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PDBsum entry 3dvg
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Immune system
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PDB id
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3dvg
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Contents |
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210 a.a.
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221 a.a.
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75 a.a.
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76 a.a.
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References listed in PDB file
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Key reference
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Title
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Ubiquitin chain editing revealed by polyubiquitin linkage-Specific antibodies.
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Authors
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K.Newton,
M.L.Matsumoto,
I.E.Wertz,
D.S.Kirkpatrick,
J.R.Lill,
J.Tan,
D.Dugger,
N.Gordon,
S.S.Sidhu,
F.A.Fellouse,
L.Komuves,
D.M.French,
R.E.Ferrando,
C.Lam,
D.Compaan,
C.Yu,
I.Bosanac,
S.G.Hymowitz,
R.F.Kelley,
V.M.Dixit.
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Ref.
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Cell, 2008,
134,
668-678.
[DOI no: ]
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PubMed id
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Abstract
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Posttranslational modification of proteins with polyubiquitin occurs in diverse
signaling pathways and is tightly regulated to ensure cellular homeostasis.
Studies employing ubiquitin mutants suggest that the fate of polyubiquitinated
proteins is determined by which lysine within ubiquitin is linked to the C
terminus of an adjacent ubiquitin. We have developed linkage-specific antibodies
that recognize polyubiquitin chains joined through lysine 63 (K63) or 48 (K48).
A cocrystal structure of an anti-K63 linkage Fab bound to K63-linked diubiquitin
provides insight into the molecular basis for specificity. We use these
antibodies to demonstrate that RIP1, which is essential for tumor necrosis
factor-induced NF-kappaB activation, and IRAK1, which participates in signaling
by interleukin-1beta and Toll-like receptors, both undergo polyubiquitin editing
in stimulated cells. Both kinase adaptors initially acquire K63-linked
polyubiquitin, while at later times K48-linked polyubiquitin targets them for
proteasomal degradation. Polyubiquitin editing may therefore be a general
mechanism for attenuating innate immune signaling.
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Figure 1.
Figure 1. Structural Analysis of Apu2.16 and Apu3.A8 Anti-K63
Linkage Fabs Bound to K63-Linked Diubiquitin
(A) The
complex between K63-linked diubiquitin (orange) and the Apu2.16
Fab fragment (heavy chain: green, light chain: magenta). Heavy
chain CDR3 (H3) contacts both ubiquitins on either side of the
isopeptide linkage. H3 side chains within 4.2 Å of
diubiquitin and ubiquitin side chains within 4.2 Å of H3
are shown as sticks. Residues mentioned in the text are in bold
for ubiquitin and italics for the Fab. K63 in the acceptor
ubiquitin is shown as a sphere.
(B) Comparison of K63-
(top) and K48-linked (bottom) diubiquitin. Lysine donor
ubiquitins are light orange and acceptor ubiquitins are bright
orange. K48-linked diubiquitin forms a more compact shape with
the chain extending perpendicular to the ubiquitin dimer, while
the K63-linked diubiquitin chain will extend in a more elongated
manner.
(C) Superposition of Apu2.16 (colored as in A) and
Apu3.A8 (heavy chain: light green, light chain: pink) showing
the location of the two changes in L2 (S52R) and H3 (S52T)
introduced in the affinity maturation process to create Apu3.A8.
The structural differences in L1 in the two Fabs are likely due
to crystal packing. L1 (as well as the N terminus) are in a
noncanonical conformation likely due to interactions with L3,
which has sequence and structural differences relative to the
phage library parent sequence (Figure S2).
(D) Charge
complementarity between Apu3.A8 (transparent) and diubiquitin
(solid). Electrostatic surfaces were calculated with PyMol.
Regions of positive potential are blue; regions with negative
potential are red. In the Apu3.A8 light chain, R52 (which is
introduced in Apu3.A8) and R66 contribute to a positive region
that is close to a negatively charged region on the ubiquitin
surface, created in part by residues D21, D58, and E18 from the
K63 acceptor ubiquitin.
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Figure 3.
Figure 3. Mass Spectrometry Confirms the Linkage Specificity
of Apu2.07 and Apu3.A8 in Immunoprecipitations
(A–D) BJAB
cell lysates were immunoprecipitated with Apu2.07, Apu3.A8, or
an isotype control antibody recognizing HER2. Mass spectrometry
was used to determine the total amount of ubiquitin
immunoprecipitated (A) as well as the polyubiquitin linkages in
the lysate (B) and immunoprecipitates (C and D).
(E) MuRF1
autoubiquitination reactions performed in vitro with WT, K48R,
or K63R ubiquitin were immunoprecipitated with Apu2.07, Apu3.A8,
or isotype control. Numbers in parentheses indicate the relevant
lanes and columns in (F)–(I).
(F) Autoubiquitination
reactions and immunoprecipitations depicted in (E) were western
blotted with a pan-ubiquitin antibody. The hatched red lines
indicate the portion of the gel that was cut out and subjected
to analysis by mass spectrometry.
(G–I) Mass spectrometry
was used to determine the polyubiquitin linkages in the
autoubiquitination reactions and immunoprecipitations depicted
in (E).
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The above figures are
reprinted
by permission from Cell Press:
Cell
(2008,
134,
668-678)
copyright 2008.
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