Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Structures of Human Host Defense Cathelicidin LL-37 and Its Smallest Antimicrobial Peptide KR-12 in Lipid Micelles

2008; Elsevier BV; Volume: 283; Issue: 47 Linguagem: Inglês

10.1074/jbc.m805533200

ISSN

1083-351X

Autores

Guangshun Wang,

Tópico(s)

Invertebrate Immune Response Mechanisms

Resumo

As a key component of the innate immunity system, human cathelicidin LL-37 plays an essential role in protecting humans against infectious diseases. To elucidate the structural basis for its targeting bacterial membrane, we have determined the high quality structure of 13C,15N-labeled LL-37 by three-dimensional triple-resonance NMR spectroscopy, because two-dimensional 1H NMR did not provide sufficient spectral resolution. The structure of LL-37 in SDS micelles is composed of a curved amphipathic helix-bend-helix motif spanning residues 2–31 followed by a disordered C-terminal tail. The helical bend is located between residues Gly-14 and Glu-16. Similar chemical shifts and 15N nuclear Overhauser effect (NOE) patterns of the peptide in complex with dioctanoylphosphatidylglycerol (D8PG) micelles indicate a similar structure. The aromatic rings of Phe-5, Phe-6, Phe-17, and Phe-27 of LL-37, as well as arginines, showed intermolecular NOE cross-peaks with D8PG, providing direct evidence for the association of the entire amphipathic helix with anionic lipid micelles. The structure of LL-37 serves as a model for understanding the structure and function relationship of homologous primate cathelicidins. Using synthetic peptides, we also identified the smallest antibacterial peptide KR-12 corresponding to residues 18–29 of LL-37. Importantly, KR-12 displayed a selective toxic effect on bacteria but not human cells. NMR structural analysis revealed a short three-turn amphipathic helix rich in positively charged side chains, allowing for effective competition for anionic phosphatidylglycerols in bacterial membranes. KR-12 may be a useful peptide template for developing novel antimicrobial agents of therapeutic use. As a key component of the innate immunity system, human cathelicidin LL-37 plays an essential role in protecting humans against infectious diseases. To elucidate the structural basis for its targeting bacterial membrane, we have determined the high quality structure of 13C,15N-labeled LL-37 by three-dimensional triple-resonance NMR spectroscopy, because two-dimensional 1H NMR did not provide sufficient spectral resolution. The structure of LL-37 in SDS micelles is composed of a curved amphipathic helix-bend-helix motif spanning residues 2–31 followed by a disordered C-terminal tail. The helical bend is located between residues Gly-14 and Glu-16. Similar chemical shifts and 15N nuclear Overhauser effect (NOE) patterns of the peptide in complex with dioctanoylphosphatidylglycerol (D8PG) micelles indicate a similar structure. The aromatic rings of Phe-5, Phe-6, Phe-17, and Phe-27 of LL-37, as well as arginines, showed intermolecular NOE cross-peaks with D8PG, providing direct evidence for the association of the entire amphipathic helix with anionic lipid micelles. The structure of LL-37 serves as a model for understanding the structure and function relationship of homologous primate cathelicidins. Using synthetic peptides, we also identified the smallest antibacterial peptide KR-12 corresponding to residues 18–29 of LL-37. Importantly, KR-12 displayed a selective toxic effect on bacteria but not human cells. NMR structural analysis revealed a short three-turn amphipathic helix rich in positively charged side chains, allowing for effective competition for anionic phosphatidylglycerols in bacterial membranes. KR-12 may be a useful peptide template for developing novel antimicrobial agents of therapeutic use. The growing drug resistance problem of pathogenic bacteria with traditional antibiotics calls for an urgent search for a new generation of antimicrobial agents. Antimicrobial peptides are ancient and potent weapons of the innate immunity of all life forms (1Boman H.G. Annu. Rev. Immunol. 1995; 13: 61-92Crossref PubMed Scopus (1526) Google Scholar, 2Sambhara S. Lehrer R.I. Expert Rev Anti Infect Ther. 2007; 5: 1-5Crossref PubMed Scopus (13) Google Scholar, 3Hancock R.E.W. Lancet. Infect. Dis. 2001; 1: 156-164Abstract Full Text Full Text PDF PubMed Scopus (850) Google Scholar, 4Zasloff M. Nature. 2002; 415: 389-395Crossref PubMed Scopus (6852) Google Scholar). The recently updated Antimicrobial Peptide Data base collects more than 1228 such peptides (5Wang Z. Wang G. Nucleic Acids Res. 2004; 32: D590-D592Crossref PubMed Google Scholar). In mammals, defensins and cathelicidins are the two major families of antimicrobial peptides. While several cathelicidins were found in animals such as sheep, cow, and pig, only one cathelicidin was identified in humans (6Zanetti M. Curr. Issues Mol. Biol. 2005; 7: 179-196PubMed Google Scholar). The precursor proteins of the cathelicidin family share a highly conserved N-terminal "cathelin" domain, but have a highly variable C-terminal antimicrobial region. Upon bacterial insult, human cathelicidin LL-37 (named based on the first two amino acids in the sequence followed by the number of residues in the peptide) is released by proteases from its precursor hCAP-18 (i.e. human cationic antimicrobial protein, ∼18 kDa). The importance of this host defense peptide to human health is now firmly established. Patients lacking this molecule are more susceptible to infections (7Pütsep K. Carlsson G. Boman H.G. Andersson M. Lancet. 2002; 360: 1144-1149Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar). While cathelicidin knock-out mice are more readily infected (8Nizet V. Ohtake T. Lauth X. Trowbridge J. Rudisill J. Dorschner R.A. Pestonjamasp V. Piraino J. Huttner K. Gallo R.L. Nature. 2001; 414: 454-457Crossref PubMed Scopus (1031) Google Scholar), expression of additional cathelicidins protects the animals from infection (9Lee P.H. Ohtake T. Zaiou M. Murakami M. Rudisill J.A. Lin K.H. Gallo R.L. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 3750-3755Crossref PubMed Scopus (110) Google Scholar). LL-37 also associates with lipopolysaccharides (or endotoxin) and protects rats from sepsis caused by bacteria (10Cirioni O. Giacometti A. Ghiselli R. Bergnach C. Orlando F. Silvestri C. Mocchegiani F. Licci A. Skerlavaj B. Rocchi M. Saba V. Zanetti M. Scalise G. Antimicrob. Agents Chemother. 2006; 50: 1672-1679Crossref PubMed Scopus (132) Google Scholar). LL-37 is also reduced in cystic fibrosis airways as a result of direct interaction with DNA and filamentous F-actin (11Bucki R. Byfield F.J. Janmey P.A. Eur. Respir. J. 2007; 29: 624-632Crossref PubMed Scopus (82) Google Scholar). As a consequence, there is a high interest in developing novel peptides of therapeutic value based on LL-37. To provide insight into the mechanism of action of LL-37, biochemical and biophysical studies lent support to the membrane targeting of the peptide. Oren et al. (12Oren Z. Lerman J.C. Gudmundsson G.H. Agerberth B. Shai Y. Biochem. J. 1999; 341: 501-513Crossref PubMed Scopus (493) Google Scholar) found that LL-37 self-associated when bound to zwitterionic lipids, but dissociated into monomers in the presence of negatively charged vesicles. CD studies indicated a helical conformation of LL-37 upon binding to membrane-mimetic models (13Johansson J. Gudmundsson G.H. Rottenberg M.E. Berndt K.D. Agerberth B. J. Biol. Chem. 1998; 273: 3718-3724Abstract Full Text Full Text PDF PubMed Scopus (520) Google Scholar). Solid-state NMR studies indicated that LL-37 is located on the surface of lipid bilayers (14Henzler-Wildman K.A. Lee D.K. Ramamoorthy A. Biochemistry. 2003; 42: 6545-6558Crossref PubMed Scopus (409) Google Scholar). Using the monolayer of phospholipids, LL-37 was demonstrated to have preferential interactions with anionic phosphatidylglycerols (PGs) 2The abbreviations used are: PG, phosphatidylglycerol; D8PG, dioctanoylphosphatidylglycerol; DPC, dodecylphosphocholine; HSQC, heteronuclear single-quantum coherence spectroscopy; MIC, minimum inhibitory concentration; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser enhancement spectroscopy; r.m.s.d., root mean square deviation. (15Neville F. Cahuzac M. Konovalov O. Ishitsuka Y. Lee K.Y.C. Kuzmenko I. Kale G.M. Gidalevitz D. Biophy. J. 2006; 90: 1275-1287Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). This lipid selection is essential for selective targeting of anionic bacterial membranes by cationic antimicrobial peptides. The three-dimensional structure of intact LL-37 in complex with bacterial membranes would be invaluable to understanding the mechanism of action, but was not available prior to our study. Although homonuclear two-dimensional NMR techniques set the record of structural determination of micelle-bound peptides and proteins up to ∼50 residues (16Wang G. Curr. Protein Pept. Sci. 2008; 9: 50-69Crossref PubMed Scopus (48) Google Scholar), we met difficulty in structural studies of this 37-residue membrane peptide in complex with SDS micelles due to heavy spectral overlap (Fig. 1A). Thus, it is necessary to utilize three-dimensional NMR techniques. For this purpose, isotope-labeled polypeptides are required. Direct expression of LL-37 in yeast gave a very low level of the peptide (17Hong I.P. Lee S.J. Kim Y.S. Choi S.G. Biotechnol. Lett. 2007; 29: 73-78Crossref PubMed Scopus (38) Google Scholar). Using bacterial expression systems, we obtained 1.7 mg of recombinant LL-37 from one liter of culture (18Li Y. Li X. Wang G. Protein Expr. Purif. 2006; 47: 498-505Crossref PubMed Scopus (65) Google Scholar), whereas Moon et al. (19Moon J.Y. Henzler-Wildman K.A. Ramamoorthy A. Biochim. Biophys. Acta. 2006; 1758: 1351-1358Crossref PubMed Scopus (73) Google Scholar) reported 0.3 mg. Subsequent studies revealed that the LL-37-containing fusion protein was essentially uncleaved by thrombin at a site adjacent to LL-37 (probably due to peptide aggregation), but could be readily digested by formic acid. Our continued improvements not only simplified the purification protocol for LL-37 but also improved the peptide yield (reviewed in Ref. 16Wang G. Curr. Protein Pept. Sci. 2008; 9: 50-69Crossref PubMed Scopus (48) Google Scholar), opening the door to structure and dynamics studies of LL-37 by three-dimensional NMR spectroscopy. As a consequence of formic acid cleavage at the Asp-Pro site, recombinant LL-37 obtained in this manner retained an extra proline residue at the N terminus. We demonstrated that the recombinant LL-37 we obtained had an identical antibacterial activity to the synthetic peptide corresponding to the native sequence (18Li Y. Li X. Wang G. Protein Expr. Purif. 2006; 47: 498-505Crossref PubMed Scopus (65) Google Scholar). Therefore, this form of recombinant LL-37 is useful for structural analysis. Because of the complex nature of bacterial membranes, structural studies of membrane-associated peptides and proteins by solution NMR are usually performed in membrane-mimetic micelles (16Wang G. Curr. Protein Pept. Sci. 2008; 9: 50-69Crossref PubMed Scopus (48) Google Scholar). This is because the faster the tumbling of the peptide/lipid complex, the narrower the spectral linewidth. Here we report the three-dimensional structure of recombinant LL-37 in SDS micelles. To provide additional insight into the interactions of human cathelicidin with anionic PGs, we also conducted the study in the presence of D8PG, a new bacterial membrane model for solution NMR (16Wang G. Curr. Protein Pept. Sci. 2008; 9: 50-69Crossref PubMed Scopus (48) Google Scholar). Using synthetic peptides, we also report the identification and structural studies of the shortest antimicrobial peptide, KR-12, corresponding to residues 18–29 of LL-37. Selective toxicity of KR-12 makes it a promising candidate for developing novel and potent antimicrobial agents. NMR Spectroscopy—15N- or 15N,13C-labeled recombinant LL-37 was expressed and purified using the established protocol (20Li Y. Li X. Wang G. Protein Expr. Purif. 2007; 54: 157-165Crossref PubMed Scopus (44) Google Scholar). The LL-37/SDS complex contains ∼0.5 mm peptide and 80-fold of deuterated SDS (Cambridge Isotope Laboratories). A complex was also made between 15N- or 15N,13C-labeled LL-37 (∼0.5 mm) and protonated D8PG (> 98%, Avanti lipids, AL) at a molar ratio of 1:30. To facilitate peptide-lipid NOE observations, 2 mm synthetic LL-37 (>95% purity, Genemed Synthesis, TX) was co-solubilized with 10 mm D8PG. A similar sample was made for KR-12 in D8PG. The pH of all NMR samples, containing 10% D2O, was measured directly in the NMR tubes using a micro-pH electrode (Wilmad-Labglass). NMR data were recorded on a Varian INOVA 600-MHz NMR spectrometer equipped with a triple-resonance cryogenic probe with a z-axis gradient capability. In two-dimensional 1H NMR spectra, the spectral width in both dimensions was 8510.6 Hz. In the case of two-dimensional HSQC spectra, the sweep width for the 15N dimension was typically set to 2200 Hz with 100 increments. For natural abundance HSQC, 30 increments were collected in the 15N dimension with 256 scans each. For temperature coefficient measurements, HSQC spectra of LL-37 were recorded at every 5 degrees from 15 to 30 °C. The temperature coefficient (in ppm × 10-3/°C) for each backbone amide proton was calculated by linear regression of chemical shifts versus temperature. Residue-specific 15N{1H} NOE values for 15N-labeled LL-37 in SDS or D8PG micelles were measured using two-dimensional (1H,15N) correlated spectroscopy with and without proton saturation as described (21Li X. Peterkofsky A. Wang G. Amino Acids. 2008; 35: 531-539Crossref PubMed Scopus (15) Google Scholar). For signal assignments, several double- and triple-resonance NMR experiments, including HNCACB, CBCA(CO)NH, HNCA, HN(CO)CA, HNCO, C(CO)NH, H(CCO)NH, HNHA, HBHA(CO)NH, HCCH-COSY, and HCCH-TOCSY (22Bax A. Grzesiek S. Acc. Chem. Res. 1993; 26: 131-138Crossref Scopus (796) Google Scholar), were performed using 13C,15N-labeled LL-37 as detailed previously (23Li X. Peterkofsky A. Wang G. J. Biomol NMR. 2003; 27: 401-402Crossref PubMed Scopus (3) Google Scholar). Typically, these three-dimensional experiments were recorded with sweep widths/increments of 1,000 Hz/28 for 15N, 12067.8 Hz/60 for aliphatic 13C, and 3770 Hz/40 for carbonyl carbon in the indirect dimensions, and with a spectral width of 8510.6 Hz and 1024 complex points in the 1H-detected dimension, respectively. The carriers for 1H, 15N, and 13C were positioned at 4.67, 118.2, and 47.3 ppm, respectively. Chemical shifts were referenced as described (23Li X. Peterkofsky A. Wang G. J. Biomol NMR. 2003; 27: 401-402Crossref PubMed Scopus (3) Google Scholar). Data were processed on a Silicon Graphics Octane work station using NMRPipe (24Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11638) Google Scholar) and analyzed by PIPP (25Garrett D.S. Powers R. Gronenborn A.M. Clore G.M. J. Magn. Reson. 1991; 95: 214-220Crossref Scopus (802) Google Scholar). Structure Calculations—For structural calculations of SDS-bound LL-37, the major restraints were derived from three-dimensional NOESY spectra (26Wüthrich K. NMR of Proteins and Nucleic Acids. Wiley, New York1986Crossref Google Scholar). For D8PG-bound KR-12, distance restraints were obtained from two-dimensional NOESY spectra. The cross peaks were integrated by PIPP (25Garrett D.S. Powers R. Gronenborn A.M. Clore G.M. J. Magn. Reson. 1991; 95: 214-220Crossref Scopus (802) Google Scholar) and converted to distance restraints 1.8–2.8, 1.8–3.8, 1.8–5.0, and 1.8–6.0 Å corresponding to strong, medium, weak, and very weak types of NOE peaks, respectively. Based on 1Hα, 15N, 13Cα, 13Cβ, and 13C carbonyl chemical shifts, backbone angles of micelle-bound LL-37 were predicted by using an updated version of TALOS (27Cornilescu G. Delaglio F. Bax A. J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2740) Google Scholar). Hydrogen bond restraints for the structured region were derived from temperature coefficients as described (21Li X. Peterkofsky A. Wang G. Amino Acids. 2008; 35: 531-539Crossref PubMed Scopus (15) Google Scholar). For KR-12 in D8PG micelles, backbone angles were predicted based on 1Hα and natural abundance 15N chemical shifts (16Wang G. Curr. Protein Pept. Sci. 2008; 9: 50-69Crossref PubMed Scopus (48) Google Scholar). An extended covalent structure was used as starting coordinates. An ensemble of structures was calculated by using the simulated annealing protocol in the Xplor-NIH program (28Schwieters C.D. Kuszewski J. Tjandra N. Clore G.M. J. Magn. Reson. 2003; 160: 65-73Crossref PubMed Scopus (1882) Google Scholar). Structures were accepted based on the following criteria: no NOE-derived distance violations greater than 0.20 Å, back dihedral angle violations less than 2°, r.m.s.d. for bond deviations from ideality less than 0.01 Å, and r.m.s.d. for angle deviations from ideality less than 5°. The structures were viewed and analyzed using PROCHECK (29Laskowski R.A. Rullmannn J.A. MacArthur M.W. Kaptein R. Thornton J.M. J. Biomol. NMR. 1996; 8: 477-486Crossref PubMed Scopus (4474) Google Scholar) and MOLMOL (30Koradi R. Billeter M. Wüthrich K. J. Mol. Graphics. 1996; 14: 51-55Crossref PubMed Scopus (6498) Google Scholar). Solution Structure of Human LL-37 in Complex with SDS Micelles—The 1H signals of LL-37 in SDS are poorly dispersed in the two-dimensional NOESY spectrum (Fig. 1A), but are well separated in different two-dimensional planes of the three-dimensional NOESY spectrum (Fig. 1B). Sequential 1H, 13C, and 15N signal assignments were then achieved by using triple-resonance NMR experiments (22Bax A. Grzesiek S. Acc. Chem. Res. 1993; 26: 131-138Crossref Scopus (796) Google Scholar) using isotope-labeled samples (23Li X. Peterkofsky A. Wang G. J. Biomol NMR. 2003; 27: 401-402Crossref PubMed Scopus (3) Google Scholar). TALOS (27Cornilescu G. Delaglio F. Bax A. J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2740) Google Scholar) analysis of 1Hα, 15N, 13Cα, 13Cβ, and 13C carbonyl chemical shifts of micelle-bound LL-37 identified a helical region covering residues 2–30. No consensus angles were predicted for residues 31–36. The three-dimensional structure of LL-37 in complex with SDS was determined based on the following NMR restraints: 345 NOE-derived distance restraints, 58 chemical shift-derived backbone angle restraints, and 19 temperature coefficient-derived hydrogen-bond restraints (Table 1). Fig. 2A presents an ensemble of 28 structures of LL-37 accepted based on the criteria defined under "Experimental Procedures." The r.m.s.d. is 0.69 Å when the backbone atoms of residues 2–30 are superimposed. According to PROCHECK analysis (29Laskowski R.A. Rullmannn J.A. MacArthur M.W. Kaptein R. Thornton J.M. J. Biomol. NMR. 1996; 8: 477-486Crossref PubMed Scopus (4474) Google Scholar) of the ensemble of structures deposited into the Protein Data Bank, 93.8% of the residues are located in the most favored region of the Ramachandran plot and 6.2% are located in the additionally allowed region, indicating high quality. A ribbon diagram of the structure (Fig. 2B) is generated using MOLMOL (30Koradi R. Billeter M. Wüthrich K. J. Mol. Graphics. 1996; 14: 51-55Crossref PubMed Scopus (6498) Google Scholar). The three-dimensional structure of LL-37 consists of a well-defined long helix covering residues 2–31 with the remaining C-terminal residues disordered. The overall structural pattern of LL-37 in SDS micelles agrees well with both heteronuclear NOE measurements (Fig. 3A) and the secondary chemical shift plot (Fig. 3B). The former indicates that residues 2–32 are ordered and the C terminus is mobile, while the latter indicates a helical region covering residues 2–31 of LL-37. A further examination found that the amphipathic helix is curved with a bend between residues Gly-14 and Glu-16 (Fig. 2, B and C). Residues 14–16 as the bend are consistent with the 1Hα secondary chemical shift plot (Fig. 3B), where residues 14 and 16 showed near zero or positive deviations. We propose that the hydrophobic packing between residues Ile-13 and Phe-17 plays an important role in causing the helical bend in the structure of LL-37. Aligned on the concave surface is a train of hydrophobic side chains: Leu-2, Phe-5, Phe-6, Leu-13, Phe-17, Ile-20, Val-21, Ile-24, Phe-27, Leu-28, and Leu-31. Note that a hydrophilic residue Ser-9 is also located on the hydrophobic surface (Fig. 2B), leading to a division of the hydrophobic surface into two regions. The aromatic-aromatic stacking between Phe-5 and Phe-6 (Fig. 2B), also observed in the structure of the N-terminal fragment of LL-37 (31Li X. Li Y. Han H. Miller D.W. Wang G. J. Am. Chem. Soc. 2006; 128: 5776-5785Crossref PubMed Scopus (224) Google Scholar), may be responsible for the upfield shift of the Hδ protons of Phe-5 at 6.92 ppm.TABLE 1Structural statistics of human LL-37 and KR-12 bound to micellesStructural restraintsLL-37 in SDSKR-12 in D8PGNOE restraints (total)34586Intra-residue12417Sequential12837Short range9432Backbone angles (ϕ/ψ)aPredicted by the updated version of the TALOS program (27)5820Hydrogen bonds19Structural qualityBackbone r.m.s.d. (Å)bCalculated by MOLMOL (30). For LL-37, residues 2–30 of an ensemble of 28 structures were superimposed; for KR-12, residues 20–28 of an ensemble of 20 structures were superimposed0.690.24Distance violations (Å)<0.2<0.2Dihedral angle violations<2°<2°Ramachandran plotcCalculated by PROCHECK (29)Residues in the most favored region93.8%90.0%Residues in the additional allowed region6.2%10.0%a Predicted by the updated version of the TALOS program (27Cornilescu G. Delaglio F. Bax A. J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2740) Google Scholar)b Calculated by MOLMOL (30Koradi R. Billeter M. Wüthrich K. J. Mol. Graphics. 1996; 14: 51-55Crossref PubMed Scopus (6498) Google Scholar). For LL-37, residues 2–30 of an ensemble of 28 structures were superimposed; for KR-12, residues 20–28 of an ensemble of 20 structures were superimposedc Calculated by PROCHECK (29Laskowski R.A. Rullmannn J.A. MacArthur M.W. Kaptein R. Thornton J.M. J. Biomol. NMR. 1996; 8: 477-486Crossref PubMed Scopus (4474) Google Scholar) Open table in a new tab FIGURE 3Heteronuclear NOE, chemical shift plots of LL-37 and its interaction with D8PG. A, 15N NOE values of LL-37 measured in SDS (peptide/SDS ratio 1:80, pH 5.4, 30 °C). B, Hα secondary shift plot of LL-37 in SDS. Secondary shifts were calculated by taking the differences between the measured chemical shifts of the peptide and those random-coil values (46Epand R.F. Mowery B.P. Lee S.E. Stahl S.S. Lehrer R.I. Gellman S.H. Epand R.M. J. Mol. Biol. 2008; 371: 38-50Crossref Scopus (151) Google Scholar). C, Hα secondary shift plot of 15N-labeled recombinant LL-37 in D8PG (peptide/D8PG 1:30, pH 5.4, 50 °C). D, intermolecular NOE cross peaks between synthetic LL-37 (2 mm) and D8PG at a peptide/D8PG molar ratio of 1:5, pH 5.4 and 30 °C. The corresponding one-dimensional slice was taken to show weak NOE cross peaks between arginines and D8PG (pointed by arrows).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Structure of LL-37 in D8PG Micelles Is Similar to That in SDS—To further understand the effect of different micelles on the structure, we also performed NMR studies of LL-37 in D8PG (32Wang G. Keifer P.A. Peterkofsky A. Protein Sci. 2003; 12: 1087-1096Crossref PubMed Scopus (46) Google Scholar, 33Wang G. Biochim. Biophys. Acta. 2007; 1768: 3271-3281Crossref PubMed Scopus (42) Google Scholar, 34Wang G. Open Magn. Reson. J. 2008; 1: 9-15Crossref Google Scholar), which has the same lipid head group as the major anionic PGs in bacterial membranes. In D8PG, spectral quality of 15N-labeled LL-37 was poor at low temperatures. However, a well-resolved spectrum was obtained at 50 °C (see supporting information, see supplemental Fig. S1). An estimation of the correlation time (τc) of the complex based on the ratio of the 15N T1/T2 relaxation times of the entire amide region (35Chou J.J. Baber J.L. Bax A. J. Biomol. NMR. 2004; 29: 299-308Crossref PubMed Scopus (122) Google Scholar) revealed that the τc of the LL-37/D8PG complex at 50 °C was 6.9 ns (similar to 7.0 ns for an 18-kDa globular protein IIAGlc in solution at 35 °C), while the τc values in SDS are 7.0, 6.83, and 6.36 ns at 25, 35, and 45 °C, respectively. Clearly, the NMR-derived correlation time of LL-37 in D8PG at 50 °C is comparable to that in SDS at ∼30 °C, indicating that the LL-37/SDS complex tumbles faster than the LL-37/D8PG complex in aqueous solution at the same temperature. Based on the chemical shift assignments of LL-37 in D8PG, we have also obtained the 1Hα secondary shifts (i.e. the measured shifts minus the random-coiled shifts). The plots of LL-37 obtained in SDS (Fig. 3B) and in D8PG (Fig. 3C) are remarkably similar, indicating a helical structure from residues 2 to 31 in both systems. Furthermore, TALOS analysis of 1Hα, 15N, 13Cα, 13Cβ, and 13C carbonyl chemical shifts of LL-37 in D8PG led to the definition of the helical region covering residues 2–30, i.e. also identical to that found in SDS. At 50 °C, the overall trend of heteronuclear NOE values of human cathelicidin in D8PG (not shown) is also similar to that in SDS (Fig. 3A). We conclude that the structures of LL-37 in SDS and D8PG micelles are very similar if not identical. The introduction of protonated D8PG, based on the comparison of a series of PGs (33Wang G. Biochim. Biophys. Acta. 2007; 1768: 3271-3281Crossref PubMed Scopus (42) Google Scholar, 34Wang G. Open Magn. Reson. J. 2008; 1: 9-15Crossref Google Scholar), also provides a good opportunity to measure the interactions between cationic LL-37 and anionic PGs. The through-space dipole-dipole interactions between protons (<5 Å) are manifested in intermolecular NOESY spectra (16Wang G. Curr. Protein Pept. Sci. 2008; 9: 50-69Crossref PubMed Scopus (48) Google Scholar, 36Wang G. Treleaven W.D. Cushley R.J. Biochim. Biophys. Acta. 1996; 1301: 174-184Crossref PubMed Scopus (66) Google Scholar). Such NOE cross peaks were found to build up as normal intramolecular NOE peaks, indicating direct dipole-dipole interactions (26Wüthrich K. NMR of Proteins and Nucleic Acids. Wiley, New York1986Crossref Google Scholar, 33Wang G. Biochim. Biophys. Acta. 2007; 1768: 3271-3281Crossref PubMed Scopus (42) Google Scholar). As shown in Fig. 3D, the aromatic rings of Phe-5, Phe-6, Phe-17, and Phe-27 of LL-37 on the concave hydrophobic surface (Fig. 2C) all displayed NOE cross peaks with the D8PG C3-C7 protons at 1.23 ppm. Similar cross peaks were also observed when one-dimensional slices were taken in the two-dimensional spectrum corresponding to lipid signals at 2.34 ppm (C2-H) and 5.25 ppm (Hβ). These lipid signals possess unique chemical shifts and do not overlap with the peptide signals under investigation. As there are NOE cross peaks from the peptide to both lipid head group and acyl chain protons, LL-37 binds to the interfacial region of the lipid micelles. Furthermore, slice analysis facilitates the viewing of some weaker NOE cross peaks such as those between arginine side-chain protons and D8PG (pointed with arrows in Fig. 3D). Such Arg-PG cross peaks, albeit being weak, were also detected using an engineered LL-37 peptide (33Wang G. Biochim. Biophys. Acta. 2007; 1768: 3271-3281Crossref PubMed Scopus (42) Google Scholar), providing long-desired evidence for Arg-PG interactions. Observations of similar NOE cross peaks from lysines to PGs would be more difficult due to fast exchange of the side chain NH protons with solvents. Identification and Structural Determination of the Shortest Antibacterial Peptide Derived from LL-37—The existence of a helical bend between residues 14–16 in LL-37 allows us to classify micelle-bound human cathelicidin into three structural and functional regions. The N-terminal region (residues 1–13, labeled as I in Fig. 2B) has been implicated in chemotaxis (37Braff M.H. Hawkins M.A. Di Nardo A. Lopez-Garcia B. Howell M.D. Wong C. Lin K. Streib J.E. Dorschner R. Leung D.Y. Gallo R.L. J. Immunol. 2005; 174: 4271-4278Crossref PubMed Scopus (255) Google Scholar), in peptide oligomerization, in conferring proteolysis resistance, and in hemolytic activity of LL-37 (12Oren Z. Lerman J.C. Gudmundsson G.H. Agerberth B. Shai Y. Biochem. J. 1999; 341: 501-513Crossref PubMed Scopus (493) Google Scholar, 13Johansson J. Gudmundsson G.H. Rottenberg M.E. Berndt K.D. Agerberth B. J. Biol. Chem. 1998; 273: 3718-3724Abstract Full Text Full Text PDF PubMed Scopus (520) Google Scholar). The C-terminal region of LL-37 (residues 32–37, III in Fig. 2B) is disordered and mobile in complex with SDS, D8PG, or lipopolysaccharides (see online supporting information, supplemental Fig. S1B) and thus plays little role in targeting bacterial membranes. Our previous study found that these C-terminal residues participate in tetramer formation of LL-37 at physiological pH (20Li Y. Li X. Wang G. Protein Expr. Purif. 2007; 54: 157-165Crossref PubMed Scopus (44) Google Scholar). It remains to be established whether the C-terminal tail of LL-37 is involved in other biological functions. Numerous studies verified that the middle region (residues 17–31, II in Fig. 2B) contains the primary antimicrobial region of LL-37 (37Braff M.H. Hawkins M.A. Di Nardo A. Lopez-Garcia B. Howell M.D. Wong C. Lin K. Streib J.E. Dorschner R. Leung D.Y. Gallo R.L. J. Immunol. 2005; 174: 4271-4278Crossref PubMed Scopus (255) Google Scholar, 38Wang G. Protein Pept. Lett. 2007; 14: 57-69Crossref PubMed Scopus (25) Google Scholar, 39Nell M.J. Tjabringa G.S. Wafelman A.R. Verrijk R. Hismstra P.S. Drijfhout J.W. Grote J.J. Peptides. 2006; 27: 649-660Crossref PubMed Scopus (140) Google Scholar, 40Sigurdardottir T. Anderson P. Davoudi M.

Referência(s)