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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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