No abstract

FMRF-NH2 peptides which contain a conserved, identical C-terminal tetrapeptide but unique N terminus modulate cardiac contractility; yet, little is known about the mechanisms involved in signaling. Here, the structure-activity relationships (SARs) of the Drosophila melanogaster FMRF-NH2 peptides, PDNFMRF-NH2, SDNFMRF-NH2, DPKQDFMRF-NH2, SPKQDFMRF-NH2, and TPAEDFMRF-NH2, which bind FMRFa-R, were investigated. The hypothesis tested was the C-terminal tetrapeptide FMRF-NH2, particularly F1, makes extensive, strong ligand-receptor contacts, yet the unique N terminus influences docking and activity. To test this hypothesis, docking, binding, and bioactivity of the C-terminal tetrapeptide and analogs, and the FMRF-NH2 peptides were compared. Results for FMRF-NH2 and analogs were consistent with the hypothesis; F1 made extensive, strong ligand-receptor contacts with FMRFa-R; Y→F (YMRF-NH2) retained binding, yet A→F (AMRF-NH2) did not. These findings reflected amino acid physicochemical properties; the bulky, aromatic residues F and Y formed strong pi-stacking and hydrophobic contacts to anchor the ligand, interactions which could not be maintained in diversity or number by the small, aliphatic A. The FMRF-NH2 peptides modulated heart rate in larva, pupa, and adult distinctly, representative of the contact sites influenced by their unique N-terminal structures. Based on physicochemical properties, the peptides each docked to FMRFa-R with one best pose, except FMRF-NH2 which docked with two equally favorable poses, consistent with the N terminus influencing docking to define specific ligand-receptor contacts. Furthermore, SDNAMRF-NH2 was designed and, despite lacking the aromatic properties of one F, it binds FMRFa-R and demonstrated a unique SAR, consistent with the N terminus influencing docking and conferring binding and activity; thus, supporting our hypothesis.

Peptidergic signaling regulates cardiac contractility; thus, identifying molecular switches, ligand-receptor contacts, and antagonists aids in exploring the underlying mechanisms to influence health. Myosuppressin (MS), a decapeptide, diminishes cardiac contractility and gut motility. Myosuppressin binds to G protein-coupled receptor (GPCR) proteins. Two Drosophila melanogaster myosuppressin receptors (DrmMS-Rs) exist; however, no mechanism underlying MS-R activation is reported. We predicted DrmMS-Rs contained molecular switches that resembled those of Rhodopsin. Additionally, we believed DrmMS-DrmMS-R1 and DrmMS-DrmMS-R2 interactions would reflect our structure-activity relationship (SAR) data. We hypothesized agonist- and antagonist-receptor contacts would differ from one another depending on activity. Lastly, we expected our study to apply to other species; we tested this hypothesis in Rhodnius prolixus, the Chagas disease vector. Searching DrmMS-Rs for molecular switches led to the discovery of a unique ionic lock and a novel 3–6 lock, as well as transmission and tyrosine toggle switches. The DrmMS-DrmMS-R1 and DrmMS-DrmMS-R2 contacts suggested tissue-specific signaling existed, which was in line with our SAR data. We identified R. prolixus (Rhp)MS-R and discovered it, too, contained the unique myosuppressin ionic lock and novel 3–6 lock found in DrmMS-Rs as well as transmission and tyrosine toggle switches. Further, these motifs were present in red flour beetle, common water flea, honey bee, domestic silkworm, and termite MS-Rs. RhpMS and DrmMS decreased R. prolixus cardiac contractility dose dependently with EC50 values of 140 nM and 50 nM. Based on ligand-receptor contacts, we designed RhpMS analogs believed to be an active core and antagonist; testing on heart confirmed these predictions. The active core docking mimicked RhpMS, however, the antagonist did not. Together, these data were consistent with the unique ionic lock, novel 3–6 lock, transmission switch, and tyrosine toggle switch being involved in mechanisms underlying TM movement and MS-R activation, and the ability of MS agonists and antagonists to influence physiology.