Báo cáo Y học: Thermolysin-linearized microcin J25 retains the structured core of the native macrocyclic peptide and displays antimicrobial activity doc

Thermolysin-linearized microcin J25 retains the structured coreof the native macrocyclic peptide and displays antimicrobial activity Alain Blond1, Michel Cheminant1, Delphine Destoumieux

Thermolysin-linearized microcin J25 retains the structured core

of the native macrocyclic peptide and displays antimicrobial activity

Alain Blond1, Michel Cheminant1, Delphine Destoumieux-Garzo´n1, Isabelle Se´galas-Milazzo2,

Jean Peduzzi1, Christophe Goulard1and Sylvie Rebuffat1

1

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

antimicro-bial 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-dimen-sional 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

anti-microbial 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

three-dimensional solution structure of MccJ25-L1)21, determined

by two-dimensional NMR, consists of a boot-shaped

hair-pin-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 anti-parallel 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 condi-tions

Keywords: antimicrobial peptide; conformational stability; microcin; molecular modeling; solution structure

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

pro-teins 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, Salmo-nellaand Shigella strains, at minimum inhibitory concen-trations (MICs) ranging from 1 to 100 nM[14,15]

MccJ25 was reported to interact with liposomes com-posed 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, Muse´um National d’Histoire Naturelle,

63 rue Buffon, 75231 Paris, Cedex05, France.

Fax : + 33 1 40 79 31 35, Tel.: + 33 1 40 79 31 18,

E-mail: rebuffat@mnhn.fr

Abbreviations: Mcc, microcin; MIC, minimum inhibitory

concentra-tion; 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)

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

three-dimensional 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

thermolysin-linearized 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

sMccJ25-L12)11 peptide (G12GAGHVPEYF21V1GIGTPISFYG11),

which sequence derives from MccJ25 precursor (Fig 1)

Despite identical sequences, the folding and activity of the

enzymatically generated MccJ25-L1)21and chemically

syn-thesized 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

antibac-terial activity

E X P E R I M E N T A L P R O C E D U R E S

MccJ25 and MccJ25-L1 )21sample preparation

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 2 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

(Cap-cell C18, 5 lm, 7.5· 250 cm; Interchim, France) under

isocratic conditions in a (61 : 39) methanol/water mixture

containing 0.05% CF3COOH Separation was performed

at a 2 mLÆmin)1flow rate, and absorbance was monitored

at 226 nm

MccJ25-L1)21was obtained by thermolysin-digestion of native MccJ25 Typically, 1 lmol MccJ25 was dissolved in

8M urea (600 lL) and incubated at 46C for 30 min, before the addition of 0.17MNH4HCO3(1200 lL), 10 mM CaCl2(200 lL) and 40 lg thermolysin (Boehringer Mann-heim) The digestion was performed at 46C, pH 8.0, for

60 min The reaction was stopped by adding 400 lL acetic acid, and MccJ25-L1)21was 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) acetonit-rile/water solution containing 0.1% CF3COOH (flow rate:

1 mLÆmin)1) Absorbance was monitored at 226 nm Purity

of MccJ25-L1)21was 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)21sample was purified in two steps on an RP-HPLC C18 column (semipreparative Capcell, 5 lm; 7.5· 250 mm; Interchim) used at a flow-rate of 2 mLÆ min)1 The first separation was performed under isocratic elution with a (27 : 73) acetonit-rile/water mixture, whereas the second separation consisted

of a (40 : 60) to (60 : 40) methanol/water linear gradient at 0.7% methanolÆmin)1 The sMccJ25-L12)11 sample was purified in two steps on the same column as sMccJ25-L1)21

at a flow-rate of 2 mLÆ min)1 The first RP-HPLC separation was performed with a biphasic gradient composed of a 10-min isocratic step in a (26 : 74) acetonitrile/water mixture, followed by a 26 : 74 to 28 : 72 acetonitrile/water flat linear gradient (0.4% acetonitrileÆmin)1) The second HPLC con-sisted of an isocratic elution with a (30 : 70) acetonitrile/ water mixture Absorbance was monitored at 226 nm Antibacterial assays

Antibacterial activity of MccJ25, MccJ25-L1)21,

sMccJ25-L1)21and sMccJ25-L12)11was assayed against two bacteria highly sensitive to MccJ25 The test microorganisms,

E coli MC4100 tolC– and S enteritidis, were kindly provided by M Lavin˜a (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

Fig 1 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)11displays

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].

bacteria diluted in PB to D600¼ 0.001 Plates were

incubated for 16 h at 30C 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

(Longjum-eau, France), using a 0.05-mm path cell The spectra were

measured for methanolic solutions at peptide

concentra-tions of 0.05–1 mM

NMR spectroscopy

Samples (0.5 mL) of 6 mM MccJ25-L1)21, sMccJ25-L1)21

and sMccJ25-L12)11in methanol (CD3OH) were placed in

5-mm Wilmad tubes for the NMR experiments Data were

acquired on Bruker AVANCE 400 and DMX 600

spec-trometers, equipped with1H-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 10C 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

1H-13C heteronuclear experiments, optimized for J-values of

135 Hz (HSQC) and 7 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 10–35C, by acquiring sixseries of

one-dimensional (1D)-1H and TOCSY spectra at 400.13 MHz,

using 5C temperature increments Exchange of amide

protons was monitored as described previously [20] Briefly,

a normal isotopic sample (either MccJ25-L1)21or

sMccJ25-L1)21) was dissolved in CD3OD at 0C It was analysed for

2 h at 0C and over 3 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)21structure calculation

were derived from NOE cross-peaks in the NOESY

spectrum recorded at 10C with sm¼ 100 ms, that in turn

were converted into distances by volume integration using

intensity between the Tyr20 Hd and He protons, which corresponds to a distance of 2.45 A˚, was used for calibra-tion To ascertain whether the contribution of zero-quan-tum 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 nonstereospe-cifically 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 10C from the 1D- and the high digital resolution DQF-COSY spectra (CD3OH), were restrained to )120 ± 25 for 3JNHCaHP 9.5 Hz (Phe9, Val17), )120 ± 45 for a3JNHCaHin the range 8.1–8.9 Hz (Ser8, Tyr20, Phe21) and)120 ± 50 for a3JNHCaH68.0 Hz (Thr5, Tyr10, Glu19) Two v1dihedral 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 else-where [20], using simulated annealing and energy minimi-zation protocols within theX-PLORversion 3.851 software [25], run either on a Silicon Graphics O2 workstation (IRIX 6.5), or a Gateway computer (SUSE LINUX7.0) 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 ambi-guous assignments were further used with the appropriate treatment in X-PLOR [27,28] During the processing, the distance restraint force was kept at 50 kcalÆmol)1ÆA˚)2and the NOE intensities were averaged with the sum option Of the 100 structures generated, 80 had a total energy less than

25 kcalÆmol)1and led in all cases to systematic distance and dihedral angle violations lower than 0.2 A˚ and 5, respect-ively Refinement of the structures was achieved using the conjugate gradient Powell algorithm with 7000 cycles of energy minimization and theCHARMM 22force 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 theX-PLOR [25],MOLMOL[30] andPROCHECK_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 devi-ation angles with respect to 180, with either: distance

< 2.7 A˚, deviation angle < 35 (NH12fi CO9,

NH10fi CO17), or distance < 3.0 A˚, deviation angle

< 40 (NH19fi CO8, NH9fi CO13, NH13fi CO9), or distance < 3.0 A˚, deviation angle < 50 (NH11fi CO9,

NH14fi 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

Stability of MccJ25 and MccJ25-L1)21to denaturing

conditions

Thermal stability of MccJ25-L1)21and MccJ25 was

exam-ined between 25 and 165C 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 25C 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 165C, 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 165C, by slowly lowering the

temperature to 25C and acquiring control 1D-1H and

TOCSY spectra The acquisition time of the NMR spectra

at each temperature was fixed at 2 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 (1–6M guanidinium

hydro-chloride or 1–8M urea in water) and temperature

conditions (25–95C) 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% CF3

COOH-con-taining (32 : 68) acetonitrile/water mixture at a flow rate

of 1 mLÆmin)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 0 : 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

R E S U L T S

Generation of MccJ25 linear variants: MccJ25-L1)21,

s MccJ25-L1)21ands 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/z¼ 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)21initially called

MccJ25-L [19] or by chemical synthesis (sMccJ25-L1)21and

sMccJ25-L12)11; Fig 1) The sequences chosen for the

synthetic peptides are identical to those of the

thermolysin-cleaved MccJ25 (sMccJ25-L1)21) and of the 21-residue

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)21and [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)11and 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 nMand 300–

600 nM, respectively This led us to examine the three-dimensional structures of these linear MccJ25 variants, with particular attention to MccJ25-L1)21, following the hypo-thesis 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)21at 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)21was evaluated by recording several CD spectra at concentrations ranging from 0.05 to 1 mM The similarity in the patterns obtained

at the various concentrations indicated the absence of aggregation below 1 mM In addition, 1D-1H and TOCSY spectra performed for concentrations between 1 and 6 mM, did not show any significant variation of the amide and a

Table 1 Minimum inhibitory concentrations of MccJ25 linear variants against S enteritidis and E coli MC4100 tolC) MICs were deter-mined in triplicate according to the liquid growth inhibition assay MICs (n M ) 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 l M

Peptide S enteritidis E coli MC4100 tolC –

proton chemical shifts, ensuring that MccJ25-L1)21will 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)21were obtained at

10C, 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 the1H-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

dimethyl-sulfoxide-d6, respectively In both sMccJ25-L1)21 and

sMccJ25-L12)11NOESY spectra, the aHi-1-dHicross-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-carbon13C 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,jand dNai,jcontacts 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 (> 8 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

complete lack of medium- and long-range NOE connectiv-ities 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

Fig 2 Comparison of NMR conformational parameters for MccJ25 (black), MccJ25L 1)21 (grey) and sMccJ25-L 1)21 (white) The intensities

of the secondary chemical shifts of the Ha protons (CSD Ha ) and Ca carbons (CSD Ca ), of the3J NHCaH coupling constants and temperature coefficients of the NH protons (Dd/DT NH ) 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

3 days), slow (S: 1–2 days), medium (M: 10–24 h), fast (F: 2–10 h) and very fast (VF: less than 1 h) exchanging NH protons; * stands for not determined.

Fig 3 Pattern of sequential, medium- and long-range NOE connectiv-ities involving the NH, a, b and d protons of MccJ25-L1)21 dAB i,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.

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 the3JNHCaHcouplings were all in

the range 6.5–7.0 Hz, except for three values around 8 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)21structure

was demonstrated by: (a) temperature coefficients of amide

protons in the range of 6–10 p.p.b.ÆK)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 2 h in

sMccJ25-L1)21vs more than 3 days in both the

thermoly-sin-generated MccJ25-L1)21and the native cyclic MccJ25

(Fig 2)

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 tempera-ture coefficients between)4 and )7.5 p.p.b.ÆK)1 Less than half of the3JNHCaHcoupling 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)21structure 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-dimen-sional structure of the thermolysin-linearized form,

MccJ25-L1)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)21solution structure is given in Table 2

From Thr5 to Tyr20, the individual backbone confor-mation 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 4 NOE distribution per residue (A) and values of / and w angles in

the 30 final structures (B) for MccJ25-L 1)21 Intraresidual, sequential,

and medium- and long-range NOEs are in black, grey and white,

respectively.

Table 2 Structural statistics for the 30 final structures of MccJ25-L 1–21 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 kcalÆmol)1ÆA˚)2 The dihedral angle potential is calculated with a force constant of 20 kcalÆmol)1Ærad)2.

E Dihedral restraint 0.00 ± 0.01 Mean rmsd from idealized covalent geometry 0.012 ± 0.001

Average rmsd values (A˚)

N, Ca, C¢, for residues 8–19 0.20 ± 0.07

N, Ca, C¢, for residues 8–10 and 17–19 0.18 ± 0.07

N, Ca, C¢, for residues 11–16 0.07 ± 0.03

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)21amino acids (Fig 4B) These

//w couples reflected a certain degree of heterogeneity in the

MccJ25-L1)21structural definition

Description of the MccJ25-L1)21three-dimensional

structure

The superimposition of the 30 final structures in Fig 5

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

two-stranded 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 (< 1 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

agree-ment 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 (NH10fi CO17, NH19fi CO8) that are

also found in the MccJ25 structure These involve the Tyr10

and Glu19 amide protons, which remained strikingly unexchanged at room temperature for more than 3 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 NH11fi CO9 and NH14fi CO12 hydrogen bonds define a reverse c-turn (Phe9-Tyr10-Gly11), with /10¼)78.8 ± 1/w10¼ +24.6 ± 3 and a c-turn (Gly12-Gly13-Ala14), with /13¼ +100.2 ± 2/

w13¼)81.9 ± 8, respectively The NH11fi CO9 is in fact a trifurcated hydrogen bond, as the acceptor atom (from the CO9carboxyl group) is shared by three protons, namely NH11, NH12 and NH13 Taken together, these results define a mixed a/b-turn (NH13fi CO9,

NH12fi CO9) The 11–16 loop is finally stabilized by the

NH9fi 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 (NH10fi CO17,

NH19fi 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 thermo-lysin-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

MccJ25-L1)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 rmsd¼ 0.53 A˚) and adopt a position close

to that observed in the cyclic MccJ25 peptide The Phe9 side chain (mean rmsd¼ 0.65 A˚) is engaged in the concave face

of the boot and is flanked by the Ile7 side chain (mean rmsd¼ 1.22 A˚) and the Pro18 ring (mean rmsd ¼ 0.35 A˚) (Fig 7) The side-chains of Tyr10, His16 and Val17 (mean rmsd¼ 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

Fig 5 Superimposition of backbone atoms (N, Ca, C¢) of the 30 final

NMR-derived lowest-energy structures of MccJ25-L 1)21 (best overlap

for residues 8–19), whose geometric and energetic statistics are given in

Table 2 The view exhibits the 8–10/17–19 antiparallel b-sheet and the

11–16 loop.

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, respect-ively 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 NH10fi CO17, NH19fi CO8hydrogen bonds des-cribed above, those hydrophobic interactions may account for the high stability of the peptide

Stability studies The preservation of the structured core shared by MccJ25 and MccJ25L1)21 in 2M urea, i.e the conditions of thermolysin cleavage, led us to investigate the stability of the linear peptide against denaturing agents and tempera-ture 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)21were therefore investigated by NMR spectro-scopy, 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)21was first treated with 1–6Mguanidinium hydrochloride or 1–8Murea for 10 h at 25C and was then separated from the denaturing agent on a C8 cartridge RP-HPLC analysis did not show any variation in MccJ25-L1)21retention 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,

MccJ25-L1)21is resistant to chaotropic agents

Fig 7 Stereoview of the mean MccJ25-L 1)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.

Fig 6 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

thermo-lysin is indicated by an arrow.

In parallel experiments, we tested the

temperature-induced 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 165C The conformation was completely

stable up to 95C 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 25C On the contrary, at

165C, the minor conformers were unable to refold When

subjected to the same protocol, MccJ25 maintained a stable

three-dimensional structure up to 115C

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 6M guanidinium hydrochloride at

65C for 16 h Treatment with 8M urea at 65C 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)

Further-more, 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 95C in 8M urea Thus, the

MccJ25-L1)21structure 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

enzymat-ically prepared MccJ25-L1)21

D I S C U S S I O N

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

open-ring 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

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)21contain a stable arrangement of cross-linking hydrogen bonds associated with very low NH–ND exchange rates (over 3 days) exhibited by several amide protons Those which stabilize the b-sheet (NH10fi CO17, NH19fi 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)21is 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

93C [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)21structure 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)21structure 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

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)21and 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

precur-sor 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

fold-ing could also involve unidentified molecular partners

Similar to the E coli microcin B17 synthase, which

copu-rifies with an uncharacterized chaperone protein [46,47], it is

possible that MccJ25 folding is also assisted by a helper

molecule

A C K N O W L E D G E M E N T S

We thank J.-P Briand (UPR 9021 CNRS, Strasbourg, France) for

peptide synthesis, A.-M Pons (Universite´ de La Rochelle, France) and

M Lavin˜a (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 Programme de Recherche

Fondamentale en Microbiologie et Maladies Infectieuses et Parasitaires

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 Re´gion Ile-de-France, the French Ministry for

Research and Technology and by CNRS (France); the 600-MHz NMR

spectrometer was funded by the Re´gion Haute-Normandie, France.

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