PDBsum entry 2joc

Go to PDB code: 
protein links
Ligase PDB id
Protein chain
37 a.a. *
* Residue conservation analysis
PDB id:
Name: Ligase
Title: Mouse itch 3rd domain phosphorylated in t30
Structure: Itchy e3 ubiquitin protein ligase. Chain: a. Fragment: ww 3 domain, sequence database residues 399-432. Engineered: yes
Source: Synthetic: yes. Other_details: the peptide was chemically synthesized. The sequence of the peptide is naturally found in mus musculus (mouse).
NMR struc: 7 models
Authors: M.J.Macias,A.Z.Shaw,P.Martin-Malpartida,B.Morales,L.Ruiz, X.Ramirez-Espain,F.Yraola,M.Royo
Key ref:
B.Morales et al. (2007). NMR structural studies of the ItchWW3 domain reveal that phosphorylation at T30 inhibits the interaction with PPxY-containing ligands. Structure, 15, 473-483. PubMed id: 17437719 DOI: 10.1016/j.str.2007.03.005
06-Mar-07     Release date:   17-Apr-07    
Go to PROCHECK summary

Protein chain
Pfam   ArchSchema ?
Q8C863  (ITCH_MOUSE) -  E3 ubiquitin-protein ligase Itchy
864 a.a.
37 a.a.*
Key:    PfamA domain  Secondary structure
* PDB and UniProt seqs differ at 4 residue positions (black crosses)


DOI no: 10.1016/j.str.2007.03.005 Structure 15:473-483 (2007)
PubMed id: 17437719  
NMR structural studies of the ItchWW3 domain reveal that phosphorylation at T30 inhibits the interaction with PPxY-containing ligands.
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.
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.
  Selected figure(s)  
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.
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.
  The above figures are reprinted by permission from Cell Press: Structure (2007, 15, 473-483) copyright 2007.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
20937913 P.A.Chong, H.Lin, J.L.Wrana, and J.D.Forman-Kay (2010).
Coupling of tandem Smad ubiquitination regulatory factor (Smurf) WW domains modulates target specificity.
  Proc Natl Acad Sci U S A, 107, 18404-18409.
PDB code: 2kxq
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB code is shown on the right.