Journal of the American Chemical Society
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sults were observed for the Nedd4ꢀ1 E554A F707A muꢀ
polyUb chains. E3 ligase inhibitors with this mechanism
of action were not previously known and this work outꢀ
lines a conceptually new design strategy for E3 ligase
inhibitors. Taken together, our studies provide fundaꢀ
mental insights into the HECT E3 enzymatic mechanism
and represent an important case study in the emerging
area of E3 ligase inhibitor discovery.19 Further studies of
Nedd4ꢀ1 inhibitors will be reported in due course.
tant (Figure S20). These experiments prove for the first
time that Nedd4-1 is processive, and when the non-
covalent interaction between the N-lobe and Ub is dis-
rupted by compound 3 or the F707A mutation the en-
zyme becomes distributive. Previously, it was assumed,
but not rigorously proven, that HECT E3s are proces-
sive and not distributive enzymes.
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Comment [S4]: Added in response to reviewer
3’s comments.
Since endogenous intracellular deubiquitinating enꢀ
zymes (DUBs) reverse protein ubiquitination, we hyꢀ
pothesized that distributive Nedd4ꢀ1 would be more susꢀ
ceptible to antagonism by DUBs than processive Nedd4ꢀ
1. To test this hypothesis, full length Nedd4ꢀ1 E554A
with or without compound 3 bound and the Nedd4ꢀ1
E554A F707A mutant were allowed to ubiquitinate Fluꢀ
Wbp2 in the presence of the DUB USP8. In vitro, USP8
can disassemble K48ꢀ and K63ꢀlinked polyUb chains,18
while Nedd4ꢀ1 predominantly makes K63ꢀlinked
chains.11 Remarkably, we observed that untreated
Nedd4ꢀ1 robustly polyubiquitinated FluꢀWbp2 in the
presence of USP8 after 30 min. However, neither comꢀ
pound 3ꢀtreated Nedd4ꢀ1 nor the F707A mutant were
able to consume FluꢀWbp2 or build ≥Ub4 chains on Fluꢀ
Wbp2 in the presence of USP8, even though they conꢀ
sumed FluꢀWbp2 in the absence of USP8 (Figure 4,
S21). Since distributive Nedd4ꢀ1 dissociates from the
substrate in between rounds of Ub addition to the growꢀ
ing polyUb chain, this provides an opportunity for the
DUB to hydrolyze the Ub chain before another Ub can
be added to it by Nedd4ꢀ1. Processive catalysis appears
to be a necessary condition for the formation of ≥Ub4
chains on the substrate in the presence of DUB.
ASSOCIATED CONTENT
Supporting information available free of charge via the
Internet at http:/pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
aꢀstatsyuk@northwestern.edu
ACKNOWLEDGMENT
Funding was provided from Northwestern University
(A.V.S.), Pew Charitable Trusts (A.V.S.), National Instiꢀ
tutes of Health Grants R01GM115632 (A.V.S.), GM58518
(A.C.R.), T32GM105538 (S.G.K.), and F32GM105339
(A.T.S.), Deutsche Forschungsgemeinschaft (DFG) Sp
1476/1ꢀ1 (I.S.), Chemistry of Life Processes Institute Lamꢀ
bert Fellowship (Z.X.), and the ACS Medicinal Chemistry
Fellowship (S.G.K.). We thank Simona Polo (IFOM, Miꢀ
lan, Italy) and Jon Huibregtse (University of Texas) for
Nedd4ꢀ1 and Wbp2ꢀCꢀK222 constructs.
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Figure 4. Ubiquitination of FluꢀWbp2 (100 nM) by a full length
Nedd4ꢀ1 (150 nM) at different time points in the presence of the
deubiquitinase USP8 (200 nM) shows that distributive Nedd4ꢀ1 is
inhibited by USP8, but processive Nedd4ꢀ1 is not. Lane 1: DMSO
treated Nedd4ꢀ1 full length E554A, lane 2: compound 3 treated
Nedd4ꢀ1 full length E554A, lane 3: Nedd4ꢀ1 full length E554A
F707A, and lane 4: no ATP control.
In summary, we report the first rigorous proof that the
HECT E3 Nedd4ꢀ1 is a processive enzyme, and that disꢀ
rupting nonꢀcovalent binding of Ub to the Nꢀlobe of
Nedd4ꢀ1 switches Nedd4ꢀ1 to a distributive enzyme.
Furthermore, we report the discovery and structural
characterization of a covalent Nedd4ꢀ1 inhibitor that
targets this processive site and switches Nedd4ꢀ1 from a
processive to a distributive mechanism. We also show
that introducing a DUB antagonist into the assay augꢀ
ments the inhibitory effect of compound 3 on Nedd4ꢀ1
while the untreated enzyme is still able to build long
(16) Kim, H. C.; Steffen, A. M.; Oldham, M. L.; Chen, J.; Huiꢀ
bregtse, J. EMBO Rep., 2011, 12, 334.
(17) Mari, S.; Ruetalo, N.; Maspero, E.; Stoffregen, M. C.;
Pasqualato, S.; Polo, S.; Wiesner, S. Structure, 2014, 22, 1639.
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