ITK
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.