A GAP-GTPase-GDP-P_i Intermediate Crystal Structure Analyzed by DFT Shows GTP Hydrolysis Involves Serial Proton...

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Cell signaling relies on small G proteins switching between ON and OFF states.
• This switch is regulated by GTP hydrolysis, but the exact timing of inorganic phosphate release remains unclear.
• The RhoA GTPase activating protein (GAP) accelerates GTP hydrolysis, yet the mechanism of product release is not fully understood.

Purpose of the Study:

• To elucidate the mechanism of inorganic phosphate release during RhoA GTP hydrolysis.
• To determine the structural basis for the interaction between RhoA, GAP, GDP, and inorganic phosphate.
• To investigate the role of proton transfer in facilitating signal termination.

Main Methods:

• Generation and X-ray crystallography of a RhoA-GAP-GDP-P_i quaternary complex at 1.75 Å resolution.
• Density Functional Theory (DFT) calculations on a selected QM core of the H-bonded network.
• Analysis of proton movement and coordination within the complex.

Main Results:

• The crystal structure reveals the precise coordination of GDP and P_i in an intermediate state.
• DFT calculations identified serial proton locations and a three-step proton transfer mechanism for stable complex formation.
• The study suggests a pathway involving two additional proton transfers for facilitating inorganic phosphate release.

Conclusions:

• The structural and computational data provide a detailed mechanistic insight into RhoA GTP hydrolysis.
• The findings clarify the role of proton transfer in inorganic phosphate release and signal termination.
• This work offers a foundation for understanding GTPase regulation and developing targeted therapeutics.