Anuran Host-Defense Peptides That Complex with Ca2+ Calmodulin and Inhibit the Synthesis of the Cell Signaling Agent Nitric Oxide by Neuronal Nitric Oxide Synthase

The Chemistry and Biological Activities of Peptides from Amphibian Skin Secretions

“…The amphipathic amphibian peptides inhibit nNOS by changing the shape of a cofactor or interacting with Ca 2+ /CaM, hindering its interaction with the CaM-docking site on nNOS and electron transfer from cofactors to heme in the reductase domain during NO formation. This mechanism is confirmed by evidences: (1) nNOS is inhibited and NO production is decreased when the peptides are added to nNOS in vitro, and nNOS activity can be partially recovered by adding Ca 2+ /CaM; (2) the function of calcineurin, another enzyme using Ca 2+ /CaM as a regulatory protein, is inhibited by frenatin 3 and citropin 1.1; , and (3) caerin 1.8, citropin 1.1, dahlein 5.6, and spendipherin form globular complexes with Ca 2+ /CaM under electrospray ionization conditions. − In addition, three-dimensional structures of frenatin 3 demonstrate amphibian peptide binding to Ca 2+ /CaM. An amphipathic α-helix of residues 1–14 forms in frenatin 3, whose 8 residues at the C-terminus are less structured and more flexible in trifluoroethanol/water mixture.…”

Structural Analysis of Calmodulin Binding by nNOS Inhibitory Amphibian Peptides

“…6−8 Additionally, the peptides may also form an integral part of the animal's selfdefense mechanisms by interfering with NO signaling in an attacking predator or pathogen. 5 A thorough and complete analysis of the amphibian integument and its roles in both physiological regulation and self-defense is vital to ensure the continued survival of declining amphibian populations worldwide.…”

“…13 A number of different CaM/peptide complexes have been shown to adopt this conformation, or some variant of it. 14 Evidence suggests that the amphibian peptides inhibit NO production by interacting with CaM (reviewed by Doyle et al 5 ). This includes in vitro studies where dose−response curves showed a Hill slope greater than 1, indicating a noncompetitive interaction is causing nNOS inhibition.…”

“…Also, it has been shown that the addition of excess CaM is able to partially rescue nNOS activity in an amphibian peptide treated sample, and that these peptides also inhibit the function of calcineurin, another CaM regulated enzyme. 5 Finally, NMR and MS data have shown that the peptides complex with CaM in a high-affinity fashion. 22,23 Together, these data suggest that attenuation of NO production is achieved by the peptide binding to CaM, rendering the protein unable to associate with nNOS and trigger NO synthesis.…”

“…2,3 A number of other peptides have also been isolated which cannot be categorized by this classification system. 5 Integrated approaches to structural biology, combining data from complementary techniques, is becoming increasingly common to overcome challenges associated with individual methods. In this work, the complementary techniques of isothermal titration calorimetry (ITC) and ion mobility-mass spectrometry (IM-MS) were used to afford low resolution structural information about the complexes formed between Ca 2+ 4 CaM and the amphibian peptides caerin 1.8, caerin 1.8.11, citropin 1.1 (mod.…”