A cooperative model for receptor recognition and cell adhesion: evidence from the molecular packing in the 1.6-A crystal structure of the pheromone Er-1 from the ciliated protozoan Euplotes raikovi.

Abstract

The crystal structure of the pheromone Er-I from the unicellular eukaryotic organism Euplotes raikovi was determined at 1.6 A resolution and refined to a crystallographic R factor of 19.9%. In the tightly packed crystal, two extensive intermolecular helix-helix interactions arrange the Er-i molecules into layers. Since the putative receptor of the pheromone is a membrane-bound protein, whose extracellular C-terminal domain is identical in amino acid sequence to the soluble pheromone, the interactions found in the crystal may mimic the pheromone-receptor interactions as they occur on a cell surface. Based on this, we propose a model for the interaction between soluble pheromone molecules and their receptors. In this model, strong pheromone-receptor binding emerges as a consequence of the cooperative utilization of several weak interactions. The model offers an explanation for the results of binding studies and may also explain the adhesion between cells that occurs during mating.Pheromones from the ciliated protozoan Euplotes raikovi are proteins of 37-40 amino acids that function both as growth factors and as signaling molecules in cellular adhesion during mating (1-3). Over a dozen different cell types of E. raikovi have been identified based on their ability to form mating pairs (1). In the presence of only one cell type, the homologous secreted Er (Er-x = E. raikovi pheromone of type x) molecules stimulate growth of the cells by binding to cell-surface receptors in an autocrine fashion. In the presence of two different cell types or just one cell type to which heterologous pheromone has been added, Er molecules also stimulate cell adhesion between mating pair partners presumably by binding to the same cell-surface receptors (2, 3) in a paracrine manner. The putative receptors of the pheromones are membranebound proteins whose extracellular C-terminal domain is identical in amino acid sequence to the soluble pheromones (4). They arise by alternate splicing of the transcripts of the same gene that carries the information for the soluble pheromone (4).Different cell types can also be distinguished by the amino acid sequence of their pheromones. Sequences from nine different cell types have been determined (5-8), yielding seven unique sequences with pairwise sequence identities ranging from about 25% to 95%. Only seven residues are conserved among all sequences; these are the N-terminal aspartic acid and six cysteines that are involved in formation of three disulfide bridges (9).The NMR structures of three of the pheromones (Er-1, Er-2, and Er-10) have been determined and compared to each other (10-13). They revealed the three-helical bundle fold of the proteins and the orientations for about two-thirds of the side chains. The crystal structure reported here offers a detailed picture of the monomer and reveals two types of interactions between molecules that provide the basis for a model for receptor recognition, cell adhesion, and signaling. (14) is 1.53 A3/Da and the solvent content of the crystals is ...

NMR structure of the protein NP_247299.1: comparison with the crystal structure

“…We thus do not address the question of comparing absolute values of D and B in the NMR structure and the crystal structure. In some ways (3) corresponds to the 'inverse' of previous approaches to derive 'pseudo-B values' from NMR displacements in attempts to obtain improved models for molecular replacement in crystal structure determinations (Weiss et al, 1995;Wilmanns & Nilges, 1996). Global r.m.s.d.…”

Structural and functional analysis of Aplysia attractins, a family of water-borne protein pheromones with interspecific attractiveness

“…In particular, the second helix of attractin, containing the IEECKTS motif, is quite similar to the third helix of Er-11 (4). This third helix of the Euplotes pheromones is involved in receptor recognition (25,26). Although Euplotes pheromone sequences differ significantly, sharing only six cysteines and an N-terminal aspartic acid, all have the same compact ''pyramid'' 3D structure of three ␣-helices (25)(26)(27)(28)(29).…”

“…This third helix of the Euplotes pheromones is involved in receptor recognition (25,26). Although Euplotes pheromone sequences differ significantly, sharing only six cysteines and an N-terminal aspartic acid, all have the same compact ''pyramid'' 3D structure of three ␣-helices (25)(26)(27)(28)(29). The pheromone Er-1 can bind to the mammalian receptor for interleukin-2 (30), raising the possibility that water-borne pheromones may be ancestors of cytokines in higher organisms.…”

Autocrine, Mitogenic Pheromone Receptor Loop of the Ciliate Euplotes raikovi : Pheromone-Induced Receptor Internalization

“…This hypothesis was suggested earlier by the finding that, like autocrine pheromone binding, antibodies binding to p15 promote cell growth (31). Considering the similarity of the p15 ectodomain and Er-1 (20,24), it is a short extrapolation to propose the hetero-oligomerization of p15 and Er-1 from the crystal structural data of Er-1, which show homo-oligomerization (33). In addition, the Er-1 molecules pack in the crystal according to a pattern of cooperative protein-protein oligomerization, which is imposed by the capacity to interact with one another by means of all their surfaces provided by their three-helix bundle conformation.…”

“…The three-helix configuration of the p15 ectodomain was extrapolated from the equivalence of the primary structure of this domain with Er-1 (20). Clustering of the p15/Er-1 complexes was based on a pattern of cooperative association determined for the Er-1 molecules in the crystal structure (33). This pattern requires that every molecule utilizes all three of its helices to associate with two other molecules: one association (dimer 1) involves helix-1/helix-1 (yellow/ yellow) and helix-2/helix-2 (red/red) interactions, and the second association (dimer 2) involves helix-3/helix-3 (green/green) interactions.…”