Solution Conformation of the Synthetic Bovine Proenkephalin-A209–237 by 1H NMR Spectroscopy

Abstract

Proenkephalin-A has been described to generate enkephalins, opoid peptides, and several derived peptides, which display various biological effects, including antinociception and immunological enhancement. Recently, we have isolated from bovine chromaffin granules a new antibacterial peptide, named enkelytin, which corresponds to the bisphosphorylated form of PEAP 209 -237 (Goumon, Y., Strub, J. M., Moniatte, M., Nullans, G., Poteur, L., Hubert, P., Van Dorsselaer, A., Aunis, D., and Metz-Boutigue, M. H. (1996) Eur. J. Biochem. 235, 516 -525). In this paper, the three-dimensional solution structure of synthetic PEAP 209 -237 was investigated by NMR. These studies indicate that this peptide, which is unstructured in water, folds into an ␣-helical structure in trifluoroethanol/water (1/1). NMR data revealed two possible three-dimensional models of PEAP 209 -237 . In both models, the proline residue Pro-227 induces a 90°h inge between two ␣-helical segments (Ser-215 to Ser-221 and Glu-228 to Arg-232) leading to an overall Lshaped structure for the molecule. The negative charge of PEAP 209 -237 and the low amphipathy of the two ␣-helical segments imply new mechanisms to explain the antibacterial activity of enkelytin.Proenkephalin-A (PEA), 1 the precursor of enkephalins (1), and several derived peptides (PEAP) (2, 3) have been described in neural, neuroendocrine, and immune cells (4). This precursor to the opioid pentapeptides is highly conserved from invertebrates (leech, mytilus) (5), Xenopus (6) to humans (7). Because relatively large amounts of enkephalins and enkephalincontaining peptides were present in adrenal medullary chromaffin granules, these organelles have been shown to represent an excellent model to study the intragranular processing of PEA (for review, see Ref. 8). Proteolytic degradation of PEA in adrenal medulla appears to start at the COOH-terminal region with the removal of peptide B, PEAP 209 -239 (9). Immunoreactive forms of peptide B can be found in various regions of brain and circulating in plasma (10). From bovine adrenal medulla, four peptide B variants were isolated corresponding to the addition of one, two, or three phosphate groups to each peptide chain (11, 12), the phosphorylation sites being clustered together at positions Ser-215, Ser-221, and Ser-223.Among the complex mixture of intragranular matrix components, we have identified enkelytin, an antibacterial peptide corresponding to the bisphosphorylated PEAP 209 -237 , which is included in peptide B (13). More recently, a new antibacterial peptide, exhibiting 98% sequence identity with bovine enkelytin, has been identified as derived from leech and mytilus PEA (14).2 In both mammals and invertebrates, opioid peptides stimulate immune cells, triggering chemotaxis as well as secretion of cytokines (4).Enkelytin is active at a concentration around the micromolar range against several Gram-positive bacteria including Staphylococcus aureus, but it is unable to inhibit the Gram-negative bacteria growth (13, 16). Furthermore, enkelytin has...

Characterization of Antibacterial COOH-terminal Proenkephalin-A-derived Peptides (PEAP) in Infectious Fluids

“…In conclusion, the antibacterial activity of enkelytin toward M. luteus is directly related to three structural parameters: (i) the length of the peptidic chain, (ii) the natural conformational constraints induced by the three proline residues Pro 212 , Pro 214 , Pro 227 , and (iii) the phosphorylation of Ser 221 and Ser 223 . Computer Model of PEAP 209 -237 -An extensive study using biophysical techniques has been carried out on PEAP 209 -237 in our laboratory (89). We refer to some of these data to draw up the computer model of PEAP 209 -237 fitting with the biological activity.…”

“…Several natural and synthetic enkelytin-derived peptides were also tested to determine the structural features necessary for the antibacterial activity. Then, the activity of these peptides was related with the ␣-helical structure, obtained from recent 1 H NMR data (89).…”

Kappacin, a Novel Antibacterial Peptide from Bovine Milk

“…The antibacterial mechanism of the chromaffin peptides has not been elucidated; however, recent 1 H nuclear magnetic resonance spectroscopic analysis of synthetic nonphosphorylated enkelytin demonstrated that the peptide adopts a proline-kinked amphipathic helical structure in 50% (vol/vol) trifluoroethanol (10). The structure of the phosphorylated form of enkelytin has not been determined; however, phosphorylation is likely to change the peptide's conformation through electrostatic repulsion or by divalent metal ion binding (5,10). The binding of divalent cations may act to stabilize the peptide structure but also may help to localize the peptide at the bacterial cell surface.…”

Animal antimicrobial peptides: An overview

“…A number of secondary structures of antimicrobial peptides, broadly representative of the above structural groups, have been determined by two-dimensional nmr, either in solution or in model membrane environments. They include cecropins, 89 -92 magainins, [93][94][95] PGLa, 96 sarcotoxin, 97 buforin, 98 caerin, 99 bactenecins, 100 enkelytin, 101 histatin, 102 ranalexin, 103 thanatin, 104 protegrin, 105 tachyplesin, 106 different types of defensins, [107][108][109][110][111][112][113][114][115] and drosomycin.…”