Counteracting effects operating on Src homology 2 domain-containing protein-tyrosine phosphatase 2 (SHP2) function drive selection of the recurrent Y62D and Y63C substitutions in Noonan syndrome

Abstract

Activating mutations in PTPN11 cause Noonan syndrome, the most common nonchromosomal disorder affecting development and growth. PTPN11 encodes SHP2, an Src homology 2 (SH2) domain-containing protein-tyrosine phosphatase that positively modulates RAS function. Here, we characterized functionally all possible amino acid substitutions arising from single-base changes affecting codons 62 and 63 to explore the molecular mechanisms lying behind the largely invariant occurrence of the Y62D and Y63C substitutions recurring in Noonan syndrome. We provide structural and biochemical data indicating that the autoinhibitory interaction between the N-SH2 and protein-tyrosine phosphatase (PTP) domains is perturbed in both mutants as a result of an extensive structural rearrangement of the N-SH2 domain. Most mutations affecting Tyr(63) exerted an unpredicted disrupting effect on the structure of the N-SH2 phosphopeptide-binding cleft mediating the interaction of SHP2 with signaling partners. Among all the amino acid changes affecting that codon, the disease-causing mutation was the only substitution that perturbed the stability of the inactive conformation of SHP2 without severely impairing proper phosphopeptide binding of N-SH2. On the other hand, the disruptive effect of the Y62D change on the autoinhibited conformation of the protein was balanced, in part, by less efficient binding properties of the mutant. Overall, our data demonstrate that the selection-by-function mechanism acting as driving force for PTPN11 mutations affecting codons 62 and 63 implies balancing of counteracting effects operating on the allosteric control of the function of SHP2.

FIGURE 1.

Structure of SHP2 in its catalytically inactive conformation and location of Tyr⁶² and Tyr⁶³. The N-SH2, C-SH2, and PTP domains of the protein (residues 2–525; PDB entry 2shp, chain A) are shown in blue, green, and red, respectively. The signature motif of the PTP active site (orange), N-SH2 blocking loop (cyan), and N-SH2 phosphopeptide-binding cleft (according to the x-ray structure of the N-SH2-peptide complex, PDB entry 1aya) (light blue) are also displayed. The side chains of residues Tyr⁶² and Tyr⁶³ are reported in green and yellow, respectively. Residues missing in the experimental crystallographic structure were reconstructed as described previously (21). The inset shows the electrostatic potential generated by the PTP domain on the N-SH2 surface in the catalytically inactive conformation of SHP2. The PTP backbone is reported as a ribbon, and the N-SH2 surface is shown. Red and blue colors indicate negative and positive potential values ranging from −40 kT/e to +40 kT/e, respectively. The side chains of residues Tyr⁶² and Tyr⁶³ are shown as above.

FIGURE 2.

ERK phosphorylation assays. 293T cells were co-transfected with HA-tagged ERK2, FLAG-Gab1, and the indicated V5-tagged SHP2 constructs. Following starvation (12 h) and EGF stimulation (30 ng/ml), cells were immunoprecipitated (IP) with anti-HA antibody and probed with phosphorylated ERK1/2 (anti-pERK1/2) or anti-HA antibodies. Aliquots of corresponding cell lysates were probed with anti-V5, anti-HA, and anti-FLAG antibodies. Representative blots (above) and mean ± S.D. densitometry values (below) of three independent experiments are shown.

FIGURE 3.

Biochemical characterization of SHP2 mutants. A, in vitro phosphatase assay of wild-type SHP2 and all mutants arising from a single-base change at codons 62 and 63. Mutants carrying the Y62E substitution and the leukemia-associated E76K change are also shown for comparison. Catalytic activity was measured as pmoles of phosphate released using p-nitrophenyl phosphate as a substrate, basally (white bars) and following stimulation with 10 μm BTAM peptide (black bars). Values are expressed as mean ± S.D. of at least three independent experiments and are normalized to the basal activity of the wild-type SHP2. Asterisks indicate the recurrent NS-causing amino acid substitutions. B, in vitro phosphatase assay of wild-type SHP2 and mutant proteins as a function of BTAM peptide concentration. Catalytic activity (mean values ± S.D. of three independent experiments) was measured as above.

FIGURE 4.

Molecular dynamics simulations. A, ribbon representation of the final structures obtained from simulations of the N-SH2 domain for wild-type (WT), Y62D, Y63C, and Y63D mutants. The blocking loop (residues 58–62) is shown in red. B, time evolution of the distance between the blocking loop and the B-helix located behind it during simulations. C, root mean square positional fluctuations (RMSF) of the C_α atoms of the wild-type (black), Y62D (red), Y63C (green), and Y63D (blue) N-SH2 domains during simulations. Magenta marks indicate residues belonging to the phosphopeptide-binding pocket, as defined previously (21). D, ribbon representation of two frames extracted from the Y63D N-SH2 simulation. Asp⁶³ is shown as red sticks. The blue circles indicate the region undergoing the main conformational transitions.

FIGURE 5.

Chemical denaturation of the isolated wild-type and mutant N-SH2 domains, as indicated by the shift in the fluorescence spectrum of Trp⁶. Protein unfolding leads to exposure of the fluorophore to the water environment, resulting in a red shift in the spectrum. Average wavelength was calculated according to the following equation: ΣⁱλⁱFⁱ/ΣⁱFⁱ, where F represents fluorescence intensity and λ represents the wavelength at which it was measured. Values are expressed as mean ± S.D. of three independent experiments.

FIGURE 6.

Phosphopeptide binding properties of wild-type and mutant N-SH2 domains. A, surface plasmon resonance analysis. Sensorgrams of the interaction of the wild-type protein (red) and the Y62D (blue), Y63C (cyan), and Y63D (pink) SHP2 mutants with the biotinylated BTAM peptide are shown. B, Two Sample Logo representation of the binding recognition specificity of wild-type and mutant N-SH2 domains. Each logo abridges the residue enrichment at the positions flanking the phosphorylated tyrosine in the best ligand peptides. At each position of the top diagram, the overall stack height indicates the sequence conservation, whereas the height of symbols within each stack indicates the relative frequency of the indicated residue. The bottom diagram illustrates the “anti-motif” (i.e. residues that are enriched at each position in the peptide sequences of the negative set) (P_{Student's t test} < 0.05). Residue positions are numbered by referring their position to that of the pY. The Pearson correlation coefficient (P. C. C.) between the wild-type N-SN2 domain and each mutant is also shown.

FIGURE 7.

Phosphopeptide recognition specificity in SPOT synthesis assay. Phosphopeptides were synthesized on a cellulose membrane, which was incubated with the indicated N-SH2 domain expressed as a GST fusion protein. The binding efficiency and affinity were revealed using an anti-GST antibody conjugated to a fluorophore. The intensity of the signal was quantified, and the -fold increase (assuming the wild-type intensity equal to 1 for each peptide) was plotted in the bar chart.