Ligand Specificity Modulated by Prolyl Imide Bond Cis/Trans Isomerization in the Itk SH2 Domain:â•› A Quantitative NMR Study

The exquisite specificity exhibited by many proteins for their respective ligands can often be attributed to particular structural features within the binding site. The Src homology 2 (SH2) domain of interleukin-2 tyrosine kinase (Itk) contains overlapping binding sites capable of mediating binding to two distinct ligands: a phosphotyrosine-containing peptide and the Itk Src homology 3 (SH3) domain 1 (Figure 1a). We have previously demonstrated that the Asn 286-Pro 287 imide bond in the Itk SH2 domain adopts both the cis and trans conformations in solution. 2,3 Exchange between the conformers is slow on the NMR time scale, leading to the appearance of doubled resonances in NMR spectra for 35 of the 109 SH2 residues (Figure 1b). The structural changes induced by isomerization of the peptidyl prolyl imide bond in the Itk SH2 domain modulate its affinity for both of its ligands. We now report a quantitative study of the equilibria governing the peptidyl prolyl cis/trans isomerization and concomitant ligand binding to the Itk SH2 domain. Hereafter, we will refer to the cis and trans imide bond containing SH2 conformers as the cis and trans conformers, respectively. The conformer-specific nature of Itk SH2 ligand recognition is evident in NMR spectra of the protein/ligand complexes. Addition of phosphopeptide to the Itk SH2 domain shifts the equilibrium to favor the trans conformer, resulting in changes in the volumes of the NMR peaks corresponding to each conformer (Figure 1c). Also, ligand-induced chemical shift changes are larger for cross-peaks corresponding to residues in the trans conformer than for those corresponding to residues in the cis conformer. In contrast, binding of the Itk SH3 domain to the SH2 domain shifts the cis/trans ratio to favor the cis conformer (Figure 1d). In this case, shifts in the positions of cross-peaks corresponding to the cis conformer (but not those of the trans conformer) are observed in the HSQC spectrum of the Itk SH2 domain to which recombinant Itk SH3 domain has been added 2. To our knowledge, this is the first demonstration of ligand recognition that is governed by the conformation of a single prolyl imide bond within a folded protein. However, a qualitative analysis of the data cannot establish that the trans SH2 conformer has no affinity for the Itk SH3 domain or that the cis conformer cannot bind phosphopeptide. We have therefore developed a method of analyzing chemical shift perturbation and cross-peak volumes to measure the …