PMC

Coomassie-stained SDS polyacrylamide gel showing purity of the wild-type SHP-2, and C459C, T42A, A72S, A72V, T73I, E76D, E76K, E139D, Y279C, I282V, N308D, T468M, and M504V mutants (from the left) expressed in E. coli Rosetta 2 (DE3) cells and utilized in phosphatase assay experiments.

Impaired phosphatase activity of the Y279C and T468M SHP-2 mutants expressed in COS1 cells. Representative experiment of the in vitro phosphatase assay of wild-type and mutated SHP-2 immune complexes immunoprecipitated from cell lysates with the use of anti-V5 antibodies (upper panel). Phosphatase activity was measured as picomoles of phosphate released, with the use pNPP as substrate in basal (white bars) and BTAM peptide–stimulated (black bars) conditions. Values are normalized to unstimulated wild-type SHP-2. Coomassie-stained SDS polyacrylamide gel showing comparable amount of the wild-type protein, and Y279C and T468M SHP-2 mutants (from the left) utilized in the assay (lower panel).

MD simulations. A, Comparison between the crystallographic structure of SHP-2 (gray) and the conformation attained at the end of simulations by the E76K (blue) and A72V (red) SHP-2 mutants. The N-SH2 domain and the signature motif of the PTP domain (residues 457–467) are depicted with a ribbon representation. B, RMSD of the N-SH2 loop (residues 58-62) from its starting position. Translational and rotational motions of the protein were removed by fitting the positions of the PTP signature motif atoms to their coordinates in the initial structure. A72V (red), A72S (green), E76K (blue), E76D (cyan), and wild-type (light and dark gray) SHP-2 proteins. C, Electrostatic energy between residue 76 and the PTP domain (upper panel) and solvent-accessible surface area of the PTP signature motif (lower panel) during simulations with the E76K (blue) and wild-type (black) SHP-2 proteins. D, Time behavior during simulations of the H-bonds between residues N58-Q506, G60-Q510, and A72-Q506, contributing to stabilize the interaction between the N-SH2 loop and the PTP active site, in the A72V (red) and wild-type (black) SHP-2 proteins.

Germline and somatic PTPN11 mutations in human disease. A, Location of mutated residues in the three-dimensional structure of SHP-2 in its catalytically inactive conformation (green, N-SH2 domain; cyan, C-SH2 domain; pink, PTP domain). Residues affected by germline (left) or somatically acquired (right) mutations are shown with their lateral chains colored according to the proposed classification (red, group I; yellow, group II; green, group III; cyan, group IV; orange, group V; violet, group VI; blue, unclassified). B, In vitro phosphatase assay of wild-type and mutated SHP-2 proteins. The C459G mutant is a catalytically inactive protein used as negative control. Activity was measured as picomoles of phosphate released, with the use of pNPP as substrate in basal (white bars) and BTAM peptide-stimulated (black bars) conditions. Values are means ±SDs of at least four independent experiments and are normalized to unstimulated wild-type SHP-2.