The adaptor protein Grb2 is a critical regulator in signaling pathways responsible for cell growth and proliferation, making it a key target in various carcinomas. Grb2’s function is intricately linked to its dynamic equilibrium between monomeric and dimeric states. This equilibrium is tightly regulated by factors such as protein concentration and post-translational modifications (e.g., Y160/Y207 phosphorylation), making it a significant challenge to biophysically isolate the monomeric form to understand its specific contributions to signaling. The dimerization interface is complex, and while several residues are involved, the specific role of W60─located at the canonical interface─in stabilizing this oligomeric state has remained unexplored. Here, we demonstrate that the W60 residue is a critical link for dimerization. We engineered a point mutation (W60A) and employed a comprehensive biophysical approach (including SAXS, NMR, and molecular dynamics) to characterize its structural and dynamic consequences. Our results are definitive: the W60A mutation successfully disrupts the dimer interface, yielding a stable, constitutively monomeric protein in solution, which adopts a more elongated conformation. Crucially, our structural analyses suggest that this mutation is highly specific and nonperturbative, disrupting dimerization while preserving the structural integrity of canonical interaction sites, including the SH3 domains (for proline-rich motifs) and the primary phosphotyrosine-binding pocket of the SH2 domain. This Grb2 W60A mutant therefore serves as a powerful new biophysical tool to uncouple dimerization from function. It provides an unprecedented platform to investigate complex regulatory mechanisms─such as the impact of phosphorylation on Grb2─in a purely monomeric context, overcoming a major challenge in dissecting its complex signaling roles.
Tedesco et al. (2026) studied this question.