GTP-Bound N-Ras Conformational States and Substates Are Modulated by Membrane and Point Mutation

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Ras proteins, including N-Ras, exist in multiple conformational states crucial for their function.
• The GTP-bound state of Ras proteins is typically classified into two main states: inactive (state 1) and active (state 2).
• Previous studies on H-Ras identified two substates within the active state, differentiated by the orientation of Tyrosine 32 (Tyr32).

Purpose of the Study:

• To identify and characterize novel conformational substates of N-Ras.
• To investigate the impact of the G12V mutation on N-Ras conformational dynamics.
• To explore the influence of the cell membrane on N-Ras conformational states and stability.

Main Methods:

• X-ray crystallography to determine protein structures.
• Molecular dynamics simulations to analyze protein dynamics and membrane interactions.
• Analysis of N-Ras wild-type and G12V mutant forms.

Main Results:

• N-Ras exhibits a previously unidentified substate of the active state (state 2), characterized by a third orientation of Tyr32.
• The G12V mutation significantly increases the sampling of this novel substate in N-Ras.
• The G12V mutation prevents the sampling of the GTPase-activating protein (GAP) binding substate, thereby promoting oncogenesis.
• Molecular dynamics simulations reveal that the cell membrane profoundly influences N-Ras conformational dynamics, stability, and substate sampling.

Conclusions:

• A novel N-Ras conformational substate involving Tyr32 has been identified.
• The G12V mutation's oncogenic potential is linked to its disruption of GAP binding via altered conformational sampling.
• Membrane interactions are critical for regulating N-Ras conformational dynamics and its role in the Ras activation/deactivation cycle involving GEFs and GAPs.