Rheb membrane orientation dynamics and functional consequences elucidated by molecular simulations, single-molecule-FRET and signaling assays.

Membrane binding and dynamics of prenylated Ras family small GTPases are being increasingly recognized as key regulators of their cellular signaling. However, previous studies have focused primarily on the plasma membrane-associated Ras proteins. The Ras homolog enriched in brain (Rheb) is predominantly found in endo-membranes, such as the endoplasmic reticulum and lysosomes where it directly activates mTORC1 to control cell growth and proliferation. Little is known about the molecular details and functional consequences of Rheb membrane engagement, interaction with lipids, and orientational dynamics. In this work, we combined molecular dynamics simulations, hidden Markov modeling and single-molecule FRET measurements to map the modes of membrane binding and orientation landscape of Rheb. We also assessed the functional consequence of these processes on mTORC1 activation through simulation-guided mutagenesis and cell signaling assays. We found that Rheb interconverts between two dominant orientation states through two intermediates, and that mutationally destabilizing some of these orientation states modulates mTORC1 activity. Together, these results establish membrane orientation dynamics as a functionally relevant phenomenon in the endo-membrane localized Rheb and suggest a broader role of orientational dynamics for the regulation of functional in lipid-modified small GTPases.