Structure‐function analysis of the Saccharomyces cerevisiae tridecapeptide pheromone using alanine‐scanned analogs

Abstract

Twenty‐six peptide analogs of the Saccharomyces cerevisiaeα‐factor, a tridecapeptide mating pheromone (W1H2W3L4Q5L6K7P8G9Q10p11M12Y13) with either l‐ or D‐alanine replacement of each amino acid residue (Ala‐scanned) and with the isosteric replacement of methionine at position 12 by norleucine, were synthesized, purified to homogeneity and assayed for biological activity and receptor binding. Two new and effective antagonists. [D‐Ala3,Nle12]α‐factor and [D‐Ala4,Nle12]α‐factor, were found among the series, and the [D‐Ala10,Nle12]α‐factor demonstrated a marked ability to increase the biological activity of [Nle12]α‐factor without having any effect by itself. One analog, the [L‐Ala1α‐factor, showed a 3‐fold increase in bioactivity over the [Nle12]α‐factor, although its binding to the α‐factor receptor was about 70‐fold less than [Nle12]α‐factor. Residues near the carboxyl terminus contributed more strongly to receptor binding than other residues, whereas those near the amine terminus of the α‐factor played an important role in signal transduction. The effect of insertion of D‐Ala residues at positions 7, 8, 9 and 10 on bioactivity and receptor binding of the peptide suggested a specific positioning role of the central loop in establishing optimal contacts between the receptor and the ends of the pheromone. We conclude that the α‐factor may be divided into segments with dominant roles in forming the biologically active pheromone conformation, in receptor binding and in initiating signal transduction. The discovery of such relationships was made possible by the systematic variation of each residue in the peptide and by the testing of each analog in highly defined biological and binding assays.

Interspecies pheromone signaling promotes biofilm formation and same-sex mating in Candida albicans

“…Notably, peptide 2 induced stronger FIG1 expression than the native C. albicans pheromone. A similar phenomenon was previously described in S. cerevisiae, where several single alanine-substituted analogs showed increased bioactivity by more than threefold (29).…”

“…The only peptide that was not active was peptide A, which is lacking the first three residues at the N terminus. Substitutions at position 11 of the α pheromone (in peptides 10 and 11) also had a very significant affect on pheromone activity, and reduced mating signaling to a larger extent than that seen with the corresponding substitution in S. cerevisiae (29). Overall, these data indicate that an important region for bioactivity exists near position 11, but the first three amino acids in particular do not contain critical amino acid contacts for signaling through C. albicans Ste2.…”

“…In this case, single alanine substitutions revealed that no side chain is absolutely required for activity (29,33). In general, alanine analogs resulted in one order of magnitude variation in bioactivity, and included analogs with both increased and decreased activity.…”

Multiple regulatory roles of the carboxy terminus of Ste2p a yeast GPCR

“…The WT STE2 gene containing a native promoter was cloned into the yeast/bacterial shuttle vector, pGA314.WT (12). For site-directed mutagenesis of STE2 (refer to Fig.…”

“…Filter discs (sterile blanks from Difco), 6 mm in diameter, were impregnated with 10-μl portions of the α-factor solutions at various concentrations and placed onto the overlay. The αfactor used in this study was [Nle12]α-factor (18), which is isosteric, equally active, and has the same binding affinity as the WT (WT) pheromone (12). The plates were incubated at 30°C for 24–36 hr and examined for the presence of clear zones (halos) around the discs.…”