Ras membrane orientation and nanodomain localization generate isoform diversity

Area of Science:

• Molecular Biology
• Cell Signaling
• Biophysics

Background:

• Ras GTPases exhibit functional diversity, but the underlying structural elements remain unclear.
• Ras G-domain orientation relative to the plasma membrane influences effector interactions and downstream signaling.
• C-Raf binding to H-ras G-domain orientation links membrane positioning to MAPK activation.

Purpose of the Study:

• To investigate how Ras G-domain orientation is recognized by other effector proteins.
• To elucidate the structural determinants of Ras G-domain orientation and its role in signaling.
• To understand the synergistic effects of G-domain orientation and nanoclustering on Ras isoform specificity.

Main Methods:

• Fluorescence lifetime imaging microscopy (FLIM)-Förster resonance energy transfer (FRET) imaging.
• Molecular dynamics simulations of Ras protein-membrane interactions.
• Mutational and functional analyses of Ras structural elements.

Main Results:

• Phosphoinositide-3-kinase-alpha and galectin-1 recognize Ras G-domain orientation.
• Two distinct, signaling-competent orientations of the Ras G domain were identified.
• Amphilicities of helix alpha4 and the C-terminal hypervariable region critically tune Ras G-domain orientation.
• Ras G-domain orientation and nanoclustering synergize to confer isoform-specific effector interactions.

Conclusions:

• Ras G-domain orientation is a key determinant of effector recognition and signaling specificity.
• Galectin-1's role in Ras signaling nanoclusters is explained by its recognition of G-domain orientation.
• Structural elements like helix alpha4 and the C-terminal region are crucial for regulating Ras G-domain orientation.