Structural and phylogenetic evidence supports a key role played by helix-3 of Euplotes pheromone structure in autocrine and heterologous pheromone/receptor interactions

Like many other organisms, ciliates rely on diffusible pheromones to socialize. In Euplotes, these cell signals form species-specific families of structurally homologous disulfide-rich globular proteins which bind their receptors on target cells in competition with one another, eliciting cell growth or mating responses according to whether the binding occurs in autocrine, or heterologous fashion, respectively. In each cell type, the receptors’ extracellular ligand binding domain is structurally identical with the soluble pheromone, as a result of a common gene determination via an intron-splicing mechanism. Given this structural context, the pheromone/receptor interactions were inquired by carrying out a comparative crystallographic analysis of pheromones Er-1 and Er-13 each specific to cells with strong mating compatibility. Er-1 and Er-13 crystals showed to markedly differ in their symmetry space groups, C2 and P43, respectively. Nonetheless, both crystals equally result from a tight association of molecules into linear chains, in which each molecule (i) rigorously takes an opposite orientation with respect to its neighborhood (as expected to be the case for pheromone and receptor molecules interacting on the cell surface), and (ii) forms contact interfaces relying on amino-acid side-chains lying in the great majority on helix-3 of its three-helical fold. This identification of helix-3 as central functional element of the pheromone molecular structure receives strong support from the tight conservation of the backbone structure that this helix shows at both intra-and inter-specific level andsuggests a parsimonious explanation for the molecular mechanisms underlying the competitive, autocrine and heterologous, pheromone/receptor binding reactions.