Biology subjects

Gorfe, A.

Publications and source records attributed to Gorfe, A..

4 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↗

bioRxiv2023

A new ferrocene derivative blocks KRAS localization and function by oxidative modification at His95.

Ras proteins are membrane-bound GTPases that regulate essential cellular processes at the plasma membrane (PM). Constitutively active mutations of K-Ras, one of the three Ras isoforms in mammalian cells, are frequently found in human cancers. Ferrocene derivatives, which elevate cellular reactive oxygen species (ROS), have shown to block the growth of non-small cell lung cancers (NSCLCs) harboring oncogenic mutant K-Ras. Here, we developed and tested a novel ferrocene derivative on the growth of human pancreatic ductal adenocarcinoma (PDAC) and NSCLC. Our compound inhibited the growth of K-Ras-dependent PDAC and NSCLC and abrogated the PM binding and signaling of K-Ras, but not other Ras isoforms. These effects were reversed upon antioxidant supplementation, suggesting a ROS-mediated mechanism. We further identified K-Ras His95 residue in the G-domain as being involved in the ferrocene-induced K-Ras PM dissociation via oxidative modification. Together, our studies demonstrate that the redox system directly regulates K-Ras PM binding and signaling via oxidative modification at the His95, and proposes a role of oncogenic mutant K-Ras in the recently described antioxidant-induced metastasis in K-Ras-driven lung cancers.

cell biology↗

bioRxiv2020

Long-range coupled motions underlie ligand recognition by a chemokine receptor

Chemokines are unusual class-A GPCR agonists because of their large size ([~]10 kDa) and binding at two distinct receptor sites: N-terminal domain (Site-I, unique to chemokines) and a groove defined by extracellular loop/transmembrane helices (Site-II, shared with all small molecule class-A ligands). Whereas binding at Site-II triggers receptor activation, the role of Site-I is not known. Structures and sequence analysis reveal that the receptor N-terminal domains (N-domains) are flexible and contain intrinsic disorder. Using a hybrid NMR-MD approach, we characterized the role of Site-I interactions for the CXCL8-CXCR1 pair. NMR data indicate that the CXCR1 N-domain becomes structured on binding and that the binding interface is extensive with 30% of CXCL8 residues participating in this initial interaction. MD simulations indicate that CXCL8 bound at Site-I undergoes extensive reorganization on engaging Site-II with several residues initially engaged at Site-I also engaging Site-II. We conclude that structural plasticity of Site-I interactions plays an active role in driving ligand recognition by a chemokine receptor.

biophysics↗