pH-Dependent Conformational Changes in Grb2 Monomer Reveal Different Binding Sites for Coumarin: An Insight for Small Molecule Drug Discovery.

Growth factor receptor-bound protein 2 (Grb2) is an essential adaptor protein that mediates activation of the RAS/mitogen-activated protein kinase (MAPK) signaling cascade by linking receptor tyrosine kinase to the guanine nucleotide exchange factor SOS (Son of Sevenless). Grb2 exists in a dynamic monomer-dimer equilibrium, with only the monomer competent for signaling. Dysregulation of this pathway is common in cancer, making Grb2 a compelling therapeutic target. Coumarin, a plant-derived compound with reported anticarcinogenic activity, was previously shown to bind the SH2 domain of dimeric Grb2. Here, we investigated the interaction between coumarin and the monomeric form of Grb2 under two physiologically relevant pH conditions (7 and 8), corresponding to healthy and cancer cell environments, respectively. Fluorescence quenching, dynamic light scattering, and STD-NMR analyses revealed that pH modulates Grb2's conformation, increasing its hydrodynamic diameter and exposing alternative ligand-binding sites. The interaction mapping identified Trp60 (buried in the dimeric form) as a key contact residue and at pH 8.0, coumarin binds Grb2 with a 10-fold higher stability. These findings demonstrate, for the first time, that coumarin interacts with monomeric Grb2 and that pH-sensitive structural dynamics modulate ligand binding. Our results highlight the importance of mimicking disease-relevant physicochemical conditions in ligand discovery efforts and support the development of pH-sensitive inhibitors targeting aberrant MAPK signaling in cancer.