Search

Trabajo presentado en el XVth Congress of the Spanish Biophysical Society, celebrado en Granada (España) del 10 al 12 de junio de 2015., Sexual development in ascomycetous fungi is initiated by the perception of diffusible peptide pheromones via G protein-coupled plasma membrane receptors (GPCRs). Binding of pheromone to the cognate GPCR elicits a range of cellular responses including transcriptional reprogramming, cell cycle arrest, shmoo formation and chemotropic growth. High-resolution NMR studies of the S. cerevisiae α-pheromone tridecapeptide (WHWLQLKPGQPMY) in solution identified a transient type II beta-turn spanning residues 7-10, which are required for activation of the cognate receptor. Here we have characterized the α-pheromone of Fusarium oxysporum, a fungal pathogen of plants and humans. The chemically synthesized decapeptide WCTWRGQPCW was shown to elicit a chemotropic response in F. oxysporum germ tubes which is dependent on the cognate GPCR Ste2. Substitution of the conserved G6 and Q7 residues by alanines abolished the biological activity of the peptide. In order to understand the structural bases of its biological properties the wild type (wt) peptide and a scrambled sequence (WRWPCCWGQT) were analysed by biophysical methods including NMR spectroscopy. By HPLC, the two peptides behave as single molecular species, with no disulphide bridges. Far-UV CD was consistent with the presence of a β-turn in the wt pheromone. 1H and 13C NMR spectra were recorded and assigned in H2O and H2O/TFE (70/30 v/v) mixtures. 13C chemical shifts confirm the oxidised state of the Cys residues in both peptides. In agreement with the CD data, the α-pheromone adopts a β-turn in H2O involving the central 4WRGQ7 sequence. By contrast, NMR data of the scrambled peptide do not provide evidence for a regular secondary structure in H2O solution. In the presence of TFE, both peptides showed stabilised secondary structure. On the basis of the chemical shifts and assigned NOEs the 3D structures were calculated. The structure of the α-pheromone is a βhairpin, containing the central 4WRGQ7 β-turn, stabilised by strong W4-W10 π/π interactions. In TFE, the scrambled sequence mainly adopts a β-turn conformation spanning the central 4PCCW7 residues. Collectively, these data indicate that the hairpin adopted by the wt pheromone could be relevant for its biological activity and for the establishment of interactions with the cognate receptor or other biological partners., Financial support from the Spanish MINECO (projects CTQ2011-22514, BIO2013- 47870-R and BFU2012-32404) is acknowledged.