Probing the Epidermal Growth Factor Receptor under Piconewton Mechanical Compressive Force Manipulations

Studying the relationship among protein structure, dynamics, and function under external compressive forces offers valuable insights. While extensive research has focused on manipulating protein dynamics and ligand-receptor interactions under pulling forces, the exploration of protein conformational changes under compressive forces has been limited. In this study, we investigate the response of unliganded epidermal growth factor receptor (EGFR) monomers, liganded EGF-EGFR monomers, and dimers when exposed to external compressive forces using a home-modified AFM setup with an ultrasoft AFM tip. We observed that both ligand-bound and unbound EGFR proteins can undergo spontaneous tertiary structural rupture under piconewton-level compressive forces, a previously hidden protein behavior that may play a significant role in protein cell signaling. The magnitudes of the threshold compressive forces obtained in our study lie in the range of tens and hundreds of piconewtons (pN), which is accessible within a live biological system. Moreover, we developed a kinetic model to exhibit that only a fraction of the uniaxial compressive force exerted by the AFM tip affects the internal tension that causes a pseudopulling force within the protein before it undergoes the tertiary structural rupture. This calculated fraction ranged from 0.45 to 0.65, depending on the protein type and the approach velocity of the AFM tip. Additionally, we employed molecular dynamics (MD) simulations, particularly Steered MD (SMD) simulations along with Umbrella Sampling (US), to investigate the dynamics of unliganded and liganded EGFR in the presence of external compressive forces. These MD simulation results offer valuable insights into the flexibilities and unfolding behaviors of both liganded and unliganded EGFR proteins when subjected to external compressive forces.