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PDBsum entry 2jo9
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References listed in PDB file
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Key reference
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Title
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Nmr structural studies of the itchww3 domain reveal that phosphorylation at t30 inhibits the interaction with ppxy-Containing ligands.
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Authors
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B.Morales,
X.Ramirez-Espain,
A.Z.Shaw,
P.Martin-Malpartida,
F.Yraola,
E.Sánchez-Tilló,
C.Farrera,
A.Celada,
M.Royo,
M.J.Macias.
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Ref.
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Structure, 2007,
15,
473-483.
[DOI no: ]
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PubMed id
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Abstract
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In this work, we study the role of phosphorylation as a regulatory mechanism for
the interaction between the E3 ubiquitin ligase ItchWW3 domain and two PPxY
motifs of one of its targets, the Epstein-Barr virus latent membrane protein 2A.
Whereas ligand phosphorylation only diminishes binding, domain phosphorylation
at residue T30 abrogates it. We show that two ItchWW domains can be
phosphorylated at this position, using CK2 and PKA kinases and/or with
stimulated T lymphocyte lysates. To better understand the regulation process, we
determined the NMR structures of the ItchWW3-PPxY complex and of the
phosphoT30-ItchWW3 variant. The peptide binds the domain using both XP and
tyrosine grooves. A hydrogen bond from T30 to the ligand is also detected. This
hydrogen-bond formation is precluded in the variant, explaining the inhibition
upon phosphorylation. Our results suggest that phosphorylation at position 30 in
ItchWW domains can be a mechanism to inhibit target recognition in vivo.
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Figure 3.
Figure 3. Solution Structure of ItchWW3 in Complex with the
PY Peptide—1′-EEPPPPYED-9′
(A) Stereo view of the
best-fit backbone (N, C^α, C′) superposition of the ten
lowest-energy structures after water refinement. The backbone is
shown in blue, with selected side chains represented in magenta
(domain) and green (peptide). Some selected residues are
labeled. Residue numbers are maintained as in the reference
(Shaw et al., 2005). L6 corresponds to L401 in the full protein
sequence. In the peptide, the conversion is such that E2′
corresponds to E55.
(B) Lowest-energy structure of the
complex with the domain shown as a solid surface representation
(in gold) and with the same orientation as above. The peptide is
shown by blue lines. Residues located in the binding site as
well as both tyrosine and proline binding grooves are labeled.
The green circle displays additional contacts observed in the
complex.
(C) Surface electrostatic representation of the
complex rotated by 90° around the x axis with respect to the
orientation shown in (B). The left green circle displays the
electrostatic complementation observed between the aspartic acid
D9′ in the peptide and both H25 and R13 in the domain. This
interaction is supported by NOEs from the peptide to the domain.
The right circle displays the potential contacts between
arginine 19 in the domain and E2′ in the peptide.
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Figure 4.
Figure 4. Titration Experiments for the Phosphorylated Ligand
Variants
(A) Bar representation showing the average
chemical-shift changes observed for the amide domain residues
upon addition of the peptides PY (brown), PY1phosYa (green), and
PY1phosYb (yellow) with respect to the free domain. PY was
measured at a ratio of 1:2.5 domain:ligand; PY1phosYa and
PY1phosYb were measured at ratios of 1:7 and 1:2.5,
respectively. The inset corresponds to the HSQC region
displaying the N[  epsilon
]H peaks of arginines (nitrogen chemical shift is folded). Black
signals correspond to the reference spectrum. The final point of
the PY titration is plotted in brown, PY1phosYa titration is
shown in green, and PY1phosYb is shown in yellow. Although the
addition of peptide PY induces changes mainly in R19 (on the
left), the presence of a tyrosine phosphorylated in PY1phosYa
also affects the resonance of R28.
(B) Amide changes
observed upon addition of peptides PY2phosY (green) and PY2phosS
(orange) corresponding to the second PPxY motif present in
LMP2A. Changes induced by PY are plotted as a reference. Changes
were not followed to saturation. The inset corresponds to the
HSQC region displaying the N[ epsilon
]H peaks of arginines as in (A). Tyrosine phosphorylation of the
second PPxY motif present in LMP2A also induces changes in R28,
but in this case the changes observed in R19 are smaller than
those observed for the PY and PY1phosYa peptides. We attribute
these differences to the absence of negatively charged residues
preceding the PPxY motif in both PY2phosS and PY2phosY peptides.
(C) Model based on the minimum-energy structure of ItchWW3
in complex with PY peptide, showing that the phosphotyrosine in
the PPxY motif can be accommodated in the complex.
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The above figures are
reprinted
by permission from Cell Press:
Structure
(2007,
15,
473-483)
copyright 2007.
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