a, Isothermal titration calorimetry of SHP099 binding to SHP2. SHP099 binds stoichiometrically to SHP2 with a dissociation constant measured at 73 ± 15 nM. b, Structural differences in the central tunnel between SHP1 and SHP2. Ribbon representation of SHP2 (multi-colour) and SHP1 (grey) X-ray structures in the closed conformation. The PTP (tan) and N-SH2 (green) domain overlay well (r.m.s.d. < 1.5 Å), however the C-SH2 (blue) domain has a significantly different orientation. c, Surface representation of SHP2–SHP099 co-crystal structure. d, Surface representation of SHP1 with SHP099 modelled on the basis of the SHP2 superimposition. Central tunnel is significantly larger in SHP1 owing to a change in orientation of the C-SH2 domain. This change repositions the linker between the two SH2 domains removing several key interactions, highlighted by residue Arg109 in SHP1 and by Arg111 in SHP2 (equivalent residues). e, Biochemical activity of wild-type SHP2, SHP2Q257L and SHP2T253M/Q257L. SHP2 activity was determined using DiFMUP in the presence of various concentrations of 2P-IRS-1. Data points along the line represent the mean of two replicate values. SHP2Q257L and SHP2T253M/Q257L retain activity regulation and 2P-IRS-1 activation potential comparable to wild-type SHP2 but are 18- and <1,000-fold less sensitive to SHP099 inhibition.