Membrane Organization of Rheb and RhoA: Roles in Function and Allosteric Druggability

Chase Martin Hutchins (Advisor: Alemayehu A. Gorfe, MS, PhD)

Small GTPases of the Ras superfamily are membrane-bound molecular switches that regulate nearly all major cellular processes, and their dysregulation drives cancers, neurological disorders, and metabolic diseases. While anchored to cellular membranes, the catalytic domains of small GTPases undergo orientation dynamics, adopting distinct configurations that can occlude or expose effector-binding surfaces and modulate signaling output. These dynamics have been characterized in the Ras oncoproteins, but whether they extend across the superfamily and influence allosteric druggability has remained unknown.

In this dissertation, I address these questions through Rheb and RhoA, two small GTPases with complementary differences in subfamily lineage, membrane localization, and effector biology. Rheb activates mTORC1, the master regulator of cell growth, from endomembrane compartments, and its aberrant activity promotes tumorigenesis. RhoA regulates cytoskeletal organization from the plasma membrane, and its dysfunction enhances metastasis and worsens neurodegenerative disease. Combining molecular dynamics simulations, single-molecule FRET in cell-derived lipid nanodiscs, simulation-guided mutagenesis, cell-based signaling assays, probe-based molecular dynamics, and high-throughput virtual screening, I found that Rheb and RhoA adopt fundamentally different membrane organizational strategies, and that disrupting orientation-stabilizing interactions altered downstream signaling in both systems in distinct ways, suggesting that membrane organization modulates function through distinct mechanisms tuned to the biology of each GTPase. Probe-based simulations further revealed that membrane binding reshapes the allosteric pocket landscape of both proteins, and virtual screening against membrane-derived conformations identified candidate inhibitors currently undergoing experimental evaluation. Collectively, these findings suggest that membrane context shapes both the signaling function and druggability of small GTPases.

Advisory Committee:

• Alemayehu A. Gorfe, MS, PhD, Chair
• James M. Briggs, PhD
• Shane R. Cunha, PhD
• Carmen W. Dessauer, PhD
• Jeffrey A. Frost, PhD