Structural dynamics of the N-terminal SH2 domain of PI3K in its free and CD28-bound states.

Protein conformational changes with fluctuations are fundamental aspects of protein-protein interactions (PPIs); understanding these motions is required for the rational design of PPI-regulating compounds. Src homology 2 (SH2) domains are commonly found in adapter proteins involved in signal transduction and specifically bind to consensus motifs of proteins containing phosphorylated tyrosine (pY). Here, we analysed the interaction between the N-terminal SH2 domain (nSH2) of the regulatory subunit in phosphoinositide 3-kinase (PI3K) and the cytoplasmic region of the T-cell co-receptor, CD28, using NMR and molecular dynamics (MD) simulations. First, we assigned the backbone signals of nSH2 on 1 H-15 N heteronuclear single quantum coherence spectra in the absence or presence of the CD28 phosphopeptide, SDpYMNMTPRRPG. Chemical shift perturbation experiments revealed allosteric changes at the BC loop and the C-terminal region of nSH2 upon CD28 binding. NMR relaxation experiments showed a conformational exchange associated with CD28 binding in these regions. The conformational stabilisation of the C-terminal region correlated with the regulation of PI3K catalytic function. Further, using 19 F- and 31 P-labelled CD28 phosphopeptide, we analysed the structural dynamics of CD28 and demonstrated that the aromatic ring of the pY residue fluctuated between multiple conformations upon nSH2 binding. Our MD simulations largely explained the NMR results and the structural dynamics of nSH2 and CD28 in both bound and unbound states. Notably, in addition to its major conformation, we detected a minor conformation of nSH2 in the CD28 bound state that may explain the allosteric conformational change in the BC loop.

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.