Solution conformation of the synthetic bovine proenkephalin-A209-237 by 1H NMR spectroscopy.

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 PEAP209-237 (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 PEAP209-237 was investigated by NMR. These studies indicate that this peptide, which is unstructured in water, folds into an alpha-helical structure in trifluoroethanol/water (1/1). NMR data revealed two possible three-dimensional models of PEAP209-237. In both models, the proline residue Pro-227 induces a 90 degrees hinge between two alpha-helical segments (Ser-215 to Ser-221 and Glu-228 to Arg-232) leading to an overall L-shaped structure for the molecule. The negative charge of PEAP209-237 and the low amphipathy of the two alpha-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 been detected in bovine wound fluid and in bovine chromaffin and human polymorphonuclear cells secretions upon stimulation (16). In invertebrates, we have established that besides lipopolysaccharide, surgical trauma and electrical stimulation induce the production of enkelytin-like peptide. 2 In a recent study we have related the antibacterial activity of enkelytin with structural features (16). Using various synthetic peptides, we were able to conclude that the antibacterial activity of enkelytin results from three structural parameters: (i) the length of the peptidic chain, (ii) the endogenous conformations of the three proline residues Pro-212, Pro-214, and Pro-227, and (iii) the phosphorylation of the two serine residues Ser-221 and Ser-223.
In order to characterize the molecular mechanism by which enkelytin inhibits bacteria growth, it is important to determine the three-dimensional structure of this antibacterial peptide. In this paper we report the three-dimensional structure of the synthetic nonphosphorylated PEAP 209 -237 in aqueous trifluoroethanol solution, which is the first requirement for the structural analysis of enkelytin. We focused on the conformational changes induced in the active molecule by the phosphorylation of the two serine residues, Ser-221 and Ser-223. This study leads to the characterization of the COOH-terminal domain of PEA whose biological significance in neuroimmuno-modulation is revealed by its intact presence in organisms 500 million years divergent in evolution (14). 2
NMR Spectroscopy-For acquisition of NMR spectra, 2.3 mg of PEAP 209 -237 was dissolved in 700 l of aqueous 20 mM sodium acetate-d 6 buffer, pH 5.0, 50 mM NaCl with addition of deuterated trifluoroethanol (trifluoroethanol-d 3 ) to yield 50% v/v solution. The final peptide concentration corresponds to 1 mM. During all NMR experiments, the temperature was set to 283 K (10°C). The 2,2-dimethyl-2silapentane-5-sulfonate was used as internal chemical shift reference (0.00 ppm).
Spin systems identification experiments, COSY (18) and HOHAHA (19), were carried out at 500 MHz ( 1 H frequency) on a Bruker AMX 500 spectrometer. A 60-ms DIPSI2 (20) isotropic mixing pulse sequence with a B1 field strength of 9.4 kHz was used in the HOHAHA experiment. The spectral width was 6024 Hz in both dimensions, 512 t1 increments were recorded for the F1 dimension, and water signal suppression was achieved by presaturating the water frequency with a low power 2.5-s pulse. A SCUBA COSY (21) pulse sequence was used to measure 3 J NH-H␣ coupling constants in order to restore the intensity of H␣, which were close to the water frequency. 4096 points/ free induction decay were recorded for this experiment leading to a 1.5 Hz/point resolution.
NOE buildup were explored by recording several NOESY spectra at 500 MHz with mixing times ranging from 100 to 500 ms. A NOESY (22,23) spectrum was recorded on a 600-MHz DRX 600 spectrometer with mixing times of 500 ms.
Processing was performed on IBM RS/6000 computer using the program FELIX V2.10 (Biosym Inc.) and on SGI INDY R5000 computer using UXNMR software (Bruker). Data were zero-filled and processed by apodization with a 90°-shifted sine squared bell window in both dimensions before Fourier transformation. The residual water signal was removed by convolution of the time domain signal with a cosine (24). The final size of matrices was 2048 ϫ 1024 points for all spectra except the COSY for which a size of 4096 ϫ 1024 points was used. 3 J NH-H␣ coupling constants were fitted from one-dimensional slices corresponding to the NH-H␣ correlations extracted from the COSY spectrum.
Structure Calculations-A set of distance restraints was obtained by classifying NOE intensities on the 600-MHz 500-ms NOESY map as strong, medium, and weak, corresponding to interproton distance restraints of 2.7, 3.7, and 5.0 Å, respectively. An additional correction of 1.0 Å was added for methylene and methyl groups. These distances were included in the XPLOR 3.1 (25) force field (topallhdg.pro and parallhdg.pro files) as upper bonds soft-square potential functions. An harmonic potential was used to specify torsion angle restraints where needed (see below). The calculations used the simulated annealing protocol proposed by Nilges et al. (26) as implemented in the XPLOR 3.1 program package. Successive rounds of structure calculations were performed in an iterative manner with incorporation of initially ambiguous restraints. An all ␣ starting structure (template) was used in initial rounds of calculations. Latest calculations used a random set of initial structures. Average structures were calculated with the final set of refined structures and further energy minimized. 400 steps of energy minimization was calculated on average structures to ensure correct local geometry. The MOLMOL program (27) was used to visualize sets of structures and to calculate and draw the electrosatic surface potential of the final three-dimensional models.

RESULTS
NMR Spectroscopy-The complete proton assignment is presented in Table I and H␣ chemical shifts, short and medium range NOE patterns, and 3 J NH-H␣ coupling constants are summerized in Fig. 1. Only one set of resonances was found for all spin systems of the peptide except for the backbone protons of Leu-213. The structural picture that emerges from the NMR data presented in Fig. 1 corresponds to a conformation with two ␣-helical segments hinged around Pro-227. The first helical segment extends from residue Ser-215 to Ser-223 and the second one from Glu-228 to Arg-232. Prolines Cis-Trans Isomerism-Two NOE patterns characteristic of the cis-and trans-isomers of the Leu-213-Pro-214 peptide bond are detected. This observation, together with the presence of two sets of resonances obtained for Leu-213, clearly indicates the presence of a slow conformational exchange between the cis-and trans-conformations for Pro-214. In contrast, this exchange is not observed for the first proline residue Pro-212, for which the trans-isomer could be unambiguously characterized. The analysis of the NOE pattern involving the Pro-227 residue is in favor of Val-226-Pro-227 peptide bond being in the cis-conformation. However, due to the peak overlap that affects the Val-226 H␣-Pro-227 H␣ cross-peak, one cannot exclude completely a possible trans-conformation for the Val-226-Pro-227 peptide bond. Therefore two three-dimensional models corresponding to both cis-and trans-isomers of Val-226-Pro-227 peptide bond are presented.
Three-dimensional Model-169 distance constraints were derived from the analysis of the 500-ms NOESY map. Among them, 91 were (i, i ϩ 1) distances, 41 (i, i ϩ 2), 26 (i, i ϩ 3), and 10 were detected between residues separated by more than 3 amino acids. Additional dihedral angle constraints were used for amino acids where NOE pattern, H␣ chemical shifts, and 3 J NH-H␣ coupling constants indicated the presence of an helical conformation. Simulated annealing calculations were carried out with both cis-and trans-topology for Pro-227, resulting in two possible conformations for PEAP 209 -237 . For cis-and transisomers of Pro-227, the 25 conformers with the lowest overall energy were selected. None of these structures had distance violations greater than 0.5 Å and showed no dihedral angle deviations greater than 10°from the target value. Average energies for both sets of selected structures are a NOE and dihedral energies were calculated using the XPLOR 3.1 force field. The final values of the target function force constants were k NOE ϭ 50 kcal ⅐ mol Ϫ1 and k tor ϭ 200 kcal ⅐ mol Ϫ1 ⅐ rad Ϫ1 .
b The Lennard-Jones van de Waals energies were calculated on energy minimized average structures using CHARMM version 22 (47).
c The Ramachadran plot parameters were taken from the PRO-CHECK-NMR software (15).

FIG. 1. Summary of the sequential and medium-range NOE, H␣ chemical shift index (Wishart), and 3 J NH-H␣ coupling constants for PEAP 209 -237 at 10°C in a 50% trifluoroethanol-d 3 solution.
Filled and open squares indicate 3 J NH-H␣ coupling constants lower than 6.5 Hz and greater than 8.5 Hz, respectively. In the Wishart chemical shift index, a value of ϩ1 is assigned for residues whose H␣ chemical shift deviates from tabulated random coil value by more than 0.1 ppm. Continuous runs of ϩ1 values are statistically observed for ␣-helical conformations (28). The NOE connectivities are indicated by black lines with thickness proportional to the NOE intensity (weak, medium, strong).
given in Table II. These results indicate that NMR experimental data define a single set of structures for each isomer and that both sets of structures are compatible with experimental distance constraints. The superimposition of the 25 lowest energy structures for each Pro-227 conformation is shown in Fig.  2. In both conformations, the two helical segments (Ser-215- Ser-223, Glu-228-Arg-232) of the peptide are bent with an angle of 90°. This orientation is defined by 8 NOE between residues from both sides of Pro-227. However, the orientation of the second helix (Glu-228-Arg-232) in regard to the first one differs, for the trans-and cis-isomers of Pro-227, by an angle of 120°.
A ribbon diagram of the average structure of the PEAP 209 -237 is presented in Fig. 3 for both isomers of Pro-227. Three and a half-canonical helical turns are found in both models, which is in agreement with CD data predicting that a third of the peptide is folded in helical conformation (data not shown). It is worth noting that all aromatic side chains are located on the same side of the helix. The NH 2 -terminal part has no regular secondary structure due to the lack of medium range NOE observed in this region. The COOH-terminal part of the peptide, which contains the Met-enkephalin motif, is likely to experience some motional averaging that affects 3 J NH-H␣ coupling constants and NOE values in a different way. Due to the 1/r 6 dependence of the dipolar interaction, the helical conformation indicated by the observation of ␣n(i ϩ 3) and nn(i ϩ 3) NOE may be overestimated in the model (Fig. 3) and might correspond to only a fraction of all possible conformations.

DISCUSSION
The structural analysis of PEAP 209 -237 demonstrates that this peptide adopts an ␣-helical fold in trifluoroethanol/water solution. The folding of peptides in the presence of trifluoroethanol is a behavior that is often found for peptides interacting with membranes and has been widely used to study the three-dimensional structure of these molecules. In relation to antibacterial peptides, trifluoroethanol has been used to study the structure of various antibacterial peptides, including cecropin A (29), pardaxin P-2 (30), magainin 2 (31), and ranalexin (32). Futhermore, comparative study of NMR structures of magainin 2 and ranalexin in the presence of phospholipid micelles confirmed the helical structure previously found in trifluoroethanol/water solution (32,33).
NMR data indicate that PEAP 209 -237 is composed of two helical segments (Ser-215-Ser-223 and Glu-228-Arg-232) separated by the loop Lys-224-Pro-227, the proline residue Pro-227 inducing a 90°bend in the structure. The analysis of PEAP 209 -237 models indicates that the helical structures are stabilized by various favorable side chain interactions. The motif Glu-220-Lys-224 located at the end of the first helical segment corresponds to an electrostatic (i, i ϩ 4) interaction that has been found to be a major contribution in stabilizing short helices (34). A similar interaction is found in the second segment, where a salt bridge between Glu-228 and Arg-232 could stabilize the helical conformation. In addition, Pro-227 located at the NH 2 -terminal end of the second helix appears to be a good helix initiator as based on the NH 2 -capping process (35,36). Furthermore, the fractional helical conformation observed in the enkephalin motif from NOE data is probably stabilized by the side chain interactions of the two COOH-terminal aromatic residues Tyr-233 and Phe-236 (37). The hydrophobic character is expected to mediate the antibacterial activity. In enkelytin, hydrophobic residues Phe-209, Phe-236, and Met-237 are located on the NH 2 -and COOH-terminal ends of the molecule and furthermore the aromatic side chain of Phe-209, Tyr-233, Phe-236, and Met-237 are located on the same helical face.
Recently, it has been demonstrated that the residue F1 in the ranalexin peptide is essential for the expression of the antimicrobial activity (32), and studies about brevinins, gaegurins, and temporin (38) suggest the importance of this residue to induce peptide insertion.
The position of the two helical segments observed for PEAP 209 -237 peptide correlates well with a previous NMR study performed in our laboratory on the synthetic PEAP 224 -237 in 70% trifluoroethanol (data not shown). In this shorter peptide, NMR data indicated the presence of a helical conformation for residues Glu-228 to Gly-234. Thus, the comparison of the NMR data obtained for the short (PEAP 224 -237 ) and longer (PEAP 209 -237 ) COOH-terminal fragments of PEA leads to the conclusion that the folding of the COOH-terminal part of the PEAP 224 -237 is not affected by the addition of the NH 2terminal sequence.
Many studies have focused on the three-dimensional structure of the Met-enkephalin sequence (YGGFM), because of its properties to be able to bind to opioid receptors and induce morphine-like effects (1). A wide range of conformations has been found for enkephalin peptides in crystal (39), in Me 2 SO solution, or bound to membranes (for review, see Ref. 40), most of them involving ␤ turn and ␥ turn conformations, but no helical conformation has yet been described. It has been reported that any sequence addition to the NH 2 terminus of enkephalin peptides leads to the inhibition of binding to the opiate receptor (41). This inhibition may either be due to a steric hindrance or result from the induction of an helical conformation by the additional NH 2 -terminal sequence, a phenomenon also suggested by our present results.
The PEAP 209 -237 sequence contains three proline residues, two of these (Pro-214, Pro-227) beeing strictly conserved in currently known PEA sequences (13,16). It has been shown that the energy difference between the cis-and trans-isomers of prolines, a residue that is found with high frequency in membrane transport proteins (42), is reduced in non-polar solvents (43). Thus, cis/trans-isomerism may play a functional role in transmembrane helices (44). The ambiguous NOE pattern observed for Pro-227, while favoring the cis-conformation, did not allow us to discard the trans-conformation and led us to propose two models for the three-dimensional structure. In PEAP 209 -237 , the isomerization of the Val-226-Pro-227 peptide bond would result in a reorientation of the second helical segment relative to the first one by an angle of 120°. This conformational change, which preserves a spatial proximity between two potentially phosphorylated serine residues (Ser-221, Ser-223), located in the first helical segment, and two glutamic residues, located in the COOH-terminal helical segment (Glu-228, Glu-230), may be involved in triggering the antibacterial activity of the peptide. The electrostatic interactions induced by the spatial proximity between phosphorylated serine and glutamate residues may also be critical in inducing the active conformation.
The antibacterial bisphosphorylated PEAP 209 -237 , named enkelytin, shares properties with some others antibacterial peptides: helix-kink-helix (30,45), hydrophobic residues located at the ends of the molecule. However, the surface potential calculated for this peptide highlights its overall negative charge (Fig. 4). This feature is in favor of a different mechanism than the one proposed previously for positively charged compounds like cecropins or defensins, which form large pores in the bacterial membrane by direct peptide-membrane electrostatic interactions (46). Moreover, the amphipatic character of the two helical segments of PEAP 209 -237 is very low compared with pore-forming peptides.
Thus, the structural features of this COOH-terminal PEAP indicate that enkelytin may express its antibacterial activity by different potential mechanisms: (i) a pore-forming or carpet-like mechanism by interaction of its three basic residues with the negative phospholipids and/or glycolipids on bacterial membrane; (ii) a linkage with potential bacterial membrane receptors, including enzymes, pumps, or transporters; (iii) the binding of di-or trivalent ions necessary for the bacteria growth. The 1 H NMR study of the synthetic bisphosphorylated PEAP 209 -237 and of (Ser 3 Glu) mutants are currently in progress in our laboratory to address the role of electrostatic interactions involved in the antibacterial activity of enkelytin.