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PDBsum entry 2v4h
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Transcription
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PDB id
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2v4h
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References listed in PDB file
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Key reference
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Title
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Darpin-Assisted crystallography of the cc2-Lz domain of nemo reveals a coupling between dimerization and ubiquitin binding.
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Authors
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O.Grubisha,
M.Kaminska,
S.Duquerroy,
E.Fontan,
F.Cordier,
A.Haouz,
B.Raynal,
J.Chiaravalli,
M.Delepierre,
A.Israël,
M.Véron,
F.Agou.
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Ref.
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J Mol Biol, 2010,
395,
89.
[DOI no: ]
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PubMed id
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Abstract
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NEMO is an integral part of the IkappaB kinase complex and serves as a molecular
switch by which the NF-kappaB signaling pathway can be regulated.
Oligomerization and polyubiquitin (poly-Ub) binding, mediated through the
regulatory CC2-LZ domain, were shown to be key features governing NEMO function,
but the relationship between these two activities remains unclear. In this
study, we solved the structure of this domain in complex with a designed ankyrin
repeat protein, which helps its crystallization. We generated several NEMO
mutants in this domain, including those associated with human diseases
incontinentia pigmenti and immunodeficiency with or without anhidrotic
ectodermal dysplasia. Analytical ultracentrifugation and thermal denaturation
experiments were used to evaluate the dimerization properties of these mutants.
A fluorescence-based assay was developed, as well, to quantify the interaction
to monoubiquitin and poly-Ub chains. Moreover, the effect of these mutations was
investigated for the full-length protein. We show that a proper folding of the
ubiquitin-binding domain, termed NOA/UBAN/NUB, into a stable coiled-coil dimer
is required but not sufficient for efficient interaction with poly-Ub. In
addition, we show that binding to poly-Ub and, to a lesser extent, to
monoubiquitin increases the stability of the NOA coiled-coil dimer.
Collectively, these data provide structural insights into how several
pathological mutations within and outside of the CC2-LZ's NOA ubiquitin binding
site affect IkappaB kinase activation in the NF-kappaB signaling pathway.
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Figure 2.
Fig. 2. Structural details and sequence conservation of the
NOA motif. (a) Alignment of the NOA sequence from human, mouse,
and Drosophila NEMO homologues and from human Optineurin and
ABIN-1, -2, and -3 proteins. Numbers correspond to the first and
last residue of the protein sequence. Identical and similar
residues are colored red and yellow, respectively. Diamonds
indicate residues linked to human pathologies: EDA-ID (blue), ID
(green), and IP (red), corresponding to the following mutations
(human numbering): D311N, E315A, R319Q, and A323P. The heptad
position (a–g) is indicated at the bottom. (b) Close-up view
of the NEMO NOA region (helices are colored as in Fig. 1).
Conserved residues are shown as ball-and-stick representations,
with O and N atoms in red and blue, respectively. Broken lines
point to residues involved in salt bridge or H-bond formation.
For clarity, only one set of conserved residues is displayed.
(c) Helical wheel representation of the NOA coiled coil,
starting from residue V293 at position c. Residues linked to
human pathologies are colored red. Blue lines indicate
intermolecular salt bridges.
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Figure 4.
Fig. 4. Folding and dimeric stability of the human
CC2-LZ[215–362] and its mutants. (a) Summary of molecular
weight average and dimerization constant of WT and mutant
CC2-LZ[215–362] from SE at 10 °C (14,000 rpm). The
molecular weights deduced from SEC/LS are also indicated. (b)
Measurements of the molecular weight of WT and A323P determined
by SEC/LS. (c) Sedimentation coefficient distribution c(S[20]^W)
of WT and E296A at 10 or 35 °C.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2010,
395,
89)
copyright 2010.
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Secondary reference #1
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Title
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Inhibition of nf-Kappab activation with designed ankyrin-Repeat proteins targeting the ubiquitin-Binding/oligomerization domain of nemo.
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Authors
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E.Wyler,
M.Kaminska,
Y.M.Coïc,
F.Baleux,
M.Véron,
F.Agou.
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Ref.
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Protein Sci, 2007,
16,
2013-2022.
[DOI no: ]
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PubMed id
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Figure 1.
Enrichment of CC2-LZ binders from a RNA library using
ribosome display. The in vitro translation mixture was applied
either to a specific surface coated with BSA and the target
CC2-LZ protein, or to a surface coated with BSA only. Enrichment
of specific binders can be evaluated by comparing the quantity
of cDNA obtained after RT-PCR between the two samples. The
unspecific binders can be detected until round three.
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Figure 3.
Protein interface evolution of DARPin binding to the
ubiquitin-binding/oligomerization domain of NEMO. The N2C
scaffold AR as well as the three DARPins 1D5, 2A1, and 2F6 with
an affinity in the nanomolar range are shown in surface
representation. (Gray) The conserved surface of N2C; (red) the
variable protein interface. Residues in each ankyrin that
contribute to an improved affinity of the CC2-LZ domain are
highlighted in different colors: (black) hydrophobic; (red)
acidic; (blue) basic; (green) polar. All figures were prepared
using the program PyMOL (DeLano 2002).
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The above figures are
reproduced from the cited reference
which is an Open Access publication published by the Protein Society
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