Ligand specificity modulated by prolyl imide bond Cis/Trans isomerization in the Itk SH2 domain: a quantitative NMR study.
The Src homology 2 (SH2) domain of interleukin-2 tyrosine kinase (Itk) binds two separate ligands: a phosphotyrosine-containing peptide and the Itk Src homology 3 (SH3) domain. Binding specificity for these ligands is regulated via cis/trans isomerization of the Asn 286-Pro 287 imide bond in the Itk SH2 domain. In this study, we develop a novel method of analyzing chemical shift perturbation and cross-peak volumes to measure the affinities of both ligands for each SH2 conformer. We find that the cis imide bond containing SH2 conformer exhibits a 3.5-fold higher affinity for the Itk SH3 domain compared with binding of the trans conformer to the same ligand, while the trans conformer binds phosphopeptide with a 4-fold greater affinity than the cis-containing SH2 conformer. In addition to furthering the understanding of this system, the method presented here will be of general application in quantitatively determining the specificities of conformationally heterogeneous systems that use a molecular switch to regulate binding between multiple distinct ligands.
Breheny
Patrick J
PJ
Department of Biochemistry, Biophysics and Molecular Biology, Program of Bioinformatics and Computational Biology, Iowa State University, Ames, Iowa 50011, USA.
Laederach
Alain
A
Fulton
D Bruce
DB
Andreotti
Amy H
AH
D007700:Kinetics; D008024:Ligands; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry*; D013379:Substrate Specificity; D018909:src Homology Domains
D008024:Ligands; D011505:Protein-Tyrosine Kinases; C080813:emt protein-tyrosine kinase