Supplementary Figure 2: G131–p-Y132 exhibits comparable binding affinity to the PLC-γ1 N-SH2 domain as does a peptide with aspartate preceding p-Y132.

a. Graphs showing the raw data and binding isotherms from isothermal titration calorimetry for representative measurements of LAT p-Y132 peptides binding to the PLC-γ1 N-terminal SH2 domain. The calorimeter cell contained the SH2 domain at a concentration of 3 μM, and the peptide (30 μM) was delivered in 16 injections. Peptide sequences are given above the graphs. b. Bar graph showing the mean binding affinities from three independent experiments as in (a). Each symbol represents one independent experimental result. ns, not significant; two-tailed Mann-Whitney test. The center values presented the mean. c. Data derived from a high-throughput binding screen using the PLC-γ1 N-terminal SH2 domain and a phospho-peptide library containing all single point mutations in LAT residues 120-139, in a Y127F background and with a phosphorylated Y132 residue. This subset of the data from the full screen shows the impact of every amino acid substitution at residues 131 (-1 position) and 135 (+3 position) on the ability of the PLC-γ1 N-terminal SH2 domain to bind to p-Y132. Data are shown on a log₁₀-scale relative to the parent (“wild-type”) sequence. A positive value indicates enhancement in binding relative to the parent sequence, a value close to zero indicates no impact on binding affinity, and a negative value indicates that the substitution reduced the binding affinity. The average effect of mutations at Y132 is shown by a red dotted horizontal line to demonstrate the magnitude of the most negatively-perturbing substitutions (that is the signal floor of the assay). The screen shows that binding to the PLC-γ1 N-terminal SH2 domain is largely unaffected by the identity of the -1 residue, whereas it has a strong preference for hydrophobic residues at the +3 position, as described previously (Songyang et al., 1995). This high-throughput screen was done once.