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

## TL;DR

This study shows that pH changes affect how a cancer-related protein interacts with a plant compound, coumarin, offering new insights for drug development.

## Contribution

First demonstration that pH-sensitive conformational changes in monomeric Grb2 modulate coumarin binding.

## Key findings

- pH 8.0 (cancer-like) increases coumarin binding stability to Grb2 by 10-fold compared to pH 7.0.
- Trp60 is identified as a key residue exposed for binding in the monomeric form of Grb2.
- pH modulates Grb2 conformation, exposing alternative ligand-binding sites.

## Abstract

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.