Surface change of Ras enabling effector binding monitored in real time at atomic resolution

Area of Science:

• Molecular biology
• Cell signaling
• Biophysics

Background:

• Ras proteins function as molecular switches controlling cell growth.
• Oncogenic Ras mutations lead to uncontrolled cell proliferation by blocking GTP hydrolysis.
• Understanding the Ras(off)-to-Ras(on) transition is key to cancer research.

Purpose of the Study:

• To monitor the Ras surface changes during the Ras(off)-to-Ras(on) transition at atomic resolution in real-time.
• To identify and characterize GTP-bound intermediates in the Ras activation cycle.
• To establish a spectroscopic assay for drug screening targeting Ras-mediated signaling.

Main Methods:

• Time-resolved Fourier-transform infrared (trFTIR) spectroscopy was employed to observe dynamic structural changes.
• Atomic resolution monitoring of the Ras protein surface during GDP-to-GTP exchange.
• Spectroscopic analysis of the C=O vibration of Thr35 to track loop movement.

Main Results:

• A novel GTP-bound intermediate, Ras(off)GTP, was identified where effector binding is prevented.
• The conformational change from Ras(off)GTP to Ras(on)GTP, involving switch I loop movement, was directly observed.
• A spectroscopic fingerprint differentiating Ras(off) and Ras(on) states was established.

Conclusions:

• The Ras(off)GTP intermediate provides new insights into the mechanism of Ras activation.
• Targeting the equilibrium towards the Ras(off)GTP state could inhibit oncogenic Ras signaling.
• The developed spectroscopic assay facilitates drug discovery for cancer therapies.