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, namedenkelytin, 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 α-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° hinge between two α-helical segments (Ser-215 to Ser-221 and Glu-228 to Arg-232) leading to an overallL-shaped structure for the molecule. The negative charge of PEAP209–237 and the low amphipathy of the two α-helical segments imply new mechanisms to explain the antibacterial activity of enkelytin. 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, namedenkelytin, 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 α-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° hinge between two α-helical segments (Ser-215 to Ser-221 and Glu-228 to Arg-232) leading to an overallL-shaped structure for the molecule. The negative charge of PEAP209–237 and the low amphipathy of the two α-helical segments imply new mechanisms to explain the antibacterial activity of enkelytin. Proenkephalin-A (PEA), 1The abbreviations used are: PEA, proenkephalin-A; PEAP, proenkephalin-A-derived peptide; HOHAHA, HOmonuclear HArtman HAhn; NOE, Nuclear Overhauser Enhancement; NOESY, NOE SpectroscopY; r.m.s., root mean square. the precursor of enkephalins (1Hughes J. Smith T.W. Kosterlitz H.W. Fothergill L.A. Morgan B.A. Morris R.H. Nature. 1975; 258: 577-579Crossref PubMed Scopus (2942) Google Scholar), and several derived peptides (PEAP) (2Lewis R.V. Stern A.S. Kimura S. Rossier J. Brink L. Gerber L.D. Stein S. Udenfriend S. Adv. Biochem. Psychopharmacol. 1980; 22: 167-179PubMed Google Scholar, 3Lewis R.V. Stern A.S. Annu. Rev. Pharmacol. Toxicol. 1983; 23: 353-372Crossref PubMed Scopus (67) Google Scholar) have been described in neural, neuroendocrine, and immune cells (4Stefano G.B. Scharrer B. Smith E.M. Hughes T.J. Magazine H.I. Bilfinger T.V. Hartman A.R. Fricchione G.L. Liu Y. Makman M.H. Crit. Rev. Immunol. 1996; 16: 109-144Crossref PubMed Google Scholar). This precursor to the opioid pentapeptides is highly conserved from invertebrates (leech, mytilus) (5Salzet M. Stefano G.B. Brain Res. 1997; 768: 224-232Crossref PubMed Scopus (62) Google Scholar), Xenopus (6Martens G.J.M. Herbert E. Nature. 1984; 310: 251-254Crossref PubMed Scopus (92) Google Scholar) to humans (7Comb M. Seeburg P.H. Adelman J. Eiden L. Herbert E. Nature. 1982; 295: 663-666Crossref PubMed Scopus (500) Google Scholar). Because relatively large amounts of enkephalins and enkephalin-containing 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. 8Dillen L. Miserez B. Claeys M. Aunis D. De Potter W. Neurochem. Int. 1993; 22: 315-352Crossref PubMed Scopus (73) Google Scholar). Proteolytic degradation of PEA in adrenal medulla appears to start at the COOH-terminal region with the removal of peptide B, PEAP209–239 (9Liston D. Patey G. Rossier J. Verbanck P. Vanderhaeghen J.J. Science. 1984; 225: 734-737Crossref PubMed Scopus (99) Google Scholar). Immunoreactive forms of peptide B can be found in various regions of brain and circulating in plasma (10Lindberg I. White L. Biochem. Biophys. Res. Commun. 1986; 139: 1024-1032Crossref PubMed Scopus (11) Google Scholar). 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 (11D'Souza N.B. Lindberg I. J. Biol. Chem. 1988; 263: 2548-2552Abstract Full Text PDF PubMed Google Scholar, 12Watkinson A. Young J. Varro A. Dockray G.J. J. Biol. Chem. 1989; 264: 3061-3065Abstract Full Text PDF PubMed Google Scholar), 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 PEAP209–237, which is included in peptide B (13Goumon Y. Strub J.M. Moniatte M. Nullans G. Poteur L. Hubert P. Van Dorsselaer A. Aunis D. Metz-Boutigue M.H. Eur. J. Biochem. 1996; 235: 516-525Crossref PubMed Scopus (80) Google Scholar). More recently, a new antibacterial peptide, exhibiting 98% sequence identity with bovine enkelytin, has been identified as derived from leech and mytilus PEA (14Stefano G.B. Salzet B. Fricchione G.L. Immunol. Today. 1998; 19: 243-246Abstract Full Text Full Text PDF Scopus (98) Google Scholar). 2M. Salzet, B. Salzet, A. Tamieski, M. Verger-Bocquet, Y. Goumon, D. Aunis, M.-H. Metz-Boutigue, M. Cadet, and G. B. Stefano, submitted for publication. In both mammals and invertebrates, opioid peptides stimulate immune cells, triggering chemotaxis as well as secretion of cytokines (4Stefano G.B. Scharrer B. Smith E.M. Hughes T.J. Magazine H.I. Bilfinger T.V. Hartman A.R. Fricchione G.L. Liu Y. Makman M.H. Crit. Rev. Immunol. 1996; 16: 109-144Crossref PubMed Google Scholar). 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 (13Goumon Y. Strub J.M. Moniatte M. Nullans G. Poteur L. Hubert P. Van Dorsselaer A. Aunis D. Metz-Boutigue M.H. Eur. J. Biochem. 1996; 235: 516-525Crossref PubMed Scopus (80) Google Scholar, 16Goumon Y. Lugardon K. Kieffer B. Lefèvre J.F. Van Dorsselaer A. Aunis D. Metz-Boutigue M.H. J. Biol. Chem. 1998; 273: 29847-29856Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Furthermore, enkelytin has been detected in bovine wound fluid and in bovine chromaffin and human polymorphonuclear cells secretions upon stimulation (16Goumon Y. Lugardon K. Kieffer B. Lefèvre J.F. Van Dorsselaer A. Aunis D. Metz-Boutigue M.H. J. Biol. Chem. 1998; 273: 29847-29856Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). 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 (16Goumon Y. Lugardon K. Kieffer B. Lefèvre J.F. Van Dorsselaer A. Aunis D. Metz-Boutigue M.H. J. Biol. Chem. 1998; 273: 29847-29856Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). 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 PEAP209–237 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 neuroimmunomodulation is revealed by its intact presence in organisms 500 million years divergent in evolution (14Stefano G.B. Salzet B. Fricchione G.L. Immunol. Today. 1998; 19: 243-246Abstract Full Text Full Text PDF Scopus (98) Google Scholar).2 PEAP209–237 was synthesized in our laboratory on an Applied Biosystems 432A peptide synthesizer, SYNERGY, using the stepwise solid-phase synthetic approach (17Merrifield R.B. J. Am. Chem. Soc. 1963; 85: 2149-2154Crossref Scopus (6751) Google Scholar) with 9-fluorenylmethoxycarbonyl (Fmoc chemistry). Peptide was further purified by reverse-phase high performance liquid chromatography on a preparative Macherey-Nagel column, Nucleosil RP 300–7C18 (10 × 250 mm). For acquisition of NMR spectra, 2.3 mg of PEAP209–237 was dissolved in 700 μl 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-2-silapentane-5-sulfonate was used as internal chemical shift reference (0.00 ppm). Spin systems identification experiments, COSY (18Aue W.P. Bartholdi E. Ernst R.R. J. Chem. Phys. 1976; 64: 2229-2246Crossref Scopus (3100) Google Scholar) and HOHAHA (19Braunschweiller L. Ernst R.R. J. Magn. Reson. 1983; 53: 521-528Google Scholar), were carried out at 500 MHz (1H frequency) on a Bruker AMX 500 spectrometer. A 60-ms DIPSI2 (20Shaka A.J. Lee C.J. Pines A. J. Magn. Reson. 1988; 77: 274-293Google Scholar) 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 (21Brown S.C. Weber P.L. Mueller L. J. Magn. Reson. 1987; 77: 166-169Google Scholar) pulse sequence was used to measure3 J NH-Hα coupling constants in order to restore the intensity of Hα, 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 (22Jeener J. Meier B.H. Bachmann P. Ernst R.R. J. Chem. Phys. 1979; 71: 4546-4553Crossref Scopus (4845) Google Scholar, 23Kumar A. Ernst R.R. Wüthrich K. Biochem. Biophys. Res. Commun. 1980; 95: 1-6Crossref PubMed Scopus (2032) Google Scholar) 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 (24Marion D. Ikura M. Bax A. J. Magn. Reson. 1989; 84: 425-430Google Scholar). The final size of matrices was 2048 × 1024 points for all spectra except the COSY for which a size of 4096 × 1024 points was used. 3 J NH-Hα coupling constants were fitted from one-dimensional slices corresponding to the NH-Hα correlations extracted from the COSY spectrum. 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 (25Brünger A.T. Xplor Software Manual,Version 3.1, Ed. Yale University Press, New Haven, CT1992Google Scholar) 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. (26Nilges M. Clore G.M. Gronenborn A.M. FEBS lett. 1988; 239: 129-136Crossref PubMed Scopus (523) Google Scholar) 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 α 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 (27Koradi R. Billeter M. Wüthrich K. J. Mol. Graph. 1996; 14: 51-55Crossref PubMed Scopus (6498) Google Scholar) was used to visualize sets of structures and to calculate and draw the electrosatic surface potential of the final three-dimensional models. The complete proton assignment is presented in Table I and Hα chemical shifts, short and medium range NOE patterns, and3 J NH-Hα 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 α-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.Table IProton assignments and 3JNH-Hα coupling constants for the PEAP209–237 peptide in a 50% trifluoroethanol-d3 solution (v/v) pH 5.0 at 10 °C.Amino acid3JNH-Hα(Hz)Chemical shifts in a new NOE of the and of the peptide are This together with the presence of two sets of resonances obtained for the presence of a conformational between the and for In this is for the first proline residue Pro-212, for which the be The analysis of the NOE the Pro-227 residue is in of peptide being in the to the that the Hα one a possible for the peptide two three-dimensional models corresponding to both and of peptide are distance were derived from the analysis of the 500-ms NOESY Among were and 10 were detected between residues by 3 angle were used for where NOE Hα chemical shifts, and3 J NH-Hα coupling constants the presence of an helical annealing calculations were carried out with both and for Pro-227, in two possible conformations for PEAP209–237. For and of Pro-227, the with the energy were of these structures distance Å and angle from the Average for both sets of structures are in Table These results indicate that NMR data a set of structures for each and that both sets of structures are with distance The of the energy structures for each Pro-227 conformation is shown in In both the two helical segments of the peptide are with an angle of This is by NOE between residues from both of Pro-227. the of the second in to the first one for the and of Pro-227, by an angle of and root mean for the of calculated structures of the PEAP209–237 peptide for the and of peptide from restraints from and were calculated using the XPLOR 3.1 force The final of the force constants were NOE 50 and and were calculated using the XPLOR 3.1 force The final of the force constants were NOE 50 and were calculated on energy average structures using were from the software in in additional is in NOE and were calculated using the XPLOR 3.1 force The final of the force constants were NOE 50 and The were calculated on energy average structures using S. M. J. Chem. 1983; Scopus Google The were from the software J.M. J. 1993; Google Scholar). in a new The is in A of the average structure of the PEAP209–237 is presented in for both of Pro-227. and a helical are found in both models, which is in with data that a of the peptide is in helical conformation is that all are on the of the The has structure to the of medium range NOE in this The COOH-terminal of the peptide, which the is to that J NH-Hα coupling constants and NOE in a to the of the the helical conformation by the of 3) and 3) NOE be in the model 3) and to a of all possible The structural analysis of PEAP209–237 that this peptide an α-helical in trifluoroethanol/water solution. The of peptides in the presence of trifluoroethanol is a that is found for peptides with and has been used to study the three-dimensional structure of these In to antibacterial peptides, trifluoroethanol has been used to study the structure of various antibacterial peptides, including A A. G. H. Gronenborn A.M. Clore G.M. 1988; PubMed Scopus Google Scholar), C.J. K. PubMed Scopus Google Scholar), D. M. Bax A. FEBS 1988; PubMed Scopus Google Scholar), and E. A. P. L. G. B. A. Eur. J. Biochem. 1998; PubMed Scopus Google Scholar). study of NMR structures of and in the presence of the helical structure found in trifluoroethanol/water solution E. A. P. L. G. B. A. Eur. J. Biochem. 1998; PubMed Scopus Google Scholar, J. M. J. NMR. 1997; PubMed Scopus Google Scholar). NMR data indicate that PEAP209–237 is of two helical segments and by the the proline residue Pro-227 a 90° in the The analysis of PEAP209–237 models that the helical structures are by various chain The at the of the first helical segment corresponds to an that has been found to be a in short S. S. A. 1987; 84: PubMed Scopus Google Scholar). A is found in the second where a between Glu-228 and the helical In Pro-227 at the of the second appears to be a as on the D. P. PubMed Scopus Google Scholar, J. Res. 1997; PubMed Scopus Google Scholar). Furthermore, the helical conformation in the from NOE data is by the chain of the two COOH-terminal residues and Scopus Google Scholar). The is to the antibacterial In enkelytin, residues and are on the and COOH-terminal of the molecule and the chain of and are on the helical Recently, it has been that the residue F1 in the peptide is for the of the activity E. A. P. L. G. B. A. Eur. J. Biochem. 1998; PubMed Scopus Google Scholar), and studies and M. G. S. R. D. Eur. J. Biochem. 1996; PubMed Scopus Google Scholar) the of this residue to induce peptide The of the two helical segments for PEAP209–237 peptide well with a NMR study performed in our laboratory on the synthetic in trifluoroethanol In this peptide, NMR data the presence of a helical conformation for residues Glu-228 to the of the NMR data obtained for the short and COOH-terminal of PEA leads to the that the of the COOH-terminal of the is by the addition of the studies have focused on the three-dimensional structure of the sequence of its to be able to to opioid and induce (1Hughes J. Smith T.W. Kosterlitz H.W. Fothergill L.A. Morgan B.A. Morris R.H. Nature. 1975; 258: 577-579Crossref PubMed Scopus (2942) Google Scholar). A range of conformations has been found for peptides in A. M. M. 1989; PubMed Scopus Google Scholar), in solution, or to (for review, see Ref. A. PubMed Scopus Google Scholar), of and but helical conformation has been has been that sequence addition to the of peptides leads to the of to the Stern A.S. J. Kimura S. Stein J. Udenfriend S. S. A. PubMed Scopus Google Scholar). This be to a or from the induction of an helical conformation by the additional a by our present The PEAP209–237 sequence three proline residues, two of these conserved in PEA (13Goumon Y. Strub J.M. Moniatte M. Nullans G. Poteur L. Hubert P. Van Dorsselaer A. Aunis D. Metz-Boutigue M.H. Eur. J. Biochem. 1996; 235: 516-525Crossref PubMed Scopus (80) Google Scholar, 16Goumon Y. Lugardon K. Kieffer B. Lefèvre J.F. Van Dorsselaer A. Aunis D. Metz-Boutigue M.H. J. Biol. Chem. 1998; 273: 29847-29856Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). has been shown that the energy between the and of a residue that is found with high frequency in C.J. S. A. 1986; PubMed Scopus Google Scholar), is in A. M. PubMed Scopus Google Scholar). a in PubMed Scopus Google Scholar). The ambiguous NOE for Pro-227, the to the and to two models for the three-dimensional In PEAP209–237, the of the peptide in a of the second helical segment to the first one by an angle of This conformational which a between two serine residues in the first helical and two residues, in the COOH-terminal helical segment be in triggering the antibacterial activity of the peptide. The induced by the between serine and residues be in the active The antibacterial bisphosphorylated PEAP209–237, enkelytin, with antibacterial C.J. K. PubMed Scopus Google Scholar, L. D. Biophys. J. 1982; Full Text PDF PubMed Scopus Google Scholar), residues at the of the molecule. the surface potential calculated for this peptide its negative charge This is in of a mechanism the one proposed for or which form large in the by B. J. R.B. D. S. A. 1988; 85: PubMed Scopus Google Scholar). the of the two helical segments of PEAP209–237 is low with the structural features of this COOH-terminal indicate that enkelytin its antibacterial activity by potential (i) a or mechanism by of its three residues with the negative on (ii) a with potential including or (iii) the of or for the bacteria The NMR study of the synthetic bisphosphorylated PEAP209–237 and of are in in our laboratory to the of in the antibacterial activity of enkelytin. We are to G. Nullans for of peptides, to for using the program and to for spectra