SH3 domains of Grb2 adaptor bind to PX[psi]PXR motifs within the Sos1 nucleotide exchange factor in a discriminate manner

The isothermal titration calorimetry is used to demonstrate the binding affinities of the nSH3 domain in the physiological range to all four sites containing PX[psi]PXR motifs, designated S1, S2, S3, and S4, and the cSH3 domain binding at the S1 site. The results obtained from site-directed mutagenesis suggested that the two additional arginine residues flanking the PX[psi]PXR motif at the S1 site contributed to free energy of binding via the formation of salt bridges with specific acidic residues in SH3 domains.