Structural evolution of the water-borne protein pheromonefamilies ruling self/not-self recognition in the ciliateEuplotes:an updated scenario.

In ciliates, self/not-self recognition mechanisms (usually reported as “mating-type mechanisms”) regulate the cell switching from the reproductive/growth stage to the sexual/mating stage of the biological cycle. Species of Euplotesare all characterized by multiple mating-type systems, in which indefinite numbers of cell types are chemically distinct from one another for producing type-specific pheromones, each determined by one of a series of multiple single-locus alleles.The three-dimensional conformations of various members of theE. nobilii,E. raikoviandE. petzipheromone families have previously been determined by NMR spectroscopy and X-ray crystallography, prompting an initial picture of their degrees of intra-and inter-specific structural polymorphisms. Using these determined structures as templates in association with the AI predictive model AlphaFold 3, we have now resolved (with high degrees of reliability) the folding of pheromone amino acid sequences deduced from their coding genes cloned in species branching in key positions ofEuplotesphylogenetic tree. An updated picture of the structural evolution of Euplotespheromones suggests, first and more in general, that the course of this evolution may properly be synthesized by quoting the popular dictum “the earlier [structure], the smaller and simpler”. Second and more circumstantially, it identifies a regular alpha-helix as the secondary structure which is more tightly 28conserved at both intra-and inter-specific level. In addition to well accounting for the central role that this helix plays in driving and stabilizing the self (autocrine) and not-self (heterologous) pheromone/receptor interactions on the cell surface, this identification also provides a parsimonious explanation for common observations of pheromone-mediated inter-species mating inductions.