Molecular modeling of Grb7 and its constituent domains reveals potential domain–domain interactions and oligomerization tendencies.

Grb7 is a member of the Grb7 protein family, which includes Grb10 and Grb14. These adaptor proteins function mainly as scaffolds in signaling pathways that depend on phosphorylation. They are known to interact with receptor tyrosine kinases and other signaling molecules, and their activity is influenced by protein-protein interactions within their conserved five-domain structure. Grb7 has also been linked to cancer progression and is thought to form dimers and undergo intramolecular interactions that may regulate its signaling functions. In this study, we focused on understanding how the SH2 domain of Grb7 interacts with its RA-PH (RAPH) region, which may help regulate its overall structure and activity. We introduced five single-site mutations (V45A, R46A, R46K, E47D, S48A) into the SH2 domain and studied their effects using biochemical, biophysical, and computational methods. Circular Dichroism (CD) spectroscopy showed that all mutants maintained the overall fold of the SH2 domain. However, some mutants, especially V45A and S48A, showed lower thermal stability. Surface Plasmon Resonance (SPR) analysis revealed that mutations at R46 led to weaker binding to the RAPH region. Computational docking using AlphaFold and ClusPro supported the experimental findings, showing slightly reduced interaction stability in the R46 mutants. Additional experiments confirmed that the RA and PH domains form a stable unit. Modeling of full-length Grb7 showed that SH2 domain dimerization, correct alignment by the BPS linker, and improved model quality by removing poly-proline disorders. These results provide new insight into the regulatory mechanisms of Grb7 that could be targeted in cancer research.