Biology subjects

Hutchins, C. M.

Publications and source records attributed to Hutchins, C. M..

3 recordsLinked to original sources

bioRxiv2026

Catalytic domain-lipid interactions negatively regulate RhoA signaling by modulating conformational dynamics

RhoA belongs to the Ras superfamily of small GTPases, regulates cytoskeletal actin organization and cell motility, and is implicated in cancer metastasis and neurodegenerative diseases. It is predominantly anchored to the cytosolic side of the plasma membrane (PM) via its prenylated C-terminus, but little is known about its PM organization, dynamics, and lipid interactions. In this work, we used extensive atomistic molecular dynamics (MD) simulations and simulations-guided experiments to study the molecular details and functional consequences of RhoA membrane targeting, interactions and dynamics. We found that both GDP- and GTP-bound RhoA adopt a well-defined, stable membrane organization and binding mode irrespective of initial conditions and bilayer lipid composition. RhoA membrane organization is determined by hydrophobic interaction of the prenylated C-terminus with the membrane, and a previously unknown, critical role of electrostatic interactions between cationic residues on the G-domain and N-terminus with anionic lipids in the PM. N-terminal interactions are mediated by a polybasic patch including residues R5, K6, and K7, and substitution of these residues with Ala increased RhoA-mediated F-actin accumulation and effector activation in cells. MD simulations suggest that the triple mutant retains the global membrane organization of wild type RhoA but alters key protein-lipid interactions. Specifically, we observe a loss of cooperative lipid engagement involving the polybasic patch and Switch II residues with amine-bearing lipids, and a rerouting of intra-protein dynamic networks that primes Switch I for effector recognition. These results suggest that the membrane serves not just as a scaffold but also as an allosteric partner of RhoA to fine-tune intra-protein dynamics in a functionally relevant manner.

biophysics↗

bioRxiv2026

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.

biophysics↗

bioRxiv2024

Intrinsically disordered membrane anchors of Rheb, RhoA and DiRas3 small GTPases: Molecular dynamics, membrane organization, and interactions

Protein structure has been well established to play a key role in determining function; however, intrinsically disordered proteins and regions (IDPs and IDRs) defy this paradigm. IDPs and IDRs exist as an ensemble of structures rather than a stable 3D structure yet play essential roles in many cell signaling processes. Nearly all Ras Superfamily GTPases are tethered to membranes by a lipid tail at the end of a flexible IDR. The sequence of these IDRs are key determinants of membrane localization, and interactions between the IDR and the membrane have been shown to affect signaling in RAS proteins through modulation of dynamic membrane organization. Here we utilized atomistic molecular dynamics simulations to study the membrane interactions, conformational dynamics, and lipid sorting of three IDRs from small GTPases Rheb, RhoA and DiRas3 in model membranes representing their physiological target membranes. We found that complementarity between lipidated IDR sequence and target membrane lipid composition is a determinant of conformational plasticity. We also show that electrostatic interactions between anionic lipids and basic residues on IDRs generate semi-stable conformational sub-states, and a lack of these residues leads to greater conformational diversity. Finally, we show that small GTPase IDRs with a polybasic domain alter local lipid composition by segregating anionic membrane lipids, and, in some cases, excluding other lipids from their immediate proximity in favor of anionic lipids.

biophysics↗