Structural basis of stepwise G protein activation by a viral chemokine receptor

G protein-coupled receptors (GPCRs) govern diverse cellular responses and are crucial drug targets. However, the sequence of structural events from G protein recognition to GDP release has remained elusive. Here, we leveraged the viral chemokine GPCR US28 to capture transient activation states of the US28-G q complex. Using cryo-electron microscopy and an engineered chemokine superagonist, we determined three distinct complex structures, capturing the initial GDP-bound TL-state, the nucleotide-free C-state, and a previously unobserved intermediate bridging the two states, the T2C-state. These structures, along with simulations and functional data, provide high-resolution snapshots of the G protein activation trajectory and reveal a stepwise conformational mechanism that orchestrates G protein activation. This activation cascade closely parallels mechanisms proposed for human GPCRs, suggesting a conserved GPCR signaling mechanism.