Eur J Biochem 269, 6212–6222 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03340.x Thermolysin-linearized microcin J25 retains the structured core of the native macrocyclic peptide and displays antimicrobial activity ´ ´ Alain Blond1, Michel Cheminant1, Delphine Destoumieux-Garzon1, Isabelle Segalas-Milazzo2, Jean Peduzzi1, Christophe Goulard1 and Sylvie Rebuffat1 Laboratory of Chemistry and Biochemistry of Natural Substances, Department of Regulation, Development and Molecular Diversity, National Museum of Natural History, Paris, France; 2IRCOF, ECOBS, UMR 6014 CNRS, IFRMP 23, University of Rouen, France Microcin J25 (MccJ25) is the single macrocyclic antimicrobial peptide belonging to the ribosomally synthesized class of microcins that are secreted by Enterobacteriaceae It showed potent antibacterial activity against several Salmonella and Escherichia strains and exhibited a compact three-dimensional structure [Blond et al (2001) Eur J Biochem., 268, 2124–2133] The molecular mechanisms involved in the biosynthesis, folding and mode of action of MccJ25 are still unknown We have investigated the structure and the antimicrobial activity of thermolysin-linearized MccJ25 (MccJ25-L1)21: VGIGTPISFY10GGGAGHVPEY20F), as well as two synthetic analogs, sMccJ25-L1)21 (sequence of the thermolysin-cleaved MccJ25) and sMccJ25-L12)11 (C-terminal sequence of the MccJ25 precursor: G12GAGHVPEYF21V1GIGTPISFYG11) The threedimensional solution structure of MccJ25-L1)21, determined by two-dimensional NMR, consists of a boot-shaped hairpin-like well-defined 8–19 region flanked by disordered N and C termini This structure is remarkably similar to that of cyclic MccJ25, and includes a short double-stranded antiparallel b-sheet (8–10/17–19) perpendicular to a loop (Gly11–His16) The thermolysin-linearized MccJ25-L1)21 had antibacterial activity against E coli and S enteritidis strains, while both synthetic analogues lacked activity and organized structure We show that the 8–10/17–19 b-sheet, as well as the Gly11–His16 loop are required for moderate antibacterial activity and that the Phe21–Pro6 loop and the MccJ25 macrocyclic backbone are necessary for complete antibacterial activity We also reveal a highly stable 8–19 structured core present in both the native MccJ25 and the thermolysin-linearized peptide, which is maintained under thermolysin treatment and resists highly denaturing conditions Since the pioneering works of the 1980s, which led to the discovery of the insect cecropins [1], the mammalian defensins [2,3] and the amphibian magainins [4], numerous antimicrobial peptides have been isolated from a wide variety of species Many bacteria produce antimicrobial peptides and proteins, including bacteriocins [5] and colicins [6], as a method of defence against other microorganisms Among them, microcins are antimicrobial peptides that are synthesized ribosomally by Enterobacteriaceae [7,8] These peptides have been reported to be active against closely related species of bacteria However, bacteria that produce microcins are resistant to their own endogenous peptides due to a mechanism called self-immunity that involves resistance proteins [7] Unlike colicins, antimicrobial proteins produced by enteric bacteria [6], microcins are not synthesized in response to SOS system-inducing agents [7], but under nutrient-poor culture conditions Microcins also differ from colicins by their lower molecular weight (generally < 10 kDa), and their resistance to extreme pH and temperature conditions The known microcins are structurally unrelated peptides that exhibit different mechanisms of action Microcin B17 blocks DNA-gyrase activity by its thiazole/oxazole rings [9,10], the nucleotide heptapeptide microcin C7 inhibits protein synthesis [11], and microcins E492 and ColV form transmembrane channels that cause lysis of the target organisms [12,13] Such diversity within one class of antimicrobial peptides is quite rare Microcin J25 (MccJ25, Fig 1) is the first macrocyclic microcin described to date It inhibits the growth of several enteric bacteria, including pathogenic Escherichia, Salmonella and Shigella strains, at minimum inhibitory concentrations (MICs) ranging from to 100 nM [14,15] MccJ25 was reported to interact with liposomes composed of zwitterionic phospholipids [16], and to act on the cytoplasmic membrane of S newport [17] However, the bacterial membrane is an unlikely target for MccJ25, as the concentrations needed for these membrane activities are, in some cases, much higher than those required for the antibiotic action In addition, a recent study showed that an E coli strain displaying a mutation in the gene encoding the RNA polymerase b¢ subunit is resistant to MccJ25, which suggests that RNA polymerase could Correspondence to S Rebuffat, Laboratoire de Chimie et Biochimie ´ des Substances Naturelles, Museum National d’Histoire Naturelle, 63 rue Buffon, 75231 Paris, Cedex 05, France Fax: + 33 40 79 31 35, Tel.: + 33 40 79 31 18, E-mail: rebuffat@mnhn.fr Abbreviations: Mcc, microcin; MIC, minimum inhibitory concentration; PB, poor broth; CSD, chemical shift deviations; RTD-1, rhesus theta-defensin-1; SFTI-1, sunflower trypsin inhibitor; TMS, tetramethylsilane (Received 22 August 2002, revised 21 October 2002, accepted 30 October 2002) Keywords: antimicrobial peptide; conformational stability; microcin; molecular modeling; solution structure Ó FEBS 2002 Structure of thermolysin-linearized microcin J25 (Eur J Biochem 269) 6213 Fig Amino acid sequences of the naturally occurring cyclic MccJ25 and its linear variants MccJ25-L1)21 is the thermolysin-linearized MccJ25, previously named MccJ25-L in [19] MccJ25-L12)11 displays the sequence of the 21 last amino acids of pre-MccJ25 (mcjA gene product) The synthetic peptides are identified by an s before their name Amino acids are numbered according to [19] be the intracellular target for the microcin [18] To date, the precise mechanism of action of MccJ25 remains unknown The 21-residue primary structure [19], and the threedimensional NMR solution structure [20] of cyclic MccJ25 have been determined The peptide forms a distorted antiparallel b-sheet, which twists and folds back on itself Residues 7–10 and 17–20 form the more regular part of the b-sheet between the Phe21–Pro6 and Gly11–His16 loops A cavity delimited by two crab pincer-like regions that encompass residues 6–8 and 18–1, confines the Val1 and Ser8 side chains The compact core structure is very well defined, stabilized primarily by the hydrogen bonds of a tightly packed b-sheet In this study, we examined the solution structure, stability, and antimicrobial activity of the thermolysinlinearized MccJ25 (MccJ25-L1)21: VGIGTPISFY10GGG AGHVPEY20F), previously generated for the structural characterization of MccJ25 [19], the synthetic analog sMccJ25-L1)21, and the synthetic 21-residue sMccJ25L12)11 peptide (G12GAGHVPEYF21V1GIGTPISFYG11), which sequence derives from MccJ25 precursor (Fig 1) Despite identical sequences, the folding and activity of the enzymatically generated MccJ25-L1)21 and chemically synthesized sMccJ25-L1)21 were completely different This finding was used as a basis to discuss the high stability of the MccJ25 structured core and its involvement in the antibacterial activity containing 0.05% CF3COOH Separation was performed at a mLỈmin)1 flow rate, and absorbance was monitored at 226 nm MccJ25-L1)21 was obtained by thermolysin-digestion of native MccJ25 Typically, lmol MccJ25 was dissolved in M urea (600 lL) and incubated at 46 °C for 30 min, before the addition of 0.17 M NH4HCO3 (1200 lL), 10 mM CaCl2 (200 lL) and 40 lg thermolysin (Boehringer Mannheim) The digestion was performed at 46 °C, pH 8.0, for 60 The reaction was stopped by adding 400 lL acetic acid, and MccJ25-L1)21 was purified by RP-HPLC on an Inertsil ODS2 column (5 lm, 4.6 · 250 mm; Interchim, France) under isocratic elution in a (31 : 69, v/v) acetonitrile/water solution containing 0.1% CF3COOH (flow rate: mLỈmin)1) Absorbance was monitored at 226 nm Purity of MccJ25-L1)21 was ascertained by MALDI-TOF MS on an Applied Biosystem Applera (USA) Voyager De-Pro system used in a positive linear mode, with sinapinic acid as a matrix Calibration was performed with a mixture of standards including bovine insulin (MH+ at m/z 5734.59), thioredoxin (MH+ at m/z 11674.48) and apomyoglobin (MH+ at m/z 16952.56) (Applied Biosystems) Peptide synthesis and purification sMccJ25-L1)21 (VGIGTPISFY10GGGAGHVPEY20F) and sMccJ25-L12)11 (G12GAGHVPEYF21V1GIGTPISFYG11) were synthesized by the classical solid-phase methodology using Fmoc-protection, as described by Neimark and Briand [21] All RP-HPLC separations were performed with solvents acidified with 0.05% CF3COOH The sMccJ25-L1)21 sample was purified in two steps on an RP-HPLC C18 column (semipreparative Capcell, lm; 7.5 · 250 mm; Interchim) used at a flow-rate of mLỈ min)1 The first separation was performed under isocratic elution with a (27 : 73) acetonitrile/water mixture, whereas the second separation consisted of a (40 : 60) to (60 : 40) methanol/water linear gradient at 0.7% methanolỈmin)1 The sMccJ25-L12)11 sample was purified in two steps on the same column as sMccJ25-L1)21 at a flow-rate of mLỈ min)1 The first RP-HPLC separation was performed with a biphasic gradient composed of a 10min isocratic step in a (26 : 74) acetonitrile/water mixture, followed by a 26 : 74 to 28 : 72 acetonitrile/water flat linear gradient (0.4% acetonitrilmin)1) The second HPLC consisted of an isocratic elution with a (30 : 70) acetonitrile/ water mixture Absorbance was monitored at 226 nm EXPERIMENTAL PROCEDURES Antibacterial assays MccJ25 and MccJ25-L1)21 sample preparation Antibacterial activity of MccJ25, MccJ25-L1)21, sMccJ25L1)21 and sMccJ25-L12)11 was assayed against two bacteria highly sensitive to MccJ25 The test microorganisms, E coli MC4100 tolC– and S enteritidis, were kindly provided by M Lavina (Facultad de Ciencias, Montevi˜ ´ deo, Uruguay) and A.-M Pons (Universite de La Rochelle, France), respectively Concentrations of peptide stock solutions were determined by amino acid composition, as described previously [19] MICs were determined in triplicate in poor broth (PB: 1% bactotryptone, 0.5% NaCl w/v) by the liquid growth inhibition assay essentially as described [22] Briefly, in a sterile microtitration plate, 10 lL peptide, or deionized water as a control, were added to 90 lL of a mid-logarithmic growth phase culture of Native MccJ25 was purified according to the procedure described previously [19] Briefly, E coli J02Mcc+ (a ´ generous gift from A.-M Pons, Universite de La Rochelle, France) was grown in L M63 minimal medium and the culture supernatant was applied onto a C8 Sep-Pak cartridge (Waters, France) Two successive elution steps were performed with (50 : 50, v/v) and (80 : 20, v/v) methanol/water mixtures MccJ25, found in the (80 : 20) methanol/water Sep-Pak fraction, was further purified on an RP-HPLC semipreparative column (Capcell C18, lm, 7.5 · 250 cm; Interchim, France) under isocratic conditions in a (61 : 39) methanol/water mixture 6214 A Blond et al (Eur J Biochem 269) bacteria diluted in PB to D600 ¼ 0.001 Plates were incubated for 16 h at 30 °C with vigorous shaking and monitored spectrophotometrically at 620 nm on a Ceres 900 (Bio-Tek Instruments) plate reader MICs are expressed as the interval of concentration [a]–[b], where [a] is the highest concentration tested at which microbial growth can be observed and [b] is the lowest concentration that causes 100% growth inhibition [23] CD spectroscopy CD spectra were recorded at room temperature from 250 to 190 nm on a Jobin-Yvon Mark V dichrograph (Longjumeau, France), using a 0.05-mm path cell The spectra were measured for methanolic solutions at peptide concentrations of 0.05–1 mM NMR spectroscopy Samples (0.5 mL) of mM MccJ25-L1)21, sMccJ25-L1)21 and sMccJ25-L12)11 in methanol (CD3OH) were placed in 5-mm Wilmad tubes for the NMR experiments Data were acquired on Bruker AVANCE 400 and DMX 600 spectrometers, equipped with 1H-broad-band reverse gradient and triple resonance 1H-13C-15N-gradient probeheads, respectively Temperature, controlled by a Bruker BCU-05 refrigeration unit and a BVT 3000 control unit on both spectrometers, was set at 10 °C unless specified otherwise Data were collected and processed on a Silicon Graphics O2 workstation, using the Bruker XWIN-NMR and AURELIA softwares 1H and 13C chemical shifts were referenced to the central component of the quintet due to the CD2HOH and the CD3OH resonances of methanol taken at 3.313 p.p.m and at 49.00 p.p.m downfield from TMS, respectively The following conventional two-dimensional homonuclear spectra were recorded: double quantum filtered (DQF) COSY, TOCSY with MLEV17 mixing period of 120 ms, NOESY with different mixing times, and H-13C heteronuclear experiments, optimized for J-values of 135 Hz (HSQC) and Hz (HMBC) Methods of spectra recording and data processing are described elsewhere [20] NOE buildup curves for MccJ25-L1)21 (mixing times of 50, 100, 150, 200 and 400 ms) showed that the correlation remained linear for the 100 ms mixing time, which was selected for distance calculation Temperature coefficients of amide protons were obtained in the range of 10–35 °C, by acquiring six series of onedimensional (1D)-1H and TOCSY spectra at 400.13 MHz, using °C temperature increments Exchange of amide protons was monitored as described previously [20] Briefly, a normal isotopic sample (either MccJ25-L1)21 or sMccJ25L1)21) was dissolved in CD3OD at °C It was analysed for h at °C and over days at 20 °C by the acquisition of a series of 1D-1H and TOCSY spectra Experimental restraints and MccJ25-L structure calculations Distance restraints for MccJ25-L1)21 structure calculation were derived from NOE cross-peaks in the NOESY spectrum recorded at 10 °C with sm ¼ 100 ms, that in turn were converted into distances by volume integration using the AURELIA software (Bruker, Karlsruhe) The NOE Ó FEBS 2002 intensity between the Tyr20 Hd and He protons, which ˚ corresponds to a distance of 2.45 A, was used for calibration To ascertain whether the contribution of zero-quantum coherence to the Tyr20 Hd–He cross-peak was negligible, the consistency of the distances obtained was assessed by referring to the Tyr10 Hd)He distance and to the Pro6 and Pro18 d-methylene distances A range of ± 25% the calculated distance was used to define the upper and lower bounds of the restraints Appropriate pseudoatom corrections were applied [24] to nonstereospecifically assigned methyl and methylene protons A total of 223 upper and lower distance restraints and 15 ambiguous restraints, derived from the NOE data, were used for the structure calculations Eight / dihedral angles, measured at 10 °C from the 1D- and the high digital resolution DQF-COSY spectra (CD3OH), were restrained to )120 ± 25° for 3JNHCaH P 9.5 Hz (Phe9, Val17), )120 ± 45° for a 3JNHCaH in the range 8.1–8.9 Hz (Ser8, Tyr20, Phe21) and )120 ± 50° for a 3JNHCaH 8.0 Hz (Thr5, Tyr10, Glu19) Two v1 dihedral angles, derived from the 3JCaHCbH coupling constants measured in the DQF-COSY (CD3OD) as well as from the intraresidue NOE intensities, were restrained to +60 ± 45° (Tyr10), and 180 ± 45° (Val17) Hydrogen bonding restraints were not included in the calculations Structures were calculated in vacuo, as described elsewhere [20], using simulated annealing and energy minimization protocols within the X-PLOR version 3.851 software [25], run either on a Silicon Graphics O2 workstation (IRIX 6.5), or a Gateway computer (SUSE LINUX 7.0) The target function was similar to that used by Nilges et al [26] Briefly, a set of 100 structures was generated using random /, w dihedral angles and extended side chains, and taking into account the distance and angle restraints The ambiguous assignments were further used with the appropriate treatment in X-PLOR [27,28] During the processing, the ˚ distance restraint force was kept at 50 kcalỈmol)1ỈA)2 and the NOE intensities were averaged with the ÔsumÕ option Of the 100 structures generated, 80 had a total energy less than 25 kcalỈmol)1 and led in all cases to systematic distance and ˚ dihedral angle violations lower than 0.2 A and 5°, respectively Refinement of the structures was achieved using the conjugate gradient Powell algorithm with 7000 cycles of energy minimization and the CHARMM 22 force field [29] The 30 best structures on the basis of their total energy including the electrostatic term with no systematic distance violation ˚ larger than 0.2 A and no dihedral angle violation greater than 5° were selected as the final structure of MccJ25-L1)21 The structures were visualized and analysed on the Silicon Graphics O2 and Gateway workstations, using the X-PLOR [25], MOLMOL [30] and PROCHECK_NMR [31] programs The hydrogen bonds present in the final structures were identified with MOLMOL, using the distances determined between donor and acceptor and the corresponding deviation angles with respect to 180°, with either: distance ˚ < 2.7 A, deviation angle < 35° (NH12 fi CO9, ˚ NH10 fi CO17), or distance < 3.0 A, deviation angle < 40° (NH19 fi CO8, NH9 fi CO13, NH13 fi CO9), or ˚ distance < 3.0 A, deviation angle < 50° (NH11 fi CO9, NH14 fi CO12) in all the selected structures The coordinates for the family of 20 refined lowest energy structures were deposited in the Brookhaven Protein Data Bank, under the accession code 1GR4 Ó FEBS 2002 Structure of thermolysin-linearized microcin J25 (Eur J Biochem 269) 6215 Stability of MccJ25 and MccJ25-L1)21 to denaturing conditions Thermal stability of MccJ25-L1)21 and MccJ25 was examined between 25 and 165 °C by NMR spectroscopy, using a 2.5 mM solution in dimethylsulfoxide-d6 In preliminary TOCSY (spin-lock time 120 ms) and NOESY (mixing time 300 ms) experiments, sequential assignments were obtained at 25 °C and similarity of the global fold in both CD3OH and dimethylsulfoxide-d6 was ascertained The NH and Ha chemical shifts of Ser8, Phe9, Tyr10, Ala14, His16, Val17 and Glu19, were selected to probe the temperature-induced conformational transitions They were determined at 25, 45, 65, 90, 115, 140 and 165 °C, from seven series of 1D-1H and TOCSY spectra Changes in the slopes of the Ha and NH proton curves obtained by plotting the chemical shifts as a function of temperature were interpreted as conformational changes The reversibility of the denaturation process was checked at 115 and 165 °C, by slowly lowering the temperature to 25 °C and acquiring control 1D-1H and TOCSY spectra The acquisition time of the NMR spectra at each temperature was fixed at h Analytical grade urea and guanidinium hydrochloride used in denaturation studies were purchased from Merck (Darmstadt, Germany) A series of experiments using different denaturing agents (1–6 M guanidinium hydrochloride or 1–8 M urea in water) and temperature conditions (25–95 °C) were used to assay MccJ25-L1)21 stability For high temperature conditions, sealed tubes were used Over the reaction time course, aliquots of the peptide/denaturant mixtures were withdrawn at different incubation times and analysed by RP-HPLC on a C18 lBondapak column (4.6 · 250 mm; Waters, France) under isocratic conditions with 0.05% CF3COOH-containing (32 : 68) acetonitrile/water mixture at a flow rate of mLỈmin)1 At the end of the incubation period, the reaction mixture was cooled to room temperature and applied onto a C8 Sep-Pak cartridge (Waters, France), which stopped the reaction by removing the chaotropic agent Elution was performed in a stepwise manner by 0.05% CF3COOH-containing : 100, 25 : 75, and 45 : 55 acetonitrile/water mixtures MccJ25-L1)21, found in the (45 : 55) fraction, was dried under vacuum and analysed by HPLC, as described above and by NMR 1D-1H, TOCSY and NOESY spectra were performed in CD3OH and compared to the original reference spectra C-terminal end of the MccJ25 precursor (sMccJ25-L12)11) The three linear peptides were purified by RP-HPLC and their purity was ascertained by MALDI-TOF MS The measured masses for all three peptides (MH+ at m/z 2126.08 for MccJ25-L1)21, 2125.70 for sMccJ25-L1)21 and [M+Na]+ at m/z 2147.47 for MccJ25-L12)11) were in agreement with the expected molecular masses at 2125 Da Antibacterial activity The antibacterial activity of MccJ25 linear variants was examined by a liquid growth inhibition assay using two Gram-negative strains chosen for their high sensitivity to the native MccJ25 Contrary to the native cyclic peptide, which displayed MIC values at 0–2 nM and 2–5 nM against S enteritidis and E coli MC4100 tolC–, respectively, the synthetic linear peptides sMccJ25-L12)11 and sMccJ25-L1)21 were completely inactive at concentrations reaching 10 lM against the two test bacteria (Table 1) By contrast, the thermolysin-linearized microcin (MccJ25-L1)21) retained significant activity against both Salmonella and Escherichia strains, as indicated by MIC values of 80–150 nM and 300– 600 nM, respectively This led us to examine the threedimensional structures of these linear MccJ25 variants, with particular attention to MccJ25-L1)21, following the hypothesis that structural features essential to MccJ25 activity had most likely been retained in this linear form Peptide solubility and aggregation state Due to insolubility of MccJ25 and its linear variants in aqueous medium in the absence of denaturing agents, CD and NMR spectroscopic analyses were performed utilizing methanol, a solvent in which MccJ25 is extremely soluble [20] The CD spectrum of MccJ25-L1)21 at 0.1 mM (data not shown) was very similar to that obtained previously for MccJ25 [20] It presented a strong negative band at 193 nm, as well as a positive band centred at 210 nm, which did not enable the assignment of any defined secondary structure The aggregation state of MccJ25-L1)21 was evaluated by recording several CD spectra at concentrations ranging from 0.05 to mM The similarity in the patterns obtained at the various concentrations indicated the absence of aggregation below mM In addition, 1D-1H and TOCSY spectra performed for concentrations between and mM, did not show any significant variation of the amide and a RESULTS Generation of MccJ25 linear variants: MccJ25-L1)21, s MccJ25-L1)21 and s MccJ2512)11 Native MccJ25 was prepared according to the procedure described previously [19] After purification, the peptide preparation analysed by MALDI-TOF MS presented a single MH+ ion at m/z ¼ 2108.40, in agreement with the calculated mass at 2107 Da for the macrocyclic MccJ25 Three linear forms of MccJ25 were obtained, either by MccJ25 thermolysin cleavage (MccJ25-L1)21 initially called MccJ25-L [19] or by chemical synthesis (sMccJ25-L1)21 and sMccJ25-L12)11; Fig 1) The sequences chosen for the synthetic peptides are identical to those of the thermolysincleaved MccJ25 (sMccJ25-L1)21) and of the 21-residue Table Minimum inhibitory concentrations of MccJ25 linear variants against S enteritidis and E coli MC4100 tolC ) MICs were determined in triplicate according to the liquid growth inhibition assay MICs (nM) are expressed as intervals of concentrations [a]–[b] were [a] is the highest concentration tested at which the microorganisms are growing and [b] is the lowest concentration that causes 100% growth inhibition [21] NA, Not active in the range 0–10 lM Peptide S enteritidis E coli MC4100 tolC MccJ25 MccJ25-L1)21 sMccJ25-L1)21 sMccJ25-L12)11 0–2 80–150 NA NA 2–5 300–600 NA NA – 6216 A Blond et al (Eur J Biochem 269) Ó FEBS 2002 proton chemical shifts, ensuring that MccJ25-L1)21 will not aggregate in methanol when performing NMR sMccJ25-L1)21 was also quite soluble in methanol, showing weak negative and positive bands centred at 190 nm, and 197 nm, respectively (data not shown) CD spectra could not be acquired for sMccJ25-L12)11, due to its poor solubility in methanol Dimethylsulfoxide-d6 was finally chosen for the NMR study of this variant, and was also used to assay the thermal stability of MccJ25- L1)21 Sequential assignments and secondary structures All proton resonances of MccJ25-L1)21 were obtained at 10 °C, to ensure a good signal separation The standard sequence-specific assignment strategy was used [32] TOC SY and DQF-COSY spectra enabled the identification of amino acid spin systems, and NOESY data provided the sequential connections between these spin systems In addition, backbone and side-chain 13C resonances were assigned from the 1H-13C HSQC and HMBC data The two proline residues found in MccJ25-L1)21 (Pro6 and Pro18), both displayed the typical NOE pattern of strong aHi-1-dHi accounting for a trans conformation of the X-Pro amide bonds This was in agreement with the c-carbon 13C chemical shifts of these proline residues Taking into account the S configuration of their a carbons, the pro-R and pro-S ring protons of Pro6 and Pro18 were assigned stereospecifically in MccJ25-L1)21 from the intraresidue NOE networks The sequential assignments of sMccJ25-L1)21 and sMccJ25-L12)11 were obtained in CD3OH and dimethylsulfoxide-d6, respectively In both sMccJ25-L1)21 and sMccJ25-L12)11 NOESY spectra, the aHi-1-dHi cross-peaks that characterize a trans conformation of the X-Pro amide bonds were observed for Pro6 and Pro18, while no contribution from cis conformation could be detected In addition, the proline c-carbon 13C chemical shifts were in agreement with two trans proline residues in sMccJ25-L1)21 The Ha and Ca secondary chemical shifts (chemical shift deviations, CSD), which represent the difference between the observed chemical shifts and the random coil values of Wishart [33,34], were determined for MccJ25-L1)21 Most of the residues had chemical shifts that differed from the random coil values by more than 0.1 p.p.m., indicative of a structured peptide The CSD did not show any clear evidence of a-helix (upfield shifts) or b-sheet (downfield shifts) structure in the peptide (Fig 2) An irregular pattern of positive and negative or null values, relevant to the presence of turns, was highly similar to that observed for the cyclic MccJ25, mainly in the region comprising residues 5– 18 [20] The pattern of sequential, medium- and long-range NOEs (Fig 3) showed a series of strong daNi,i+1, very few dNNi,i+1, several daNi,j and dNai,j contacts involving the residues belonging to the 8–10 and 16–19 regions, and a strong daa9,18, which was in agreement with an antiparallel two-stranded b-sheet such as that characterized previously in MccJ25 In addition, the large 3JNHCaH coupling constants ( Hz) measured for Ile7, Ser8, Phe9, > His16, Val17, Glu19 and Tyr20 were consistent with such regions of extended b-type structure These parameters were in agreement with a structured 8–19 region, while the N-terminal 1–6 and C-terminal 20–21 extremities of MccJ25-L1)21 appeared disordered, considering the Fig Comparison of NMR conformational parameters for MccJ25 (black), MccJ25L1)21 (grey) and sMccJ25-L1)21 (white) The intensities of the secondary chemical shifts of the Ha protons (CSDHa) and Ca carbons (CSDCa), of the 3JNHCaH coupling constants and temperature coefficients of the NH protons (Dd/DTNH) are given by appropriate scales on the figure and indicated by bars The NH–ND exchange rates are expressed by bars of increasing lengths for very slow (VS: over days), slow (S: 1–2 days), medium (M: 10–24 h), fast (F: 2–10 h) and very fast (VF: less than h) exchanging NH protons; * stands for not determined Fig Pattern of sequential, medium- and long-range NOE connectivities involving the NH, a, b and d protons of MccJ25-L1)21 dABi,j indicates the NOE connectivity between the proton types A and B located in the amino acids i and j The NOE intensities are classified into three categories (strong, medium, weak) based on the cross-peak volumes and are indicated by the bar heights complete lack of medium- and long-range NOE connectivities in these two parts The sequential assignments obtained for sMccJ25-L1)21 were completely different from those for its enzymatically generated equivalent, MccJ25-L1)21 This strongly suggests that despite an identical sequence, the two peptides adopt distinct conformations Indeed, a small chemical shift Ó FEBS 2002 Structure of thermolysin-linearized microcin J25 (Eur J Biochem 269) 6217 The conformational parameters of sMccJ25-L12)11 obtained in dimethylsulfoxide-d6 (data not shown) also argued in favour of an unstructured peptide, with Ha and NH chemical shifts in the random coil range and temperature coefficients between )4 and )7.5 p.p.b.ỈK)1 Less than half of the 3JNHCaH coupling constants could be measured due to strong signal overlapping These constants were  7.5–8 Hz, thus in favour of regions of extended structure Calculation and evaluation of the MccJ25-L1)21 structure The lack of structure as well as an insufficient number of distance constraints obtained for sMccJ25-L1)21 and sMccJ25-L12)11, led us to determine only the three-dimensional structure of the thermolysin-linearized form, MccJ25L1)21 A set of 100 structures was calculated using 223 distance restraints including 103 intraresidual, 71 sequential, 49 medium-range and long-range restraints (distributed as shown in Fig 4A), 15 ambiguous restraints and 10 dihedral angle restraints All simulated annealing runs converged to produce structures, with a common fold, which were in good agreement with all experimental data The standard covalent geometry had low total energies and did not exhibit significant deviation from ideal covalent geometry The 80 structures with the lowest energy were used in the last run of energy minimization An evaluation of the quality and precision of the 30 lowest energy structures chosen to represent the MccJ25-L1)21 solution structure is given in Table From Thr5 to Tyr20, the individual backbone conformation of all nonglycine residues was located in the energetically allowed regions of the /, w space Glycine residues assembled either in the specific glycine-allowed Fig NOE distribution per residue (A) and values of / and w angles in the 30 final structures (B) for MccJ25-L1)21 Intraresidual, sequential, and medium- and long-range NOEs are in black, grey and white, respectively dispersion of the NH protons ( 0.8 p.p.m) was observed, < the Ha and Ca chemical shift deviations were close to the random coil values and the 3JNHCaH couplings were all in the range 6.5–7.0 Hz, except for three values around Hz at the C terminus (Glu19, Tyr20 and Phe21) (Fig 2) All specific NOEs characterizing the MccJ25-L1)21 structure were absent from the sMccJ25-L1)21 NOESY spectrum (data not shown) Only a few sequential NOEs of low intensity, a series of daNi,i+1 (or dadi,i+1 in the case of prolines) and a few dNNi,i+1 (Gly4–Thr5, and Gly15–His16) and dbNi,i+1 (Ile7–Ser8, Phe9–Tyr10, Ala14–Gly15, His16–Val17) were observed The absence of the 8–10/17–19 b-sheet in the sMccJ25-L1)21 structure was demonstrated by: (a) temperature coefficients of amide protons in the range of 6–10 p.p.b.ỈK)1, as usually found in unstructured peptides; and (b) rapid NH–ND exchange rates of all the amide protons, including those of Phe9, Tyr10 and Glu19 that could be observed at less than h in sMccJ25-L1)21 vs more than days in both the thermolysin-generated MccJ25-L1)21 and the native cyclic MccJ25 (Fig 2) Table Structural statistics for the 30 final structures of MccJ25-L1–21 The van der Waals’ energy is calculated with a switched Lennard– Jones potential and the electric energy with a switched Coulomb potential and a dielectric constant e ¼ 32.7 The experimental NOE energy is calculated with a square-well potential and a force constant of ˚ 50 kcalỈmol)1ỈA)2 The dihedral angle potential is calculated with a force constant of 20 kcalỈmol)1Ỉrad)2 Parameter kcalỈmol)1 ETotal EBond EAngle EDihedral EImproper EVdW EElectrostatic ENOE restraint EDihedral restraint Mean rmsd from idealized covalent geometry ˚ Bond (A) Angle (deg) Dihedral (deg) Improper (deg) ˚ Average rmsd values (A) N, Ca, C¢, for residues 8–19 N, Ca, C¢, for residues 8–10 and 17–19 N, Ca, C¢, for residues 11–16 109 12.5 36 73 1.2 )19 2.2 3.5 0.00 0.012 0.012 2.10 57.6 2.5 ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.5 0.2 0.3 0.6 0.01 0.001 0.001 0.06 0.4 0.2 0.20 ± 0.07 0.18 ± 0.07 0.07 ± 0.03 6218 A Blond et al (Eur J Biochem 269) regions (Gly12, Gly13), or in the b-turn region (Gly11) together with Tyr10, His16 and Glu19, or in the extended region (Gly15) where residues 5, 7–9, 14, 17, 18 and 20 were also located The //w couples were very dispersed for the residues at positions 1–4, in contrast with those observed for the remaining MccJ25-L1)21 amino acids (Fig 4B) These //w couples reflected a certain degree of heterogeneity in the MccJ25-L1)21 structural definition Description of the MccJ25-L1)21 three-dimensional structure The superimposition of the 30 final structures in Fig illustrates the presence of two different regions in the linear MccJ25-L1)21 As shown by the average rmsd for the backbone heavy atoms (Table 2), the region encompassing residues 8–19 adopts a well-defined structure The global fold results in a hairpin-like structure, including a short twostranded antiparallel b-sheet connected by several turns that results in the Gly11–His16 loop By contrast, the N-terminal (Val1–Pro6) and C-terminal (Tyr20 and Phe21) ends are disordered, showing a number of different orientations The faster exchange rates ( h) observed for residues 1–3 are < in agreement with the absence of hydrogen bonds in the N terminus Identification of potential hydrogen bonds using the MOLMOL software, revealed that the MccJ25-L1)21 structure is stabilized by seven hydrogen bonds, in agreement with the NH temperature coefficients and NH–ND exchange rates (Fig 2) The NH temperature coefficients were particularly low for the residues 9–10 and 15–19 within the b-sheet This region appears to be stabilized by two hydrogen bonds (NH10 fi CO17, NH19 fi CO8) that are also found in the MccJ25 structure These involve the Tyr10 Fig Superimposition of backbone atoms (N, Ca, C¢) of the 30 final NMR-derived lowest-energy structures of MccJ25-L1)21 (best overlap for residues 8–19), whose geometric and energetic statistics are given in Table The view exhibits the 8–10/17–19 antiparallel b-sheet and the 11–16 loop Ó FEBS 2002 and Glu19 amide protons, which remained strikingly unexchanged at room temperature for more than days, a feature also reported for the native cyclic MccJ25 (Fig 2) These two hydrogen bonds are therefore believed to be particularly strong Residues 11–16 are folded into a loop (Fig 5) This region, which contains a series of turns, is strongly stabilized by five hydrogen bonds that involve amide protons exhibiting medium to very slow exchange rates (Fig 2) The NH11 fi CO9 and NH14 fi CO12 hydrogen bonds define a reverse c-turn (Phe9-Tyr10Gly11), with /10 ¼ )78.8 ± 1°/w10 ¼ +24.6 ± 3° and a c-turn (Gly12-Gly13-Ala14), with /13 ¼ +100.2 ± 2°/ w13 ¼ )81.9 ± 8°, respectively The NH11 fi CO9 is in fact a trifurcated hydrogen bond, as the acceptor atom (from the CO9 carboxyl group) is shared by three protons, namely NH11, NH12 and NH13 Taken together, these results define a mixed a/b-turn (NH13 fi CO9, NH12 fi CO9) The 11–16 loop is finally stabilized by the NH9 fi CO13 hydrogen bond, which enables a tight connection between the 11–14 region and the 8–10 strand Interestingly, despite the preservation of MccJ25 global structure in this region of MccJ25-L1)21, the hydrogen bond network does not fit between the two peptides, except for the two bonds that stabilize the b-sheet (NH10 fi CO17, NH19 fi CO8) This was expected from the NH–ND exchange rates and temperature coefficients of the amide protons in the Ile7–Gly15 region that differ from those found for the cyclic MccJ25, while they fit very closely for the residues belonging to the b-sheet (Fig 2) The slow exchange rates exhibited by some other amide protons, chiefly by Ser8 and Val17, can probably be likely attributed to their low level of accessibility as a result of nearby hydrophobic and aromatic side chains The resulting highly stable fold adopted by the thermolysin-linearized MccJ25 thus results in a boot-shaped hairpin-like structure (Fig 5) The 21–7 loop observed in the original MccJ25 structure is completely absent from the MccJ25-L1)21 structure due to opening of the peptide backbone Similarly, the cavity and the two bordering crab pincer-like regions described in the MccJ25 structure [20] are not maintained in the linear form Thermolysin cleaves the Phe21–Val1 bond, thus the loss of the bigger crab-pincer, which includes the residues 18–21 and 1, was expected However, the smaller crab-pincer, including residues 6–8 that are away from the thermolysin cleavage site, does not persist either The structure in the 8–19 region of MccJ25L1)21 is highly similar to that of the cyclic microcin, as ˚ indicated by an rmsd value of 0.55 A between the backbone atoms of residues 8–19 of the two peptides (Fig 6) Most of the side chains in the Phe9–Glu19 region are well ˚ defined (mean rmsd ¼ 0.53 A) and adopt a position close to that observed in the cyclic MccJ25 peptide The Phe9 side ˚ chain (mean rmsd ¼ 0.65 A) is engaged in the concave face of the boot and is flanked by the Ile7 side chain (mean ˚ ˚ rmsd ¼ 1.22 A) and the Pro18 ring (mean rmsd ¼ 0.35 A) (Fig 7) The side-chains of Tyr10, His16 and Val17 (mean ˚ rmsd ¼ 0.89, 1.21 and 0.26 A) form a cluster at the bottom of the loop (Fig 7) As was observed previously with MccJ25, the hydrophobic side chains are not packed in a core, but are distributed over the periphery of the structure, as is often found in larger proteins In addition, the aromatic residues are not stacked Several hydrophobic side chains adopt a specific location on the two strands of the b-sheet Ó FEBS 2002 Structure of thermolysin-linearized microcin J25 (Eur J Biochem 269) 6219 The side chains of Ile7, Phe9 and Tyr10 located on one strand are facing those of Phe21 (in half of the selected structures), Pro18 and Val17 on the other strand, respectively The resulting hydrophobic interactions supported by a few long-range NOEs seem to maintain MccJ25-L1)21 structure in a zipper-like fashion Together with the two strong NH10 fi CO17, NH19 fi CO8 hydrogen bonds described above, those hydrophobic interactions may account for the high stability of the peptide Stability studies Fig Superimposition of the solution structures of the cyclic MccJ25 (cyan) and the linear MccJ25-L1)21 (blue) The N and C termini are labelled on MccJ25-L The MccJ25-specific cleavage site by thermolysin is indicated by an arrow The preservation of the structured core shared by MccJ25 and MccJ25L1)21 in M urea, i.e the conditions of thermolysin cleavage, led us to investigate the stability of the linear peptide against denaturing agents and temperature CD was not used to probe the conformational changes because of the great similarity of the spectra of MccJ25 and all its linear variants The conformational transitions of MccJ25-L1)21 were therefore investigated by NMR spectroscopy, as well as RP-HPLC, which allows an efficient separation of the linear variants For comparison, the thermal denaturation of MccJ25 was also probed by NMR MccJ25-L1)21 was first treated with 1–6 M guanidinium hydrochloride or 1–8 M urea for 10 h at 25 °C and was then separated from the denaturing agent on a C8 cartridge RP-HPLC analysis did not show any variation in MccJ25-L1)21 retention time during the entire incubation In addition, the 1H-1D, TOCSY and NOESY spectra recorded after removal of the denaturing agents were identical to those of the untreated reference Thus, MccJ25L1)21 is resistant to chaotropic agents Fig Stereoview of the mean MccJ25-L1)21 structure showing the position and orientation of the side-chains The main chain is in grey, aromatic residues are in magenta, negatively charged or polar Glu19 and Ser8 are in orange, hydrophobic Val17 and Ile7 are in blue, His16 is in green and the Pro6 and Pro18 heterocycles are in black 6220 A Blond et al (Eur J Biochem 269) In parallel experiments, we tested the temperatureinduced denaturation of MccJ25-L1)21 After verifying the similarity of the MccJ25-L1)21 global peptide fold in CD3OH and dimethylsulfoxide-d6, NMR data were acquired in dimethylsulfoxide-d6, a solvent that enables the use of high temperatures The variations of the NH and Ha proton chemical shifts, and particularly those of Ser8, Phe9, Tyr10, Ala14, His16, Val17 and Glu19 selected to probe the conformational transitions, were followed between 25 and 165 °C The conformation was completely stable up to 95 °C At this temperature, minor conformers appeared, but the process was reversible, as indicated by a TOCSY spectrum identical to that of the reference, after the temperature was lowered to 25 °C On the contrary, at 165 °C, the minor conformers were unable to refold When subjected to the same protocol, MccJ25 maintained a stable three-dimensional structure up to 115 °C The combined action of temperature and denaturing agents was finally examined by using HPLC and NMR protocols similar to those defined for chaotropic agents only MccJ25-L1)21 was recovered in its original form after treatment with M guanidinium hydrochloride at 65 °C for 16 h Treatment with M urea at 65 °C for 16 h resulted in the coupling of one urea molecule at the peptide N terminus, but did not induce conformational changes Indeed, the NOE contacts were maintained between residues of the 8–10 and 17–19 regions, including the da,a9)18 NOE typical of the b-sheet present in both MccJ25 and MccJ25-L1)21 (data not shown) Furthermore, those strongly denaturing conditions showed only poor effect on the antibacterial activity (data not shown) Complete denaturation of MccJ25-L1)21 could not be obtained up to 40 h at 95 °C in M urea Thus, the MccJ25-L1)21 structure is highly resistant to both chemical denaturants and temperature The natural cyclic MccJ25, subjected to similar denaturing conditions, also maintained its three-dimensional structure These data argue for a high thermodynamic stability of both MccJ25 and the enzymatically prepared MccJ25-L1)21 DISCUSSION In the current study, we have studied the conformation and the antibacterial activity of microcin J25 variants that lack the macrocyclic backbone of the native peptide Among the three linear analogues studied, only the peptide obtained by enzymatic cleavage (MccJ25-L1)21) was antimicrobially active against the two test bacteria Indeed, although the peptide antimicrobial activity dropped by an average of two orders of magnitude upon thermolysin cleavage, the openring peptide retained significant bioactivity, with MICs < 0.6 lM For comparison, most of the antimicrobial peptides, such as magainins [4], cecropins [1], mammalian defensins [2,3], but also the cyclic bacteriocin AS48, cyclotides, rhesus theta-defensin-1 (RTD-1) and synthetic linear analogues [35–38], all exhibit MICs in the range of 0.5–20 lM Thermolysin cleavage of MccJ25 specifically occurs at the Phe21–Val1 peptide bond This area was shown in the MccJ25 three-dimensional structure to be less protected by both the side chains and the compactness of the structure This cleavage results in the complete disruption of the Phe21–Pro6 loop, and is accompanied by a net decrease in Ó FEBS 2002 antibacterial activity However, the remaining portion of the linear MccJ25-L1)21 structure is remarkably unaltered as compared with that of MccJ25 In particular, the region 8–19 shows a very well defined structure, with an irregular double-stranded antiparallel b-sheet folded into a twisted b-hairpin Both MccJ25 and MccJ25-L1)21 contain a stable arrangement of cross-linking hydrogen bonds associated with very low NH–ND exchange rates (over days) exhibited by several amide protons Those which stabilize the b-sheet (NH10 fi CO17, NH19 fi CO8) are identical in both peptides The structure of both forms is also stabilized by hydrophobic interactions involving mainly the aromatic side chains of Phe9, Tyr10 and Phe21 facing the Pro18 ring and the hydrophobic side chains of Val17 and Ile7, respectively Therefore, the peptide region that contains both the 8–10/17–19 b-sheet and the Gly11–His16 loop is critical for antibacterial activity The Phe21–Pro6 loop, as well as the cavity present in the MccJ25 structure [20] and the macrocyclic backbone, which are both disrupted upon linearization, are necessary to allow full activity to be reached The high stability of the three-dimensional structure of MccJ25-L1)21 is reminiscent of that encountered in globular proteins from extremophiles [39,40], but is quite exceptional among nonextremophile peptides and proteins Among antimicrobial peptides, the highest stabilities have been reported for peptides presenting a macrocyclic backbone The 70-residue bacteriocin AS-48 is highly resistant to proteases and shows a thermal denaturation temperature of 93 °C [35] On the basis of their original work on cyclotides and of studies on other circular bioactive peptides [36,38,41,42], Craik et al have proposed that the cyclization process has evolved to confer advantage to the producing organisms by increasing the resistance to proteolysis and improving the thermodynamic stability of their gene products From our results, the circular backbone is not essential to the preservation of MccJ25 active structure, as both MccJ25 and MccJ25-L1)21 structure and activity are resistant to highly stringent conditions (high temperature, chaotropic agents, proteolysis) However, the circular backbone is needed to reach the highly potent antibacterial activity of MccJ25 It could also play an essential role in the resistance to the numerous exoproteases encountered in the gut microflora ecosystem where MccJ25 is naturally encountered Considering the absence of activity of the synthetic variant sMccJ25-L1)21, which lacks the MccJ25 zipper-like structured core, it is tempting to speculate that the very stable hydrogen bonding of MccJ25 is involved in both the peptide structure and activity This structured-core makes MccJ25 very different from other cyclic antimicrobial peptides The mammalian antimicrobial peptide RTD-1, and the trypsin inhibitor from sunflower seeds SFTI-1 [38,42] instead contain disulfide bridges to stabilize the double-stranded antiparallel b-sheet To date, no general rule as to which factors lead to increased protein stability has emerged, except that cumulative effects of hydrogen bonding, hydrophobic, coulombic and van der Waals’ interactions are all involved [43,44] Most likely, the stability of the MccJ25-L1)21 structure is ensured by both (a) the hydrophobic interactions that involve the aliphatic and aromatic residues on opposing strands of the b-sheet and (b) the hydrogen bond network that stabilizes the b-sheet and Ó FEBS 2002 Structure of thermolysin-linearized microcin J25 (Eur J Biochem 269) 6221 were shown to be maintained in the thermolysin-linearized form In the cyclic MccJ25, the stability is likely to be reinforced by the constraining strength of the circular backbone Interestingly, the stable three-dimensional structures of both RTD-1 and SFTI-1 can be recovered from their synthetic linear analogues [38,42] By contrast, the two synthetic peptides (sMccJ25-L1)21 and sMccJ25-L12)11) are not folded and are devoid of antibacterial activity This strongly suggests that the structure conservation between MccJ25 and MccJ25-L1)21 does not result from the sequence of the linear MccJ25 itself, and consequently that the active conformation of MccJ25 cannot be acquired spontaneously by the native peptide These results raise the question of how MccJ25 adopts a mature and functional conformation from its linear precursor In a recent study, an elegant ligation method was used to complete the chemical synthesis of the cyclic MccJ25 [45] However, the folding of the synthetic cyclic peptide obtained was not investigated To date, the molecular mechanisms involved in MccJ25 folding remain unstudied Bioactive cyclic MccJ25 results from a biosynthetic pathway that should involve one or two enzymes needed to perform (a) the removal of the N-terminal 37-amino acid propiece of the MccJ25 propeptide, and (b) the head-to-tail ligation of the 21-amino acid C-terminal part of the propeptide The propiece together with the processing enzymes might be involved in structural maturation However, MccJ25 folding could also involve unidentified molecular partners Similar to the E coli microcin B17 synthase, which copurifies with an uncharacterized chaperone protein [46,47], it is possible that MccJ25 folding is also assisted by a helper molecule ACKNOWLEDGEMENTS We thank J.-P Briand (UPR 9021 CNRS, Strasbourg, France) for ´ peptide synthesis, A.-M Pons (Universite de La Rochelle, France) and M Lavina (Facultad de Ciencias, Montevideo, Uruguay) for providing ˜ the bacterial strains used in this study, and L Dubost for MS measurements We are grateful to B Gilquin (CEA, Saclay, France) for helpful and stimulating discussions and to A Cole (University of California, Los Angeles, USA) for critical reading of the manuscript This work was supported in part by the ÔProgramme de Recherche Fondamentale en Microbiologie et Maladies Infectieuses et ParasitairesÕ of the French Ministry for Research and Technology The 400-MHz NMR spectrometer and the mass spectrometer used in this study were ´ funded jointly by the Region Ile-de-France, the French Ministry for Research and Technology and by CNRS (France); the 600-MHz NMR ´ spectrometer was funded by the Region Haute-Normandie, France REFERENCES Steiner, H., Hultmark, D., Ergstrom, A & Boman, H (1981) Sequence and specificity of 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Proc Natl Acad Sci USA 97, 2962–2964 Yan, L.Z & Dawson, P.E (2001) Synthesis of peptides and proteins without cysteine residues by native chemical ligation combined with desulfurization J Am Chem Soc 123, 526–533 Li, Y.M., Milne, J.C., Madison, L.L., Kolter, R & Walsh, C.T (1996) From peptide precursors to oxazole and thiazole-containing peptide antibiotics: microcin B17 synthase Science 274, 1188– 1193 Milne, J.C., Roy, R.S., Eliot, A.C., Kelleher, N.L., Wokhlu, A., Nickels, B & Walsh, C.T (1999) Cofactor requirements and reconstitution of microcin B17 synthetase: a multienzyme complex that catalyzes the formation of oxazoles and thiazoles in the antibiotic microcin B17 Biochemistry 38, 4768–4781 SUPPLEMENTARY MATERIAL The following material is available from http://www blackwellpublishing.com/products/journals/suppmat/EJB/ EJB3340/ EJB3340sm.htm Table S1 The 1H and 13C chemical shifts in CD3OH of the thermolysin-linearized MccJ25-L1–21 Table S2 The 1H and 13C chemical shifts in CD3OH of the synthetic peptide having the same sequence (sMccJ25L1)21)  ... interactions may account for the high stability of the peptide Stability studies Fig Superimposition of the solution structures of the cyclic MccJ25 (cyan) and the linear MccJ25-L1)21 (blue) The N and C... MccJ25-L The MccJ25-specific cleavage site by thermolysin is indicated by an arrow The preservation of the structured core shared by MccJ25 and MccJ25L1)21 in M urea, i.e the conditions of thermolysin... antibacterial activity of microcin J25 variants that lack the macrocyclic backbone of the native peptide Among the three linear analogues studied, only the peptide obtained by enzymatic cleavage (MccJ25-L1)21)