Insights into the molecular basis of self/non-self recognition in the ciliate Euplotes from the determination of pheromone crystal structures

Species-specific families of protein pheromones, each encoded by one of a series of co-dominant alleles at the same genetic locus (the mat locus), are constitutively secreted by species of Euplotes and used as autocrine (autologous, or self) signals that promote the cell vegetative growth and paracrine (heterologous, or non-self) inducers of cell-cell unions in mating pairs. In E. raikovi, the structures of a number of pheromones (designated Er-1, Er-2, Er-3 and so forth) have been determined by solution NMR spectroscopy, and the structure of pheromone Er-1 has additionally been solved also by X-ray crystallography. In each cell type, the soluble pheromone finds a structural counterpart with the extracellular ligand binding domain of its membrane receptor, both the molecules being encoded by the same gene through a mechanism of intron-splicing. Given this genetic context, the protein-protein interactions in the crystals can be assumed to mimic those underlying the pheromone/receptor interactions on the cell surface. We used non-conventional ab initio crystal structure determination methods, that exploit the high-resolution of collected diffraction data, to compare the crystal structures of two pheromones, Er-1 (structure re-determined at a resolution of 0.7 Å) and Er-13 (structure de novo determined at a resolution of 1.4 Å), that are secreted by two strongly mating compatible cell types. In spite of sharing the same disulphide bond pattern and the same up-down-up three-helix fold, Er-1 and Er-13 differ markedly in the arrangement of the molecules in the crystals. The resulting intermolecular contacts assign Er-1 and Er-13 to distinct crystallographic space groups (C2 and P41, respectively), suggesting that the autocrine pheromone/receptor interactions on the cell surface are likely regulated by the capability of each pheromone to homo-oligomerize with an its own specific pattern. In addition, although adopting distinct crystal structures, Er-1 and Er-13 make a common use of their helix 3 to stabilize the inter-molecular crystal contacts, which implies that this helix likely plays a central role in allowing cells to establish paracrine pheromone/receptor interactions.