The NMR solution structure of the pheromone Er‐11 from the ciliated protozoan Euplotes raikovi

Abstract

The NMR solution structure of the pheromone Er-11, a 39-residue protein from the ciliated protozoan Euplotes ruikovi, was calculated with the distance geometry program DIANA from 449 NOE upper distance constraints and 97 dihedral angle constraints, and the program OPAL was employed for structure refinement by molecular mechanics energy minimization in a water bath. For a group of 20 conformers used to characterize the solution structure, the average of the pairwise RMS deviations from the mean structure calculated for the backbone heavy atoms N, C a , and C' of residues 2-38 was 0.30 A. The molecular architecture is dominated by an up-down-up bundle of three short helices with residues 2-9, 12-19, and 22-32, which is closely similar to the previously determined structures of the homologous pheromones Er-1, Er-2, and Er-10. This finding provides structural evidence for the capability shown by these pheromones to compete with each other in binding reactions to their cell-surface receptors.

Structural characterization of a protein pheromone from a cold‐adapted (Antarctic) single‐cell eukaryote, the ciliate Euplotes nobilii

“…However, similar predictions for E. raikovi pheromones have been disproved by results from crystallographic and nuclear magnetic resonance analyses [15,17]. Not only have these analyses established that the structure of these molecules exclusively consists of a bundle of three α‐helices tightly associated by three disulfide bonds [6,14–16], or (limitedly to the case of pheromone E r ‐23) of a bundle of five helices interconnected by five disulfide bonds [17]; but they have also provided evidence that a key role in enforcing and stabilizing these configurations is played by the high density and strategic disposition of the disulfide bonds [17]. The fact that six of the eight E n ‐2 cysteines find their precise counterparts in the ‘standard’ six cysteines of E. raikovi pheromones (Fig.…”

“…Multiple sequence alignment between E n ‐2 and the E. raikovi pheromones was based on the CLUSTAL algorithm [25]. Only E. raikovi pheromones with six cysteines and a known three‐dimensional structure have been considered [6,14–16]. Cysteine residues (printed in bold) located in conserved positions between E n ‐2 and E. raikovi pheromones are marked by asterisks and paired by lines to denote the disulfide arrangement originally determined in E. raikovi pheromones E r ‐1 and E r ‐2 [26].…”

Cold-adaptation in Sea-water-borne Signal Proteins: Sequence and NMR Structure of the Pheromone En-6 from the Antarctic Ciliate Euplotes nobilii

“…1,2 For Blepharisma japonicum, Euplotes octocarinatus and Euplotes raikovi, which secrete their pheromones into the extracellular environment, these molecules have been chemically characterized, [3][4][5] and for six members of the E. raikovi pheromone family (collectively denoted "Er" and distinguished by a number) the three-dimensional structures were determined by NMR in solution [6][7][8][9][10][11] and by X-ray crystallography. 12 These data provided a platform for investigations of the functional biology of these molecules, 13,14 and led to the identification of structural relationships between the Er pheromones and another family of water-borne signal proteins, the "attractins", which control mate attraction and breeding aggregations in species of the marine gastropod Aplysia.…”

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

“…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.…”