Fig. 3. Characterisation of Grb2_Y160E binding to Shp2_PTP domain.

a MST measurement of Shp2_PTP binding to fluorescent labelled Grb2_Y160E (0.1 µM). Both PTP domain and tandem 2SH2 domains show similar affinity to Grb2_Y160E. Data are presented as (mean ± SD) of technical triplicates. b GST pull-down experiment using GST-tagged individual Grb2 domain (NSH3, SH2 and CSH3) to precipitate Shp2 PTP domain. The result clearly indicates the interaction is mediated through Grb2 CSH3 domain. The blot represents three independent experiments. Inset: densitometric analysis of GST pull-down results. Results are represented as mean ± SD. Statistics were determined using an unpaired Student’s t test. c Sequence analysis identifies six potential PxxP or R/KxxK (x is any amino residue) motifs for Grb2 CSH3 domain interaction. Synthesised peptides were used to test the interactions with Grb2 CSH3 domain (Atto 488 labelled, 0.1 µM) using MST. The results show ³²²KPKK³²⁵, ³⁶⁶KCVK³⁶⁹ and ⁴²⁹PSDP⁴³² interact with Shp2 PTP domain. Motif ⁴²⁹PSDP⁴³² binds to Shp2 PTP domain with the highest affinity of 18 µM. See Table for detailed information and peptide sequences. Data are presented as (mean ± SD) of technical replicates (peptide 322–325 and peptide 429–432) or triplicates. d ITC was used to corroborate the two binding events between Shp2 and Grb2_Y160E. Grb2_Y160E (200 µM) was titrated into Shp2_Δ69 (25 µM). Twelve 3 µl injections of Grb2_Y160E were titrated into Shp2_Δ69. Top panel: baseline-corrected power versus time plot for the titration. Bottom panel: the integrated heats and the molar ratio of the Grb2_Y160E to Shp2_Δ69.