Structural mechanism associated with domain opening in gain-of-function mutations in SHP2 phosphatase

Abstract

The SHP2 phosphatase plays a central role in a number of signaling pathways were it dephosphorylates various substrate proteins. Regulation of SHP2 activity is, in part, achieved by an intramolecular interaction between the PTP domain of the protein, which contains the catalytic site, and the N-SH2 domain leading to a "closed" protein conformation and autoinhibition. Accordingly, "opening" of the N-SH2 and PTP domains is required for the protein to become active. Binding of phosphopeptides to the N-SH2 domain is known to induce the opening event, while a number of gain-of-function (GOF) mutants, implicated in Noonan's Syndrome and childhood leukemias, are thought to facilitate opening. In the present study, a combination of computational and experimental methods are used to investigate the structural mechanism of opening of SHP2 and the impact of three GOF mutants, D61G, E76K, and N308D, on the opening mechanism. Calculated free energies of opening indicate that opening must be facilitated by effector molecules, possibly the protein substrates themselves, as the calculated free energies preclude spontaneous opening. Simulations of both wild type (WT) SHP2 and GOF mutants in the closed state indicate GOF activity to involve increased solvent exposure of selected residues, most notably Arg362, which in turn may enhance interactions of SHP2 with its substrate proteins and thereby aid opening. In addition, GOF mutations cause structural changes in the phosphopeptide-binding region of the N-SH2 domain leading to conformations that mimic the bound state. Such conformational changes are suggested to enhance binding of phosphopeptides and/or decrease interactions between the PTP and N-SH2 domains thereby facilitating opening. Experimental assays of the impact of effector molecules on SHP2 phosphatase activity against both small molecule and peptide substrates support the hypothesized mechanism of GOF mutant action. The present calculations also suggest a role for the C-SH2 domain of SHP2 in stabilizing the overall conformation of the protein in the open state, thereby aiding conformational switching between the open active and closed inactive states.

FIGURE 1

Images of the crystal structure of the SHP2 protein in cartoon representation. A and B represents two orientations of the protein. The PTP domain is cyan, the N-SH2 domain is blue and the C-SH2 domain is tan. Residues shown include Cys459 (yellow), Asn92 (purple, on BG loop), Tyr66 (orange, on EF loop), Arg362 (green, on FH loop) and Asp425 (red, on LF loop) in licorice and the three mutant residues in CPK are Asp61 (red, D61G), Glu76 (green, E76K) and Asn308 (purple, N308D). Images of the crystal structure in the region of the C) D61G, D) E76K, and E) N308D mutations. The respective residues undergoing mutation are in red CPK format and selected residues in the vicinity of the mutated residues are in licorice, atom type coloring with the exception of Cys459 in yellow licorice in C and E, Asp61 in red licorice in E, Asn92 in purple licorice in C and Arg362 in green in C.

FIGURE 2

Potential of mean force for opening of the N-SH2 domain from the PTP domain. A) Free energy (kcal/mol) as a function of distance (RCOM, Å) between the center of mass the PTP and N-SH2 domains as calculated from the last 500ps of sampling in each window. B) Expanded view of the minima in the PMF for all 4 systems.

FIGURE 3

Solvent accessible surface area (SASA, Ų) with the 1.4 Å probe sphere radius of the catalytic site (residues 458 to 466) as a function of RCOM (Å) from the four PMF calculations. The inset is the SASA calculated for the WT with the 5 Å probe sphere radius as a function of RCOM (Å). The values are calculated from the last 500ps of sampling in each window.

FIGURE 4

Images of the opening of WT SHP2 taken from the PMF calculation at 30 top), 40 (middle) and 50 (bottom) Å using the time frame at the first 500 ps from the respective windows. The PTP (cyan), N-SH2 (blue) and C-SH2 (blue2) domains are shown in cartoon format. Residues shown include Cys459 (yellow), Asn92 (purple, on BG loop), Tyr66 (orange, on EF loop), Arg362 (green, on FH loop) and Asp425 (red, on LF loop).

FIGURE 5

Solvent accessible surface area (SASA, Ų) for Arg362 as calculated over the course of the 20ns MD simulations.

FIGURE 6

COM distances as calculated form the trajectory generated from the lowest mode1575 between A) the BG loop, residues 91–93 and EF loop, residues 66–68 and B) the FH loop, residues 361–363 and LF loop, residues 425–427. The WT SHP2 is represented in black and the mutants are: D61G in red, E76K in blue and N308D in green.

FIGURE 7

In vitro phosphatase activity of full-length SHP2, the PTP domain of SHP2, the E76K full-length SHP2 mutant and the D61G full-length SHP2 mutant with both A) pY-EGFR and B) pNPP as substrates. Phosphatase activity of SHP2 in WT and SHP2^D61G/D61G mutant mouse embryonic fibroblasts stimulated with 15% fetal calf serum. Both C) pY-EGFR and D) pNPP were used as substrates.