Extended Data Fig. 8: Position of conserved residues in Ste2 conformational states.

a, In Ste2•Ant, a water molecule makes a polar interaction with the main chain of Asn205^5x29, whilst in the Ste2^AL•Ag state this site is occupied by Tyr266^6x58, which undergoes a rotamer change to form a polar interaction with the side chain of Asn205^5x29. The rotamer change in Tyr266^6x58 is triggered by its interaction with Trp1 of α-factor. Atomic models of Ste2 in the antagonist (map threshold 0.013) or agonist-bound active-like state (map threshold 0.560) are shown in pale brown, and the antagonist or agonist α-factor is shown as sticks in purple. b, Two putative sterol molecules, putatively assigned as CHS (purple), were found juxtaposed to H7 in the inactive state Ste2•Ant (map threshold 0.010) but there was no density in this region in the active state Ste2^AL•Ag (map threshold 0.560). Note that there were densities corresponding to sterols in other positions adjacent to the intracellular half of the receptor, but these were not modelled as the densities were not sufficiently resolved. c, On activation, changes in the dimer interface are accompanied by a relative shift of protomer Ste2_B by ~3 Å towards the protomer Ste2_A. The models of Ste2•Ant, Ste2_A•Ag•G and Ste2_B•Ag•G are shown in grey, blue and green, respectively. d, The conserved LPLSSMWA motif (residues 289^7x49-296^7x56) on H7 is kinked inwards towards the receptor core in Ste2•Ant but forms an α-helix in Ste2•Ag•G. e, The inactive state (Ste2•Ant) is stabilized by a hydrogen bond formed between Q149^3x50 and S292^7x52. Upon activation (Ste2•Ag•G), Leu289^7x49 packs against Ile80^2x42 allowing Leu289^7x49 to interact with Ile471^H5.25 of the G protein α-subunit Gpa1. A hydrophobic core formed by Ile80^2x42, Leu289^7x49, and Ile471^H5.25 replace the polar lock present in the inactive state. f, The conserved Pro258^6x50 causes a kink in H6 in the inactive state. g, The side chain of Gln253^6x45 interacts with Trp295^7x55 of the LPLSSMWA motif in the inactive state, but undergoes a 180° flip upon activation as Gln253^6x45 moves towards Ser288^7x48 and Trp295^7x55 moves away to enable H7 to interact with H1 and H7 of the adjacent protomer. h, The conserved positively charged residues in TM5 (Lys225^5x49 and Arg233^5x57) undergo side chain changes upon activation to enable Gpa1 α5-helix binding and forms polar and ionic interactions with Gpa1.