Fig. 2
Identification of the Shp1-cSH2 domain residues involved in GroPIns4P binding. a, b Overlay of the 1H-15N Heteronuclear Single-Quantum Coherence (HSQC) spectra of the 15N-labeled cSH2 domain in the absence and presence of different amount of GroPIns4P. Protein alone (black) and in the presence of GroPIns4P at the ratios of 1:1 (blue), 1:2 (green), 1:3 (magenta), 1:4 (brown), 1:5 (red) are reported. c Normalised weighted average chemical shift differences (Δav/Δmax) between the GroPIns4P-bound and free forms of the Shp1 cSH2 domain plotted against the residue number for the amide proton and nitrogen resonances. The horizontal bold line at 0.3 indicates the average value plus one standard deviation. d Chemical shift perturbation mapped onto the cSH2 structure of Shp1 (PDB code: 2B3O). The amino acid residues affected by GroPIns4P binding (Δav/Δmax ≥ 0.3) are depicted in red. e Chemical structure of GroPIns4P. f Ribbon representation of the complex, in which the cSH2 domain is in blue and GroPIns4P in red. Residues involved in the interaction are presented as sticks and are labelled. g Close view of S118 and R138 of the cSH2 domain that interact with the phosphate group of GroPIns4P. h Close view of S140, S142 and K170 of the cSH2 domain that interact with the 4′-phosphate group of GroPIns4P. Similar experiments on the nSH2 domain of Shp1 were hampered by the poor stability of the isolated fragment. i Functional validation of the Shp1 S118A/R138E/S140A mutant. Dose-response effect of GroPIns4P on Shp1 S118A/R138E/S140A mutant fluorescence emission (ΔF) at 332 nm (fluorescence emission spectra of Shp1 upon addition of the indicated μM concentrations of GroPIns4P are shown in supplementary Additional file 1 Figure S3)