Extended Data Fig. 1: Starting model and sample snapshots of the β₂AR-G_s^GDP simulations, together with overview of relevant crystal structures.

A1) In the starting model, inactive Gs^GDP (PDB ID: 6EG8) is placed below the receptor with α5 in an orientation as the 14-mer-peptide derived from the C-terminus of α5, co-crystallized with the β₂AR (PDB ID: 6E67)¹⁶. Notably, Y391^α5 and H387^α5, that interact with the receptor in the crystal structure of β₂AR-Gs^empty (PDB ID: 3SN6, inset A4) are still buried (inset A1), while E392^α5 and R389^α5, that bind to the receptor in β₂AR-Gs^GDP (inset A2) and in the β₂AR-α5-peptide complex⁶ (inset A3), are solvent exposed and ready to interact. To provide sufficient space for structural adaptations that occur upon complex formation, in the initial model, the side chains of R389^α5 and E392^α5 were put at 23.5 and 15.0 Å away from the side chains of D130^3.49 and R131^3.50, respectively. A2) In the β₂AR-Gs^GDP complex, α5 adopts a similar orientation and binding mode as in the β₂AR-α5-peptide complex. A3) Crystal structure of the β₂AR-α5-peptide complex (PDB ID: 6E67)¹⁶, without showing the stabilizing modifications. The minor deviations observed when comparing this complex with the β₂AR-Gs^GDP complex (inset A2), were expected, because of the stabilizing modifications of the β₂AR-α5-peptide complex. A4) In β₂AR-Gs^empty (PDB ID 3SN6) α5 binds differently to the receptor when compared to the previous complexes. The inset shows that Y391^α5 and H387^α5, that are initially buried in Gs^GDP (inset A1), engage the receptor in the crystal structure of β₂AR-Gs^empty, while E392^α5 and R389^α5, that engage the receptor in the other two complexes, point away from the interface. B) G protein labels for subdomains as typically used in G-protein nomenclature (CGN¹³). C) Different orientations of α5 (the remainder of the G protein is hidden) relative to the receptor in β₂AR-G_s^GDP when compared to β₂AR-G_s^empty. Top view of α5 from the cytoplasmic side reveals that α5 is rotated by ca. 40° when the two complexes are compared. The side view highlights the different binding angles of α5 relative to the membrane normal.