Cold‐adapted signal proteins: NMR structures of pheromones from the antarctic ciliate Euplotes nobilii

Abstract

SummaryCell type-specific signal proteins, known as pheromones, are synthesized by ciliated protozoa in association with their self/nonself mating-type systems, and are utilized to control the vegetative growth and mating stages of their life cycle. In species of the most ubiquitous ciliate, Euplotes, these pheromones form families of structurally homologous molecules, which are constitutively secreted into the extracellular environment, from where they can be isolated in sufficient amounts for chemical characterization. This paper describes the NMR structures of En-1 and En-2, which are members of the cold-adapted pheromone family produced by Euplotes nobilii, a species inhabiting the freezing coastal waters of Antarctica. The structures were determined with the proteins from the natural source, using homonuclear 1 H NMR techniques in combination with automated NOESY peak picking and NOE assignment. En-1 and En-2 have highly homologous global folds, which consist of a central three-a-helix bundle with an up-down-up topology and a 3 10 -helical turn near the N-terminus. This fold is stabilized by four disulfide bonds and the helices are connected by bulging loops. Apparent structural specificity resides in the variable C-terminal regions of the pheromones. The NMR structures of En-1 and En-2 provide novel insights into the cold-adaptive modifications that distinguish the E. nobilii pheromone family from the closely related E. raikovi pheromone family isolated from temperate waters. IUBMB Life, 59: 578-585, 2007

Characterization of the pheromone gene family of an Antarctic and Arctic protozoan ciliate, Euplotes nobilii

“…In the case of the en-ant1, en-ant2 and en-ant6 genes, the predicted mature sequences fully match the sequences of the En-1, En-2 and En-6 pheromones that have previously been determined by chemical analysis of the native proteins [10,11,20,21]. This matching thus strongly corroborates the identification of the ATG codon in position 362 with the effective site of initiation of transcription.…”

“…Previously we determined the conformations of a set of these E. nobilii pheromones by analysis of nuclear magnetic resonance of native protein preparations, and showed the relationships of structural homology that make these molecules able to bind their target cell receptors in competition with one another [10,11]. Now we have determined the full-length sequences of the functional genes that, in form of individual and multiple "gene-sized DNA molecules" [12, as a review], develop from the chromosomic locus mat of the micronucleus and determine the synthesis of these pheromones in the transcriptionally active genome of the somatic nucleus (macronucleus).…”

“…Three genes (designated en-ant1, en-ant2 and en-ant6) encode the pheromones En-1, En-2, and En-6 of Antarctic origin, that have already been isolated from preparations of culture supernatant and structurally characterized [10,11]; the four other genes (designated en-arc1, en-arc2, en-arc3, and en-arc4) specify the pheromones En-A1, En-A2, En-A3, and En-A4 of Arctic origin, that have not yet been purified and analyzed chemically. …”

Antarctic and Arctic populations of the ciliate Euplotes nobilii show common pheromone-mediated cell-cell signaling and cross-mating

“…3A). However, despite this low extent of amino acid sequence identity, close homology among the three-dimensional NMR solution structures of the three Antarctic pheromones was previously established (20)(21)(22). We have now investigated that this structural homology extends to the pheromones of the Arctic strains, by purifying the pheromone En-A1 from the strain 4Pyrm4 (which can be grown in large cultures more easily than any of the other Arctic strains) and by determining its NMR structure in solution (Table S4).…”

“…The horizontal lines at the top indicate the disulfide bonds, which are common to both groups of pheromones. The boxes identify the locations of helical secondary structures in the four pheromones for which three-dimensional structures are available (20)(21)(22). (B) Ribbon presentations of the previously determined En-1, En-2 and En-6 pheromone solution NMR structures from the Antartic E. nobilii strains AC-1 and AC-4 (20,21).…”