Analogues of
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-factor,
Saccharomyces cerevisiae tridecapeptide mating pheromone (H-Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr-OH), containing
p-benzoylphenylalanine (Bpa), aphotoactivatable group, and biotin as a
tag, were synthesized using solid-phase methodologies on a
p-benzyloxybenzyl alcohol polystyrene resin. Bpa was inserted at positions 1, 3, 5, 8, and 13 of
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-factorto generate a set of cross-linkable analogues spanning the pheromone. The biological activity (growtharrest assay) and binding affinities of all analogues for the
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-factor receptor (Ste2p) were determined.Two of the analogues that were tested, Bpa
1 and Bpa
5, showed 3-4-fold lower affinity than the
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-factor,whereas Bpa
3 and Bpa
13 had 7-12-fold lower affinities. Bpa
8 competed poorly with [
3H]-
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-factor forSte2p. All of the analogues tested except Bpa
8 had detectable halos in the growth arrest assay, indicatingthat these analogues are
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-factor agonists. Cross-linking studies demonstrated that [Bpa
1]-
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-factor, [Bpa
3]-
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-factor, [Bpa
5]-
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-factor, and [Bpa
13]-
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-factor were cross-linked to Ste2p; the biotin
tag on the pheromonewas detected by a NeutrAvidin-HRP conjugate on Western blots. Digestion of Bpa
1, Bpa
3, and Bpa
13cross-linked receptors with chemical and enzymatic reagents suggested that the N-terminus of thepheromone interacts with a binding domain consisting of residues from the extracellular ends of TM5-TM7 and portions of EL2 and EL3 close to these TMs and that there is a direct interaction between theposition 13 side chain and a region of Ste2p (F55-R58) at the extracellular end of TM1. The resultsfurther define the sites of interaction between Ste2p and the
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-factor, allowing
refinement of a model forthe pheromone bound to its receptor.