Rheb membrane orientation dynamics and functional consequences elucidated by molecular simulations,...

Area of Science:

• Molecular and Cellular Biology
• Biophysics
• Biochemistry

Background:

• Prenylated Ras GTPases regulate cellular signaling, with prior focus on plasma membrane proteins.
• Ras homolog enriched in brain (Rheb) localizes to endo-membranes, activating mTORC1 for cell growth.
• Limited understanding of Rheb's membrane interactions, lipid binding, and orientational dynamics exists.

Purpose of the Study:

• To investigate the membrane binding modes and orientation landscape of Rheb.
• To determine the functional consequences of Rheb's membrane dynamics on mTORC1 activation.
• To explore the role of orientational dynamics in lipid-modified small GTPase regulation.

Main Methods:

• Molecular dynamics simulations
• Hidden Markov modeling
• Single-molecule Förster Resonance Energy Transfer (smFRET) measurements
• Simulation-guided mutagenesis and cell signaling assays

Main Results:

• Rheb exhibits interconversion between two dominant membrane orientation states via two intermediate states.
• Destabilization of specific orientation states through mutation impacts mTORC1 activity.
• Identified distinct membrane binding modes and orientation landscape of Rheb.

Conclusions:

• Membrane orientation dynamics are functionally significant for endo-membrane localized Rheb.
• Rheb's orientational dynamics directly influence mTORC1 pathway activation.
• Suggests a broader regulatory role for orientational dynamics in prenylated small GTPases.