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PDBsum entry 2bf8
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References listed in PDB file
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Key reference
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Title
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Sumo modification of the ubiquitin-Conjugating enzyme e2-25k.
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Authors
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A.Pichler,
P.Knipscheer,
E.Oberhofer,
W.J.Van dijk,
R.Körner,
J.V.Olsen,
S.Jentsch,
F.Melchior,
T.K.Sixma.
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Ref.
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Nat Struct Mol Biol, 2005,
12,
264-269.
[DOI no: ]
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PubMed id
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Abstract
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Post-translational modification with small ubiquitin-related modifier (SUMO)
alters the function of many proteins, but the molecular mechanisms and
consequences of this modification are still poorly defined. During a screen for
novel SUMO1 targets, we identified the ubiquitin-conjugating enzyme E2-25K
(Hip2). SUMO attachment severely impairs E2-25K ubiquitin thioester and
unanchored ubiquitin chain formation in vitro. Crystal structures of
E2-25K(1-155) and of the E2-25K(1-155)-SUMO conjugate (E2-25K(*)SUMO) indicate
that SUMO attachment interferes with E1 interaction through its location on the
N-terminal helix. The SUMO acceptor site in E2-25K, Lys14, does not conform to
the consensus site found in most SUMO targets (PsiKXE), and functions only in
the context of an alpha-helix. In contrast, adjacent SUMO consensus sites are
modified only when in unstructured peptides. The demonstration that secondary
structure elements are part of SUMO attachment signals could contribute to a
better prediction of SUMO targets.
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Figure 2.
Figure 2. Sumoylation of E2-25K inhibits ubiquitin thioester
formation. (a) Ubiquitin (Ub) chain formation. SUMO-modified
(*S) or unmodified E2-25K (1.5  g;
arrowheads), 8 g
ubiquitin and 100 ng ubiquitin-E1 were incubated for indicated
times with an ATP-regenerating system at 37 °C. Analysis was
done by Coomassie blue staining. (b) Ubiquitin thioester
formation. SUMO-modified or unmodified E2-25K (1 g),
2 g
ubiquitin, 130 ng ubiquitin-E1 and ATP were incubated for 1 h at
30 °C. Analysis was done by immunoblotting. Left, nonreducing
conditions allow detection of thioester; Right, reducing
conditions. (c) Ubiquitin transfer. Equal amounts of E2-25K and
E2-25K*SUMO ubiquitin thioesters were generated by incubating 10
g
E2-25K, 10 g
ubiquitin K48R and 1.25 g
ubiquitin-E1 with ATP at 37 °C for 12 and 70 min, respectively.
Ubiquitin-E1 was inhibited by EDTA, and wild-type ubiquitin was
added to allow di-ubiquitin formation. Immunoblotting with
anti-E2-25K (top) or anti-ubiquitin (bottom) followed. (d)
Ubiquitin thioester formation of full-length and truncated
E2-25K. 1 g
of SUMOylated or unmodified full-length (top) or truncated
E2-25K(1 -155) (bottom) was incubated with ATP, 1 g
ubiquitin and indicated concentrations of ubiquitin-E1 for 30
min at 30 °C. Analysis under nonreducing conditions was done by
immunoblotting. Asterisk, ATP-independent unspecific band. (e)
Experiment was done as in d but in a time course using 6 ng E1
for E2-25K(1 -155) and 100 ng E1 for full-length E2-25K.
Asterisk, isopeptide linked ubiquitin to E2-25K thioester; #,
di-ubiquitin.
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Figure 4.
Figure 4. SUMO target sites are defined by their structural
context. (a) Position of four lysines in the N terminus of
E2-25K. (b) In the folded protein, Lys14 is the preferred
substrate. Wild type (WT) or indicated E2-25K mutant (500 ng), 1
g
SUMO1, 300 ng Aos1 -Uba2, 500 ng Ubc9 and ATP were incubated for
30 min at 30 °C. (c) In an unfolded peptide, Lys10 is preferred.
Indicated peptide (20 g)
1.5 g
SUMO-E1, 340 ng Ubc9, 15 g
SUMO and ATP were incubated for 0 -5 h. (d) In folded E2-25K
protein, Lys14 is preferred. Wild type or indicated F2-25K
mutant (7.5 g)
12 g
SUMO-E1, 425 ng Ubc9, 20 g
SUMO and ATP were incubated for 0 -2 h at 37 °C.
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The above figures are
reprinted
by permission from Macmillan Publishers Ltd:
Nat Struct Mol Biol
(2005,
12,
264-269)
copyright 2005.
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