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.

Structural dynamics of a DNA-binding protein analyzed using diffracted X-ray tracking.

Diffracted X-ray tracking (DXT) is one of methods for the real-time evaluation of protein structural dynamics by detecting the movement of a gold-nanocrystal attached to a target protein. However, one of the technical concerns is the size of the gold-nanocrystals, which are larger than the protein. In our previous results of mean square angular displacement curves in DXT analysis, dynamical movements of the DNA-binding protein, c-Myb R2R3, were observed in only one population in either DNA-unbound or -bound state, and was found to decrease upon DNA binding.

Structural and functional properties of Grb2 SH2 dimer in CD28 binding.

Growth factor receptor-bound protein 2 (Grb2) is an adaptor protein that plays a critical role in cellular signal transduction. It contains a central Src homology 2 (SH2) domain flanked by two Src homology 3 (SH3) domains. Binding of Grb2 SH2 to the cytoplasmic region of CD28, phosphorylated Tyr (pY) containing the peptide motif pY-X-N-X, is required for costimulatory signaling in T cells.

DNA-binding induced conformational change of c-Myb R2R3 analyzed using diffracted X-ray tracking.

Previous structural analyses have shown that R2R3, the minimum unit of the DNA-binding domain of the transcriptional factor c-Myb, is largely flexible in solution, and changes to a more rigid structure upon DNA binding. In this study, we evaluated the structural dynamics using the diffracted X-ray tracking method, in correlation with DNA-binding abilities under different salt conditions, and compared them with the previous results. The resultant curve of the mean square angular displacements (MSD) clearly showed that the flexibility of R2R3 was decreased upon DNA binding, and the DNA-binding energies determined using the angular diffusion coefficients were in good agreement with those determined using isothermal titration calorimetry.