 50 s loop.
See Figure S4 for supporting material.

poly-ubiquitination, consistent with the data for the Escherichia
coli-derived material (Figure 5A).
Finally, we investigated whether A20 ZnF4 is directly involved
in substrate recognition. Because A20 modifies K63-linked ubiq-
uitinated RIP1 to target it for proteosomal degradation, there
probably is a RIP1 recognition site in A20 that would distinguish
RIP1 from other cellular factors. In our previous work, we showed
that full-length A20, but not constructs that lack A20 ZnF4 or
have mutations to A20 ZnF4, can facilitate RIP1 ubiquitination
leading to proteasome-mediated degradation. To determine
whether A20 ZnF4 contributes to recognition of RIP1 in addition
to facilitating ubiquitination, we conducted binding studies
between endogenous RIP1 and various A20 deletion fragments.
These studies determined the region around and including A20
ZnF1 (residues 386–453) to be essential for RIP1 binding (Figures
5B and 5C). The OTU domain and A20 ZnF2-3 segment also
contribute to RIP1 binding, but to a lesser extent. Thus, different
regions of A20 make distinct and essential contributions to the
Ub editing process: (1) The OTU domain confers DUB activity,
(2) ZnF1 and surrounding residues promote RIP1 binding, (3)

554 Molecular Cell 40, 548–557, November 24, 2010 ª2010 Elsevier Inc.
Molecular Cell
Ubiquitin Binding to A20 ZnF4

A WB: anti-biotinylated Ub B OTU ZnF4

A2 -N
C
MT MT ΔN ΔC1 ΔC2 ΔC3 ΔC4 A20-N

W tor
WT

0-
0
A2T
c
E2 enzyme: UbcH5A UbcH5B UbcH5C UbcH7

ve
IP: FLAG 98-
A20 construct: – – – – WB: RIP1

98-
full length IP: FLAG
WB: FLAG 64-
98-
50-

C-terminus

50- C D
H5A
A20 WT 1 OTU 1 2 3 4 5 6 7 790
A20 OTU MT 1 OTU C->A 1 2 3 4 5 6 7 790 Ub 7
ZnF4-7 Zn7
A20 ZnF4 MT 1 OTU 1 2 3 4 5 6 7 790
A20 ΔN 68 OTU 1 2 3 4 5 6 7 790 OTU 1
Zn1 4
Zn4 6
Zn6

22- 5
Zn5
A20 ΔC1 1 OTU 1 2 3 4 659 2
Zn2 3
Zn3

A20 ΔC2 1 OTU 1 2 3 587

A20 ΔC3 1 OTU 1 2 521
ZnF1-4
A20 ΔC4 1 OTU 1 453

36- A20-N 1 OTU 386 E

A20-C 370 1 2 3 4 5 6 7 790
RIP1 Ub
ZnF3-4 OTU
1 4 7
16- 2 3 5 6

Figure 5. The RIP Recognition Site Is Distinct from A20 ZnF4

(A) Selectivity for UbcH5A resides in the ZnF5-7 region of A20. Auto-ubiquitination of full-length A20 and various ZnF domain segments (recombinantly produced
in E. coli) was tested in the presence of UbcH5A, UbcH5B, and UbcH5C E2 proteins in vitro. At right, the same fragments of A20 (expressed in 293T cells) with
UbcH7 are shown.
(B) A20 ZnF1 is essential for RIP1 binding. Immunoprecipitation assays of the association of endogenous RIP with various FLAG-tagged A20 deletion fragments
are shown. The N terminus containing the OTU domain and A20 ZnF1-3 segments contribute to RIP binding, with residues 386–453 that contain ZnF1 being
indispensable.
(C) Schematic representation of A20 constructs (deletion mutations) used in RIP immunoprecipitations shown in (B).
(D) Cartoon representation of the A20 interaction with charged UbcH5A; ZnF5-7 contacts UbcH5A while ZnF4 interacts with Ub. ZnFs are shown as circles and
are numbered 1 to 7.
(E) Cartoon representation of the active RIP recognition by A20; ZnF1 binds substrate directly while ZnF4 interacts with the K63-linked Ub chain (yellow). ZnFs are
shown as circles and are numbered 1 to 7.

ZnF4 binds Ub and facilitates poly-ubiquitination, and (4) ZnF5-7
regulates UbcH5A specificity (Figures 5D and 5E).
DISCUSSION
Numerous cellular processes are regulated through ubiquitina-
tion and deubiquitination and thus are critically dependent on
the interaction between Ub and its binding partners (Harper
and Schulman, 2006). Precise regulation of the NF-kB pathway,
which is essential for proper cellular homeostasis, is critically
dependent on the presence of active A20 to facilitate Ub editing.
Our studies reveal that A20 ZnF4 is a K63-linkage recognition
module and also show that mutations to this module result in
the inability of A20 to properly regulate NF-kB signaling.
The crystal structure of A20 ZnF4 bound to mono-Ub, NMR,
and other solution-phase characterization of A20 ZnF4 interac-
tions with defined-linkage poly-Ub reveal an extended three-
interface binding site for K63-linked tri-Ub. One of the interfaces
engages the Ub surface surrounding the TEK-box, revealing
a role for this surface in mediating protein-protein interactions.
The interaction of A20 ZnF4 with K63-linked poly-Ub is of higher
affinity and differs from observations made in the structural anal-
ysis of NF-kB essential modulator (NEMO) interaction with linear
and K63-linked di-Ub, where a-helical elements from NEMO
contact the tandem Ubs (Lo et al., 2009; Rahighi et al., 2009).
The recent complex structures of TAB2 and TAB3 Npl4 zinc
finger (NZF) with K63-linked di-Ub show that some ZnFs are
capable of binding more than one Ub; however, the mode of
interaction differs significantly from that seen here with A20
ZnF4 (Kulathu et al., 2009; Sato et al., 2009). In the case of
TAB2 and TAB3, both distal and proximal Ubs use the I44
surface to interact with distinctive surfaces on NZF and the
binding modes differ from UIM and IUIM because NZF lacks
an a-helix. The interaction with the distal Ub is mediated by
zinc-coordinating loops through a conserved Thr-Phe motif,
whereas the proximal Ub binds to the TAB2 NZF surface around
L681 (Kulathu et al., 2009; Sato et al., 2009). Therefore, A20 ZnF4
and TAB NZF, as well as the K63-linkage-specific antibody
(Newton et al., 2008), recognize K63-linked chains by binding
specific Ub surfaces that are present in this linkage form, rather
than directly interacting with the K63-linked isopeptide bond.
Our data also show that ZnF4 does not directly interact with
UbcH5A and that the adjacent regions (ZnF5-7) are required
for E2 selectivity. Previous studies suggest that A20 operates
in vivo as part of a multiprotein complex that includes TAXBP-1
and the E3 ligases Itch and RNF11 (Coornaert et al., 2008; Iha

Molecular Cell 40, 548–557, November 24, 2010 ª2010 Elsevier Inc. 555

et al., 2008; Shembade et al., 2007, 2008, 2009), implicating an
additional level of complexity in A20 modulation of the NF-kB
pathway. A recent study linking A20 ZnF4-dependent downre-
gulation of Ubc13 and UbcH5C to inhibition of NF-kB signaling
(Shembade et al., 2010) is consistent with our observations
regarding the importance of A20 ZnF4.
In summary, structural and functional data presented here link
A20 ZnF4 directly to three of the unique functions of A20: K63-
linked poly-Ub chain recognition, A20-mediated generation of
poly-Ub chains, and NF-kB signal modulation. In contrast, we
show that A20 ZnF4 is not required for other critical aspects of
the Ub editing process such as interaction with RIP1 and depo-
lymerization of poly-Ub chains, or for direct interactions with E2
enzymes. The affinity of A20 ZnF4 for K63-linked poly-Ub chains
suggests that this interaction may recruit A20 to K63-linked poly-
ubiquitinated substrates within activated signaling complexes.
Subsequent disassembly of the K63-linked poly-Ub chains by
the A20 OTU domain could facilitate the resynthesis of degrada-
tive poly-Ub chains on substrates. Additional interactions with
other reported binding partners may also modulate A20 activity.
Our studies provide a molecular basis to explain why appropriate
regulation of NF-kB signaling is dependent on functional A20 and
why Ub editing, which is central to A20 function, is critically
dependent on A20 ZnF4.
EXPERIMENTAL PROCEDURES
A detailed description of all experimental procedures is provided in the
Supplemental Information.
Protein Chemistry, In Vitro Ubiquitination, and Linkage Analysis
by Mass Spectrometry
Recombinant proteins were expressed in E. coli as described in the Supple-
mental Information. Simultaneous in vitro ubiquitination and deubiquitination
assays were performed at 30 C for the indicated time periods. Samples
were western-blotted for A20 or for K48- or K63-linked poly-Ub chains.
Peptides were separated by reverse-phase high-performance liquid chroma-
tography (HPLC), and analyses were performed using either multiple-reaction
monitoring on a QTrap4000 mass spectrometer or narrow-window-extracted
ion chromatograms from an LTQ-Orbitrap. Binding studies employing NMR
spectroscopy used 15N,1H-HSQC spectra of isotope-labeled A20 ZnF4, A20
ZnF3-4, Ub, or UbcH5A and an unlabeled binding protein partner.
Structure Determination
Crystals of the A20 ZnF4-Ub and A20 ZnF4-Ub-UbcH5A complexes diffracted
to 2.5 Å and 3.4 Å resolution, respectively (Table 1). Data were collected at
Advanced Light Source (ALS) beamlines 5.0.1 and 5.0.2. The structure of the
ZnF4-Ub complex was solved using the refined coordinates of Ub (PDB ID
code 1UBQ) (Vijay-Kumar et al., 1987) and Rabex-5 ZnF (PDB ID code 2FID)
(Lee et al., 2006). The asymmetric unit contained eight A20 ZnF4 molecules
and eight Ub molecules, with each A20 ZnF4 contacting three Ubs. For the
A20 ZnF4-Ub-UbcH5A complex, the refined coordinates of ZnF4, Ub (PDB ID
code 1UBQ), and UbcH5C (PDB ID code 1X23) were used as search models.
The crystallographic asymmetric unit contained two copies of the ternary
complex. All structural figures were made with PyMOL (http://www.pymol.org/).
ACCESSION NUMBERS
Atomic coordinates for the reported crystal structures have been deposited
with the PDB and assigned accession codes of 3OJ3 (A20 ZnF4-Ub complex)
and 3OJ4 (A20 ZnF4-Ub-UbcH5A complex).
556 Molecular Cell 40, 548–557, November 24, 2010 ª2010 Elsevier Inc.
Molecular Cell
Ubiquitin Binding to A20 ZnF4
SUPPLEMENTAL INFORMATION
Supplemental Information includes four figures and Supplemental Experi-
mental Procedures and can be found with this article online at doi:10.1016/j.
molcel.2010.10.009.
ACKNOWLEDGMENTS
We thank our colleagues Deanne Compaan, Ping Wu, Karen M. O’Rourke, Fer-
nando Bazan, Andreas Lingel, Jeremy Flinders, Brent Appleton, Till Maurer,
Erin Dueber, Anna Fedorova, and Allison Bruce for reagents and technical
advice. The Advanced Light Source and the Berkeley Center for Structural
Biology are supported by the Department of Energy, National Institutes of
Health, and the National Institute of General Medical Sciences. All authors
are or were employees of Genentech, Inc.
Received: December 18, 2009
Revised: July 7, 2010
Accepted: August 27, 2010
Published: November 23, 2010
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