Interdomain interactions in Grb2 revealed by the conformational stability and CD28 binding analysis.

Growth-factor receptor-bound protein 2 (Grb2) is an adaptor protein involved in signal transduction. The protein contains a central SH2 domain with N-terminal and C-terminal SH3 domains. We analyzed the SH3 involvement in Grb2 binding to the cytoplasmic region of CD28 receptor, using Grb2 and its SH3-deletion mutants, Grb2_nSH3del and Grb2_cSH3del, as the monomer state. The CD28 binding affinity of Grb2_nSH3del determined from surface plasmon resonance experiments was similar to that of Grb2_cSH3del, which was lower than that of Grb2 and higher than that of Grb2 SH2. The findings indicated that one of the SH3 domains is involved in CD28 binding. We also analyzed the thermal stabilities of Grb2 and its SH3-deletion mutants using differential scanning calorimetry, showing that the order of stability was Grb2_nSH3del < Grb2 < Grb2_cSH3del. Folding thermodynamics results indicated that SH3 domains, particularly nSH3, contribute to stabilizing the structure of Grb2, possibly due to the interdomain interaction.