1KC2

structure of the triple (Lys(beta)D3Ala, Asp(beta)C8Ala, AspCD2Ala) mutant of the Src SH2 domain bound to the PQpYEEIPI peptide


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.10 Å
  • R-Value Free: 0.267 
  • R-Value Work: 0.229 
  • R-Value Observed: 0.229 

wwPDB Validation   3D Report Full Report


This is version 1.3 of the entry. See complete history


Literature

Dissection of the energetic coupling across the Src SH2 domain-tyrosyl phosphopeptide interface.

Lubman, O.Y.Waksman, G.

(2002) J Mol Biol 316: 291-304

  • DOI: https://doi.org/10.1006/jmbi.2001.5362
  • Primary Citation of Related Structures:  
    1KC2

  • PubMed Abstract: 

    Src Homology (SH2) domains play critical roles in signaling pathways by binding to phosphotyrosine (pTyr)-containing sequences, thereby recruiting SH2 domain-containing proteins to tyrosine-phosphorylated sites on receptor molecules. Investigations of the peptide binding specificity of the SH2 domain of the Src kinase (Src SH2 domain) have defined the EEI motif C-terminal to the phosphotyrosine as the preferential binding sequence. A subsequent study that probed the importance of eight specificity-determining residues of the Src SH2 domain found two residues which when mutated to Ala had significant effects on binding: Tyr beta D5 and Lys beta D3. The mutation of Lys beta D3 to Ala was particularly intriguing, since a Glu to Ala mutation at the first (+1) position of the EEI motif (the residue interacting with Lys beta D3) did not significantly affect binding. Hence, the interaction between Lys beta D3 and +1 Glu is energetically coupled. This study is focused on the dissection of the energetic coupling observed across the SH2 domain-phosphopeptide interface at and around the +1 position of the peptide. It was found that three residues of the SH2 domain, Lys beta D3, Asp beta C8 and AspCD2 (altogether forming the so-called +1 binding region) contribute to the selection of Glu at the +1 position of the ligand. A double (Asp beta C8Ala, AspCD2Ala) mutant does not exhibit energetic coupling between Lys beta D3 and +1 Glu, and binds to the pYEEI sequence 0.3 kcal/mol tighter than the wild-type Src SH2 domain. These results suggest that Lys beta D3 in the double mutant is now free to interact with the +1 Glu and that the role of Lys beta D3 in the wild-type is to neutralize the acidic patch formed by Asp beta C8 and AspCD2 rather than specifically select for a Glu at the +1 position as it had been hypothesized previously. A triple mutant (Lys beta D3Ala, Asp beta C8Ala, AspCD2Ala) has reduced binding affinity compared to the double (Asp beta C8Ala, AspCD2Ala) mutant, yet binds the pYEEI peptide as well as the wild-type Src SH2 domain. The structural basis for such high affinity interaction was investigated crystallographically by determining the structure of the triple (Lys beta D3Ala, Asp beta C8Ala, AspCD2Ala) mutant bound to the octapeptide PQpYEEIPI (where pY indicates a phosphotyrosine). This structure reveals for the first time contacts between the SH2 domain and the -1 and -2 positions of the peptide (i.e. the two residues N-terminal to pY). Thus, unexpectedly, mutations in the +1 binding region affect binding of other regions of the peptide. Such additional contacts may account for the high affinity interaction of the triple mutant for the pYEEI-containing peptide.


  • Organizational Affiliation

    Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 South Euclid Ave., Saint Louis, MO 63110, USA.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Src Tyrosine kinase103Rous sarcoma virusMutation(s): 3 
EC: 2.7.1.112
UniProt
Find proteins for P00524 (Rous sarcoma virus subgroup A (strain Schmidt-Ruppin))
Explore P00524 
Go to UniProtKB:  P00524
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP00524
Sequence Annotations
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  • Reference Sequence

Find similar proteins by:  Sequence   |   3D Structure  

Entity ID: 2
MoleculeChains Sequence LengthOrganismDetailsImage
PQpYEEIPI peptide8N/AMutation(s): 0 
Sequence Annotations
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  • Reference Sequence
Small Molecules
Ligands 1 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
CO
Query on CO

Download Ideal Coordinates CCD File 
C [auth A]COBALT (II) ION
Co
XLJKHNWPARRRJB-UHFFFAOYSA-N
Modified Residues  1 Unique
IDChains TypeFormula2D DiagramParent
PTR
Query on PTR
B
L-PEPTIDE LINKINGC9 H12 N O6 PTYR
Binding Affinity Annotations 
IDSourceBinding Affinity
PTR BindingDB:  1KC2 -TΔS: -1.81e+1 (kJ/mol) from 1 assay(s)
ΔG: -2.14e+1 (kJ/mol) from 1 assay(s)
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.10 Å
  • R-Value Free: 0.267 
  • R-Value Work: 0.229 
  • R-Value Observed: 0.229 
  • Space Group: I 4 2 2
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 62.447α = 90
b = 62.447β = 90
c = 133.68γ = 90
Software Package:
Software NamePurpose
CNSrefinement
DENZOdata reduction
SCALEPACKdata scaling
CNSphasing

Structure Validation

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Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2002-04-17
    Type: Initial release
  • Version 1.1: 2008-04-27
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Version format compliance
  • Version 1.3: 2021-11-10
    Changes: Database references, Derived calculations