Rotation of the N-terminal helix triggers Gα activation

Heterotrimeric G proteins, activated by G-protein-coupled receptors (GPCRs), are central mediators of cellular signaling. Activation requires GDP release from the Gα subunit, a step attributed largely to the C-terminal α5 helix, the primary GPCR-interacting region. Contributions from other Gα regions have remained unclear. Here we show that the N-terminal helix (αN) of Gαs promotes GDP release through a rotational conformational change. Diffracted X-ray tracking revealed that engagement with an active GPCR increases αN conformational displacement. Biochemical assays and molecular dynamics simulations showed that counterclockwise rotation of αN, viewed from the extracellular side, facilitates GDP release, whereas clockwise rotation does not. Mechanistically, this αN-mediated pathway acts synergistically with α5 conformational changes and propagates through the β-sheet network within the Ras homology domain to the P-loop and the αG helix. These findings identify an αN-driven activation pathway and extend the mechanistic framework for GPCR-triggered G protein activation. Receptor engagement rotates the N-terminal helix of the G protein alpha subunit, and this motion is relayed through the protein to release bound GDP, revealing an activation pathway that works together with the long-studied C-terminal route.