Figure 3.

The binding interface and affinity of SHIP2-SH2 for Y1356-phosphorylated c-MET revealed by NMR titration. (A) Overlay of 2D ¹H-¹⁵N HSQC spectra of the apo SHIP2-SH2 (red) and the mixtures of SHIP2-SH2 with Y1356-phosphorylated c-MET peptide at the ratio of 1:0.5 (orange), 1:1 (green), 1:2 (purple). Residue D25 and S49 are zoomed in respectively. For clarity, only four points of the titration are shown. (B) The chemical shift perturbation (CSP) of each residue during NMR titrations were calculated and shown with the secondary elements on top. The residues that had significant CSPs but were untraceable in ¹H-¹⁵N HSQC spectra during the titration are labeled with red stars. Prolines are labeled with black asterisks. The broken line represents the cutoff level (CSP ≥ 0.15 ppm) for significant perturbations. (C) The residues affected significantly by the titrations are mapped onto a cartoon representation of the SHIP2-SH2 structure. Residues with CSPs greater than the cutoff level are highlighted in orange with black text, and the residues that disappeared during the titration are highlighted in yellow with blue text. The peak intensities for R16, S84 and G86 were poor, and therefore their CSP values could not be definitely determined. (D) Molecular surface representation of SHIP2-SH2 structure with residue and text colors the same as those in (C) is show. Only the residues on the protein surface are labeled. (E) Global fitting of the NMR titration data for thirteen residues. The selected residues were all suitable for fitting to the fast-exchange equation.