Induced opening of conformational switch I in <scp>GTP</scp> ‐bound Ran <scp>GTPase</scp>

Ran is a small GTPase that regulates nucleocytoplasmic transport by cycling between an inactive GDP-bound and active GTP-bound state. Structurally, Ran consists of a globular G-domain and a C-terminal region that includes a C-terminal helix. The switch I and II regions of the G-domain undergo major conformational changes upon GTP hydrolysis, with switch I shifting from a closed to an open conformation, regulating cargo loading/unloading. In this study, we describe an induced conformational change in RanGTP that drives switch I from its closed, active state (state 2) to a fully open, inactive state, resembling the RanGDP conformation. This transition is consistently triggered by disruption of two key interactions: Thr42-Mg²⁺ coordination (leading to state 1) and the Phe35-GTP-Lys152 triad, the latter due to Lys152 reorientation. These findings identify Lys152, alongside Thr42, as critical for stabilizing the active Ran conformation, and show that disruption of their interactions promotes full opening of switch I. This structural mechanism highlights a previously unrecognized mode of RanGTP inactivation and may provide a conceptual basis for considering therapeutic approaches targeting dysregulated Ran and potentially other small GTPases, particularly in cancers driven by mutations that lock switch I in its active conformation.