Disordered C-Terminus Plays a Critical Role in the Activity of the Small GTPase Ran

Ran is a small GTPase of the Ras superfamily that governs nucleocytoplasmic transport, including that of miR-126, a microRNA that supports the homeostasis and expansion of leukemia stem cells (LSCs). Ran binds to Exportin 5 to facilitate the transport of precursor (pre)-miR-126 across the nuclear membrane for its maturation. Our goal is to inhibit Ran to prevent transport of pre-miR-126 to the cytoplasm. Like other Ras family proteins, targeting Ran with small molecules is challenging due to its relatively flat surface and lack of binding cavities. Ran's activity is regulated by a long and disordered C-terminus that provides opportunities for identifying cryptic binding pockets to target. We used a combination of molecular dynamics simulations and experiments and uncovered the critical role of the ensemble of the C-terminal conformations that enable the transition of Ran from the GTP-bound "on state" to its GDP-bound "off-state". We also showed that the Ran C-terminus allosterically modulates the conformations of residues in the nucleotide binding site and in the functionally relevant Switch 1 and 2 regions. Through computational deep mutational scans and experiments, we identified four residue hotspots L182, Y197, D200, and L201 at the core-C-terminus interface and four residue mutations V27A, E70D, N122A, and N122Y that mediate the allosteric communication between the core and switch regions. This information paves the way for our next step in the design of novel allosteric modulators for Ran.