nNOS inhibition, antimicrobial and anticancer activity of theamphibian skin peptide, citropin 1.1 and synthetic modificationsThe solution structure of a modified citropin 1.1

Jason Doyle1, Craig S. Brinkworth2, Kate L. Wegener2, John A. Carver3, Lyndon E. Llewellyn1,Ian N. Olver4, John H. Bowie2, Paul A. Wabnitz2 and Michael J. Tyler5

1Australian Institute for Marine Science, Townsville MC, Queensland, Australia; 2Department of Chemistry,

The University of Adelaide, Australia; 3Department of Chemistry, University of Wollongong, Wollongong, Australia;4Oncology Department, Royal Adelaide Hospital and Department of Medicine, The University of Adelaide,

South Australia, Australia; 5Department of Environmental Biology, The University of Adelaide, South Australia, Australia

A large number of bioactive peptides have been isolatedfrom amphibian skin secretions. These peptides have avariety of actions including antibiotic and anticancer acti-vities and the inhibition of neuronal nitric oxide synthase.We have investigated the structure–activity relationship ofcitropin 1.1, a broad-spectrum antibiotic and anticanceragent that also causes inhibition of neuronal nitric oxidesynthase, by making a number of synthetically modifiedanalogues. Citropin 1.1 has been shown previously to forman amphipathic a-helix in aqueous trifluoroethanol. Theresults of the structure–activity studies indicate the terminalresidues are important for bacterial activity and increasing

the overall positive charge, while maintaining an amphi-pathic distribution of residues, increases activity againstGram-negative organisms. Anticancer activity generallymirrors antibiotic activity suggesting a commonmechanismof action. The N-terminal residues are important for inhi-bition of neuronal nitric oxide synthase, as is an overallpositive charge greater than three. The structure of one of themore active synthetic modifications (A4K14-citropin 1.1)was determined in aqueous trifluoroethanol, showing thatthis peptide also forms an amphipathic a-helix.

Keywords: citropin; antibacterial; anticancer; nNOS activity.

Amphibians have rich chemical arsenals that form anintegral part of their defence systems, and also assist withthe regulation of dermal physiological action. In response toa variety of stimuli, host defence compounds are secretedfrom specialized glands onto the dorsal surface and into thegut of the amphibian [1–4]. A number of different types ofbioactive peptides have been identified from the glandularskin secretions of Australian anurans of the Litoria genus,including (a) smooth muscle active neuropeptides of thecaerulein family [5–8], and (b) wide-spectrum antibiotics,e.g., the caerin peptides from green tree frogs of the genusLitoria [6–8], the citropins from the tree frog, L. citropa[9,10], and the aureins from the bell frogs, L. aurea andL. raniformis [11]. Among the most active of the antibioticpeptides are caerin 1.1, citropin 1.1 and aurein 1.2:caerulein 1.1 pEQGY(SO3)TGWMDF-NH2; caerin 1.1

GLLSVLGSVAKHVLPHVVPVIAEHL-NH2; citropin 1.1GLFDVIKKVASVIGGL-NH2; aurein 1.2 GLFDIIKKIAESF-NH2.Aurein 1.2 contains only 13 amino acid residues and is

the smallest peptide from an anuran reported to havesignificant antibiotic activity. The aurein peptides have alsobeen shown to exhibit modest anticancer activity in testscarried out by the National Cancer Institute (Washington,WA, USA) [12].The solution structures of the antibiotic (and anticancer

active if appropriate) peptides shown above have beeninvestigated by NMR spectroscopy. In d3-trifluoroethanol/water mixtures, caerin 1.1 adopts two well-defined helices(Leu2–Lys11 and Val17–His24) separated by a hinge regionof less-defined helicity and greater flexibility, with hydro-philic and hydrophobic residues occupying well definedzones [13]. The central hinge region is necessary for optimalantibiotic activity [13]. Similar NMR studies of citropin 1.1[9] and aurein 1.2 [11] show that these peptides adoptconventional amphipathic a-helical structures, a featurecommonly found in membrane-active agents [1–4,8]. Inter-action occurs at the membrane surface with the charged,and normally basic peptide adopting an a-helical confor-mation and attaching itself to charged, and normallyanionic sites on the lipid bilayer. This ultimately causesdisruption of normal membrane function leading to lysis ofthe bacterial or cancer cell [14–16].Many Australian anurans that we have studied conform

to the model outlined above in that they have a variety of

Correspondence to: J. H. Bowie, Department of Chemistry,

The University of Adelaide, South Australia, Australia.

Fax: +61 8303 4358, Tel.: +61 88303 5767,

E-mail: john.bowie@adelaide.edu.au

Abbreviations: MIC, minimum inhibitory concentration; NADPH,

nicotinamide adenine nucleotide phosphate, reduced form;

eNOS, endothelial nitric oxide synthase; iNOS, inducible NOS;

nNOS, neuronal NOS; RMD, restrained molecular dynamics;

SA, simulated annealing.

(Received 23 September 2002, revised 28 November 2002,

accepted 15 January 2003)

Eur. J. Biochem. 270, 1141–1153 (2003) � FEBS 2003 doi:10.1046/j.1432-1033.2003.03462.x

host defence peptides in the skin (and gut) glands including aneuropeptide that acts on smooth muscle and at least onepowerful wide-spectrum antibiotic and/or anticancer activepeptide like those described above [8]. However there aresome species of anuran that divert markedly from thisscenario. For example, the Australian stony creek frog(L. lesueuri) [17] and the giant tree frog (L. infrafrenata) [18]bothproduce the neuropeptide, caerulein, but lack anywide-spectrum antimicrobial peptide. The major peptides in theskin secretions of these two Litoria species have been namedlesueurin and frenatin 3, respectively: their sequences areshownbelow:LesueurinGLLDILKKVGKVA-NH2;Fren-atin 3 GLMSVLGHAVGNVLGGLFKPKS-OH. Neitherlesueurin nor frenatin 3 show any significant antibiotic oranticancer activity, but in tests carried out at the AustralianInstitute of Marine Science (Townsville, Queensland,Australia), both peptides were shown to inhibit the forma-tion of nitric oxide by the neuronal isoform of nitric oxidesynthase (nNOS)with IC50 values atlMconcentrations [17].Further nNOS testing on other peptides isolated from treefrogs of the Litoria genus showed that each species has atleast one major skin peptide that inhibits nNOS and thatthere are (at least) three groups of peptides that inhibitnNOS. Inhibitor group 1 includes citropin type peptides(that are also antimicrobial and anticancer agents); for thesequence of citropin 1.1 see above. The second groupcomprises peptides with sequence similarity to frenatin 3:these peptides show no significant antimicrobial or antican-cer activity. The third inhibitor group includes the caerin 1peptides (see the sequenceof caerin 1.1 above): these peptidesalso show powerful antimicrobial and antifungal activity.The three nitric oxide synthases, namely neuronal, endo-

thelial (eNOS) and inducible (iNOS), are highly regulatedenzymes responsible for the synthesis of the signal molecule,nitric oxide. They are among the most complex enzymesknown (e.g., for nNOS see [19,20]). By a complex sequenceinvolving binding sites for a number of cofactors includingheme, tetrahydrobiopterin, FMN, FAD and NADPDH,nNOS converts arginine to citrulline, releasing the short-lived but reactive radical NO [21,22]. Nitric oxide synthasesare composed of two domains: (a) the catalytic oxygenasedomain that binds heme, tetrahydrobiopterin and thesubstrate arginine, and (b) the electron supplying reductasedomain that binds NADPH, FAD and FMN. Communi-cation between the oxygenase and reductase domains isdetermined by the regulatory protein calmodulin whichinteracts at a specific site between the two domains. In thecases of nNOS and eNOS isoforms, but not for iNOS,calmodulin is regulated by intracellularCa2+ [23–26].Dime-rization of the oxygenase domain is necessary for catalyticactivity [21,22]. The amphipathic amphibian peptides inhibitnNOS by interacting with Ca2+-calmodulin, changing theshape of the regulatory enzyme, thus impeding its inter-action at the calmodulin binding site on nNOS [17]. Thereare other examples of small helical peptides inhibitingnNOS in this way [27,28].The amphibian may have two possible uses for a peptide

that inhibits nNOS. First, on attack by a predator, theamphibian may use the nNOS inhibitor to regulate its ownphysiological state. The second scenario is that the nNOSinhibitors are front-line defence compounds. A predatoringesting even a small amount of the nNOS inhibitor could

be seriously affected if only part of its NO messengercapability is reduced. All animals produce NOS isoforms,and it has been reported that bacteria also produce NOS[29–32].The citropin 1 group of peptides has significant anti-

biotic, anticancer and nNOS activity, despite being com-prised of only 16 amino-acid residues. In this paper wedescribe our investigations into the structure/activity rela-tionships for the amphibian peptide citropin 1.1. Theactivities of citropin 1.1 are compared with those of anumber of synthetically modified citropins 1 and otherrelated molecules to gain insight into the sequence require-ments for activity. The 3D solution structure of one of themost potent of the synthetically modified citropins has beendetermined using 1H-NMR procedures. This structure iscompared with that already determined for citropin 1.1 [9].

Methods

Preparation of synthetic peptides

All peptides listed in Tables 1 and 4 were synthesized (byMimotopes, Clayton, Victoria, Australia) using L-aminoacids via the standard N-a-Fmoc method (full detailsincluding protecting groups and deprotection have beenreported recently [33]). Synthetic versions of naturallyoccurring peptides were shown to be identical to the nativeform by electrospray mass spectrometry and HPLC.

Bioactivity assays

Bioactivity testing was carried out on citropin 1.1,D-citropin 1.1 and A4K14-citropin of both 95% and 80%purities. The activities were the same range for each pair ofsamples. Activity tests on all other synthetic modificationswere performed with samples which had � 80% purity asadjudged by HPLC.

Antimicrobial testing

Synthetic peptides were tested for antibiotic activity by theMicrobiology Department of the Institute of Medical andVeterinary Science (Adelaide, Australia) by a standardmethod [34]. The method involved the measurement ofinhibition zones (produced by the applied peptide) on a thinagarose plate containing the microorganisms listed inTable 2. Concentrations of peptide tested were 100, 50,25, 12.5, 6, 3 and 1.5 lgÆmL)1. The maximum error in theantibiotic results listed in Table 2 is ±1 dilution factor:e.g., if the MIC is 3 lgÆmL)1, the maximum possible rangeis 1.5–6 lgÆmL)1.

Anticancer activity testing

Synthetic peptides were tested in the human tumour linetesting program of the US National Cancer Institute [12].All compounds were tested initially against three tumourlines (breast, lung and CNS cancers), and if activity wasindicated, the peptide was then tested in vitro against 60human cell lines. If a particular peptide failed the first stageof the test program it is indicated as inactive (even though itmay have shown some activity). Full test data are not

1142 J. Doyle et al. (Eur. J. Biochem. 270) � FEBS 2003

provided in this paper. The summary data recorded inTable 3 indicate the particular groups of cancers tested, theaverage IC50 concentration of the peptide against that groupof cancers and the number of tumours, out of 60 tested, thatwere affected by the particular peptide. For details of howthe IC50 value is determined from graphical data see [12].

Neuronal nitric oxide synthase inhibition

Inhibition of nNOS was measured by monitoring theconversion of [3H]arginine to[3H]citrulline. In brief, thisinvolved incubation of a homogenate of rat cerebella (whichhad endogenous arginine removed by ion exchange chro-matography) in a reaction buffer (33 mM Hepes, 0.65 mMEDTA, 0.8 mM CaCl2, 3.5 lgÆmL)1 calmodulin, 670 lMb-NADPH, 670 lM, dithiothreitol, pH 7.4) containing20 nM [3H]arginine (NEN Life Sciences, Boston, MA,USA). The nNOS inhibitor, Nx-nitro-L-arginine (1 mM)was used to measure background radioactivity. Reactionswere terminated after 10 min with 50 lL of 0.3 M EGTA.An aliquot (50 lL) of this quenched reaction mixture wastransferred to 50 lL of 500 mM Hepes (pH 5.5). AG50W-X8 (Na+ form) resin (100 lL) was added to separate[3H]arginine from [3H]citrulline. After repeated vortexing,this slurry was centrifuged at 1200 g for 10 min, and 70 lLof supernatent was removed and the [3H]citrulline measuredby scintillation counting. Peptides selected for furtherexamination to determine the mechanism of inhibition wereassayed in the same reaction buffer as used for initialscreening except that it contained 30 nM [3H]argininesupplemented with 0.3–13.3 mM arginine.

Data analysis for nNOS studies

Peptide inhibition curves were fitted using the curve-fittingroutine of SIGMAPLOT (SPSS, Chicago, IL, USA) using avariation of the Hill equation: fmols [3H]citrulline produc-tion ¼ 1/(1 + [inhibitor]/ICn

50), where IC50 is the concen-tration at which the peptide causes 50% inhibition and n isthe slope of the curve and can be considered as a pseudoHillcoefficient [35]. Lineweaver–Burk plots [36] were generatedusing SIGMAPLOT (SPSS, Chicago, IL, USA). The meanerror in the IC50 results listed in Table 4 is ±1.3%.

NMR spectroscopy of citropin synthetic modification(A4K14-citropin 1.1)

NMR experiments were performed on a solution of 5.7 mgof A4K14-citropin 1.1 dissolved in a mixture of water(0.35 mL) and d3-trifluoroethanol (0.35 mL), that had afinal concentration of 4.9 mM and a measured pH of 4.12.NMR spectra were acquired on a Varian Inova-600 NMRspectrometer at a 1H frequency of 600 MHz and 13C

Table 1. Citropin 1.1 and synthetic modifications. Modifications are

shown in bold.

Citropin Sequence

Relative

molecular mass

1.1 GLFDVIKKVASVIGGL-NH2 1614

1.1.2 DVIKKVASVIGGL-NH2a,b 1297

Modified peptide

1 GlfdvikkvasviGGl-NH2 1614

2 GLADVIKKVASVIGGL-NH2b 1537

3 GLFAVIKKVASVIGGL-NH2 1570

4 GLFDVIAKVASVIGGL-NH2b 1557

5 GLFDVIKAVASVIGGL-NH2a 1557

6 GLFDVIAAVASVIGGL-NH2a,b 1500

7 GLFDVIKKVAAVIGGL-NH2 1599

8 GLFDVIKKVASVIGGA-NH2b 1572

9 GLFEVIKKVASVIGGL-NH2b 1628

10 GLFDVIKKVASKIGGL-NH2b 1643

11 GLFDVIKKVASVIKGL-NH2 1685

12 GLFDVIKKVASKIKGL-NH2b 1714

13 GLFDVIKKVASVIKKL-NH2 1756

14 GLFDVIAKVASVIKKL-NH2 1699

15 GLFAVIKKVASVIKGL-NH2 1655

16 GLFAVIKKVASVIKKL-NH2 1712

17 GLFAVIKKVAAVIKKL-NH2 1696

18 GLFAVIKKVAAVIRRL-NH2 1752

19 GLFAVIKKVAKVIKKL-NH2 1753

20 KLFAVIKKVAAVIGGL-NH2b 1625

21 KLFAVIKKVAAVIRRL-NH2b 1823

22 GLFKVIKKVASVIGGL-NH2 1627

23 GLFKVIKKVAKVIKKL-NH2 1810

Retro

1.1 LGGIVSAVKKIVDFLG- NH2 1614

a These compounds show no antibiotic activity against the listed

bacteria in Table 2 at MIC ¼ 100 lgÆmL)1. b Compounds somarked failed the initial NCI tests against three cancer types. Many

of these compounds do show activity, but not below concentrations

of 10)4 M. For NCI test results, see Table 3.

Table 2. Antibiotic activites of Citropin 1.1 and synthetic analogues [MIC values (lgÆmL)1)]. The absence of a figure means the activity is

>100 lgÆmL)1. For error range see Methods.

1.1 1.1D 2 3 4 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Retro

Bacillus cereus 50 50 50 25 100 50 50 50 25 12 25 25 100 50 25 100 50 50 100

Escherichia colia 100 100 50 50 50 100 100 100 100

Leuconostoc lactis 6 3 100 3 25 6 25 6 12 3 6 3 6 3 3 1.5 12 3 12 12 6 6 12

Listeria innocua 25 25 100 25 25 50 25 100 50 25 12 6 12 12 25 50 100 25 100

Micrococcus luteus 12 25 50 12 100 12 50 25 50 12 6 25 25 50 25 100 12 25 100

Pasteurella multocidaa 100 100 100 100 100 100 100 100 100 100

Staphyloccus aureus 25 25 100 25 100 25 100 25 100 6 12 25 25 25 25 50 25 50 50

Staphylococcus epidermidis 12 12 100 12 100 25 50 100 25 100 12 6 6 12 3 12 12 25 100 12 12 100

Streptococcus uberis 25 12 100 25 100 25 100 50 100 25 100 50 12 25 25 12 50 50 100 100 50 50 50

a Gram-negative organism.

� FEBS 2003 Solution structure of a modified citropin 1.1 (Eur. J. Biochem. 270) 1143

frequency of 150 MHz. All NMR experiments wereacquired at 25 �C. 1H-NMR resonances were referencedto the methylene protons of residual d3-trifluoroethanol(3.918 p.p.m). The 13C (F1) dimensions of the heteronuclearsingle-quantum coherence (HSQC) and heteronuclear mul-tiple-bond correlation (HMBC) spectra were referenced tothe 13CD2 (60.975 p.p.m) and

13CF3 (125.9 p.p.m) reso-nances of d3-trifluoroethanol, respectively.Double-quantum-filtered correlation spectroscopy

(DQF-COSY) [37]; total correlation spectroscopy (TOCSY)[38]; and nuclear Overhauser effect spectroscopy(NOESY) [39]; were all collected in the phase-sensitivemode using time proportional phase incrementation [40]in t1. Two hundred and fifty-six t1 increments were usedfor each experiment. Thirty-two scans were time averagedfor each increment in the TOCSY and NOESY experi-ments, while 16 scans were averaged in the DQF-COSYexperiment. The free induction decay in t2 consisted of2048 data points over a spectral width of 5555.2 Hz. Thetransmitter frequency was centred on the water resonanceand conventional low power presaturation from the samefrequency synthesizer was applied during a 1.5-s relaxa-tion delay to suppress the large water signal in theTOCSY and NOESY spectra. Gradient methods forwater suppression were used in the DQF-COSY spectrum[41]. The TOCSY spectrum was acquired with the pulsesequence used by Griesinger et al., 1988 [42] whichminimizes cross relaxation effects, employing a 70-msMLEV-17 spin-lock. NOESY spectra were acquired withmixing times of 80, 150 and 250 ms.An HSQC experiment [43] was performed to assign the

a-13C resonances via correlations to their attached protons.The interpulse delay was set to 1/2JCH (3.6 ms correspond-ing to JCH ¼ 140 Hz). Two hundred and fifty-six t1increments, each comprising 64 time averaged scans, wereacquired over 2048 data points and 5555.2 Hz in the directlydetected (1H, F2) dimension. The spectral width in the

13C(F1) dimension was 24133 Hz. An HMBC spectrum [44]was collected to assign the carbonyl-13C resonances viacorrelations through two and three bonds to a, b and NHprotons (with an interpulse delay of 1/2JCH ¼ 62.5 ms forJCH ¼ 8 Hz). For this experiment, 400 t1 increments, eachcomprising 64 scans, were acquired over 4096 data points

and 5555.2 Hz in the 1H (F2) dimension. The spectral widthfor the 13C (F1) dimension was 36216 Hz.All 2D NMR spectra were processed on a Sun Micro-

systemsUltra Sparc 1/170workstation using VNMR software(version 6.1 A). The data matrices were multiplied by aGaussian function in both dimensions, then zero-filled to2048 data points in F1 prior to Fourier transformation(4096 data points for the HMBC). Final processed 2DNMR matrices consisted of 2048 · 2048 or 4096 · 4096real points.

Structural restraints

Cross-peaks in the NOESY (mixing time ¼ 250 ms) spec-trumwere assigned using the program SPARKY (version 3.98)[45]. The cross-peak volumes were converted to distancerestraints using themethod of Xu et al., 1995 [46]. Briefly, inthis procedure, the weakest and strongest peaks are calibra-ted at 5.0 and 1.8 A, respectively, in order to calculateintensity-dependent proportionality factors. These factorswere then used to determine the upper bound restraints forthe remaining peaks. To be conservative, the final restraintswere increasedby10percent fromthese calculatedvalues.Alllowerbound restraintswere set to 1.8 A.For each symmetricpair of cross-peaks, the peak of smaller volume was used.This procedure generated 264 distance restraints, including115 intraresidue restraints, 52 sequential (i,i + 1) restraintsand 65 medium range restraints (from 2–4 residues distant).Thirty-two additional restraints were ambiguous. 3JNHCaH

values were measured from a 1D 1HNMR spectrum, wherethe free induction decay had been multiplied by a sine-bellwindow function to enhance the resolution. Dihedralangles were restrained as follows: 3JNHCaH 6 5 Hz,/¼ )60 ± 30�; 5 < 3JNHCaH 6 6 Hz, /¼ )60 ± 40�.Where 3JNHCaH > 6 Hz, phi angles were not restrained. Atotal of 13 dihedral angle restraintswere used in the structurecalculations.

Structural calculations

Structures were generated on a Sun Microsystems Sparc 1/170 workstation using X-PLOR software (version 3.851)[47,48]. The restrained molecular dynamics (RMD) and

Table 3. Anticancer activites of citropin 1.1 and synthetic analogues (IC50 values). Averaged concentration for a particular group of cancers,

e.g. 5 means 10)5 M. The number on the bottom line (total) indicates to how many human cancers (out of the test number of 60) that peptide is

cytotoxic.

Cancer 1.1 1.1D 3 5 7 11 13 14 15 16 17 18 19 22 23 retro

Leukaemia 5 5 >6 >4 5 5 5 5 5 5 5 >5 5 >5 >5 >5

Lung 5 5 6 5 5 5 5 5 5 5 5 5 5 5 5 5

Colon 5 5 6 5 5 5 5 5 5 5 5 5 5 >5 5 >5

CNS 5 5 6 5 5 5 5 5 5 5 5 5 5 5 5 5

Melanoma 5 5 6 5 5 5 5 5 5 5 5 5 6 5 5 5

Ovarian 5 5 6 5 5 5 5 5 5 5 5 5 6 5 5 >5

Renal 5 5 6 5 5 5 5 5 5 5 5 5 5 5 5 5

Prostate 5 5 6 5 5 5 5 5 5 5 5 5 5 5 5 5

Breast 5 5 6 5 5 5 5 5 5 5 5 5 5 5 5 >5

Total 55 56 53 43 59 59 60 57 53 60 56 38 58 46 49 18

1144 J. Doyle et al. (Eur. J. Biochem. 270) � FEBS 2003

dynamical simulated annealing (SA) protocol was used [49],which included the use of floating stereospecific assignments[50]. Sum-averaging was employed to take care of theambiguous restraints. The all hydrogen distance geometry(ALLHDG) force field (version 4.03) was employed for allcalculations [51]. Initially, a family of 60 structures wasgenerated with random / and w dihedral angles. Thesestructures were subjected to 6500 steps (19.5 ps) of hightemperature dynamics at 2000 K. The Knoe and Krepel forceconstants were increased from 1000–5000 kcalÆmol)1Ænm)2

and 200–1000 kcalÆmol)1Ænm)4, respectively. This wasfollowed by 2500 steps (7.5 ps) of cooling to 1000 K withKrepel increasing from 1000–40000 kcalÆmol

)1Ænm)4 and theatomic radii decreased from 0.9 to 0.75 times those in theALLHDG parameter set. The last step involved 1000 steps(3 ps) of cooling from 1000–100 K. Final structures were

subjected to 200 steps of conjugate gradient energy mini-mization. The 20 structures produced with the lowestpotential energies were selected for analysis. 3D structureswere displayed using INSIGHT II software (version 95.0,MSI) and the program MOLMOL [52].

Results

Biological testing

The antibiotic activities [as minimum inhibitory concentra-tion (MIC) values in lgÆmL)1] of two natural citropins (1.1and 1.1.2) and 23 synthetic modifications of citropin 1.1,against nine pathogens, are listed in Table 2; summarized inTable 3 are the IC50 values of the same peptides in in vitroanticancer tests against 60 human tumour lines as

Table 4. nNOS activities of citropin peptides, citropin synthetic modifications, and some related peptides. IC50 mean error ± 1.3%. Citropin 1.1

modification 6 has a charge of zero, is hydrophobic,and shows minimal solubility in water thus testing was carried out in dimethyl sulfoxide as

solvent, and is not reproducible. Three tests gave IC50 values of 29.6, 33.7 and 39.5 lgÆmL)1, hence we give the IC50 range as 30–40 lgÆmL)1.Qualitatively, this compound shows less nNOS inhibition than modifications 4 and 5. Modifications are shown in bold.

Peptide Sequence

Relative molecular

mass

IC50Hill

slope ChargelgÆmL)1 lM

Citropin 1.1 GLFDVIKKVASVIGGL-NH2 1614 13.3 8.2 2.0 +2

Citropin 1.1.2 DVIKKVASVIGGL-NH2 1297 >100 +2

Modified peptide

1 GlfdvikkvasviGGl-NH2 1614 49.5 30.7 1.0 +2

2 GLADVIKKVASVIGGL-NH2 1537 5.1 3.3 1.6 +2

3 GLFAVIKKVASVIGGL-NH2 1570 4.3 2.7 2.1 +3

4 GLFDVIAKVASVIGGL-NH2 1557 3.8 2.4 3.4 +1

5 GLFDVIKAVASVIGGL-NH2 1557 7.0 4.5 2.1 +1

6 GLFDVIAAVASVIGGL-NH2 1500 30–40 20–26 0

7 GLFDVIKKVAAVIGGL-NH2 1599 8.0 5.0 1.4 +2

8 GLFDVIKKVASVIGGA-NH2 1572 12.4 7.9 1.7 +2

9 GLFEVIKKVASVIGGL-NH2 1628 6.8 4.2 1.8 +2

10 GLFDVIKKVASKIGGL-NH2 1643 11.5 7.0 2.3 +3

11 GLFDVIKKVASVIKGL-NH2 1683 6.8 4.0 3.0 +3

12 GLFDVIKKVASKIKGL-NH2 1714 5.0 2.9 2.1 +4

13 GLFDVIKKVASVIKKL-NH2 1756 3.5 2.0 2.5 +4

14 GLFDVIAKVASVIKKL-NH2 1699 1.6 0.9 4.0 +3

15 GLFAVIKKVASVIKGL-NH2 1655 1.6 1.0 2.3 +4

16 GLFAVIKKVAAVIKKL-NH2 1696 1.9 1.1 3.8 +5

17 GLFAVIKKVAAVIRRL-NH2 1752 1.9 1.1 4.6 +5

18 GLFAVIKKVAKVIKKL-NH2 1753 2.1 1.2 2.2 +6

19 KLFAVIKKVAAVIGGL-NH2 1625 2.1 1.3 3.0 +3

20 KLFAVIKKVAAVIRRL-NH2 1823 1.9 1.0 4.4 +5

21 GLFKVIKKVASVIGGL-NH2 1646 3.4 2.1 2.1 +4

22 GLFKVIKKVAKVIKKL-NH2 1810 2.2 1.2 3.3 +7

Retro

23 LGGIVSAVKKIVDFLG-NH2 1614 24.2 15.0 1.3 +2

Lesueurin

Modified peptide

1 GLLDIIKKVGKVA-NH2 1353 17.8 13.2 2.0 +3

2 GLLDIIKKVGQVA-NH2 1353 49.0 36.2 2.0 +2

3 GLLDIIKKVGEVA-NH2 1354 >100 +1

Citropin 1.2.3 GLFDIIKKVAS-NH2 1188 24.4 20.5 2.2 +2

Aurein 1.1 GLFDIIKKIAESI-NH2 1444 49.1 34.0 2.0 +1

Dahlein 1.1 GLFDIIKNIVSTL-NH2 1430 >100 +1

Dahlein 1.2 GLFDIIKNIFSGL-NH2 1434 >100 +1

� FEBS 2003 Solution structure of a modified citropin 1.1 (Eur. J. Biochem. 270) 1145

determined by the National Cancer Institute. The NCI listsanticancer activities in molar concentrations and these arethe units used here. In Table 3 (anticancer activities), thenumbers 5 and 6 refer to 10)5 and 10)6 M, respectively. Tenof the peptides failed the first stage of the anticancer testingprogram and are specified as �inactive�: essentially thismeansthat no anticancer activity is noted at peptide concentrationsless than 1 · 10)4 M.Table 4 lists the data for nNOS inhibition by 32 peptides.

Twenty-five of these peptides are citropin 1.1 and synthe-

tically modified analogues. The other seven peptides arerelated to citropin 1.1, but have fewer residues. Theseinclude lesueurin [17], dahleins 1.1 and 1.2 [53] and somesynthetic modifications of lesueurin.

The solution structure of citropin 1.1 syntheticmodification (A4K14-citropin 1.1)

The solution structure of the basic peptide citropin 1.1, asdetermined by 2D NMR, is that of a well defined a-helicaland amphipathic peptide [9]. A number of syntheticallymodified citropin peptides have significantly greater anti-cancer and antibacterial activity (and also nNOS activity)than citropin 1.1 itself. We have chosen to investigate the

7.407.607.808.008.208.408.608.80

7.40

7.60

7.80

8.00

8.20

8.40

8.60

8.80A10

V12

V5

K8

I13

D4

I6K7

F3

SllV9

L2

G14

G15

F3

D4

V5

V9K8

A10

I13

G14

F2(ppm)

F1(ppm)

V12Sll

K7I6

Fig. 1. NH to NH region of the NOESY spectrum (mixing time ¼250 ms) of A4K41-citropin 1.1 in 50% (v/v) d3-trifluoroethanol in water.

NOEs between sequential NH protons are indicated.

Fig. 2. Summary of NOEs used in structure calculations for A4K14-

citropin 1.1 in 50% (v/v) d3-trifluoroethanol in water. The thickness of

the bars indicates the relative strength of the NOEs (strong <3.1 A,

medium 3.1–3.7 A or weak >3.7 A). Grey shaded boxes represent

NOEs that could not be assigned unambiguously. The 3JNHaCH values

obtained are also shown. The error here is ±0.5 Hz. A cross-hatch (#)

indicates the coupling constant could not be determined reliably due to

overlap. Due to overlap with the diagonal, the dNN(i,i + 1) NOE

between I6 and K7 could not be determined with certainty, and is not

included in this figure.

Fig. 3. Deviation from random coil chemical shifts [59]. (A) 1H a-CHresonances, (B) 13C a-C resonances, and (C) 1H NH resonances. Solidline, A4K14-citropin 1.1 (GLFAVIKKVASVIKGL-NH2). Dotted

line, citropin 1.1 (GLFDVIKKVASVIGGL-NH2). A negative chemi-

cal shift difference indicates an upfield chemical shift compared to

random coil, while a positive chemical shift difference indicates a

downfield shift. Deviation values for the a-CH resonances weresmoothed over a window of n ¼ ±2 residues [60].

1146 J. Doyle et al. (Eur. J. Biochem. 270) � FEBS 2003

structure of one of the more active synthetic modificationsof citropin 1.1 – A4K14-citropin 1.1 (number 15 inTables 1–4) – by CD and NMR spectroscopy in order tosee whether there is any major difference between thesolution structure of this peptide and that of citropin 1.1.

NMR spectroscopy

NMR experiments were performed on the syntheticallymodified citropin analogue in which the Asp4 residue wasreplaced with Ala and the Gly14 residue was replaced withLys (A4K14-citropin 1.1). NMR studies were performedusing a 50% d3-trifluoroethanol/H2O solution of A4K14-citropin 1.1 as the parent peptide citropin 1.1 has maximalhelicity in this solvent system, as judged by circulardichroism [9]. d3-Trifluoroethanol is widely thought of asa helix-inducing solvent, however, Sonnichsen et al., 1992[54] found that for peptides in trifluoroethanol/H2O solu-tions, helical structure was only observed where there was ahelical propensity in the sequence. In addition, examples ofb-turn [55] and b-sheet [56,57] structures have been observedin aqueous trifluoroethanol mixtures, demonstrating thattrifluoroethanol does not enforce helical structure butmerely enhances it if the propensity exists. Thus trifluoro-ethanol/H2O was deemed a suitable solvent system forstructural studies on the citropin 1.1 peptides. The NMRexperiments were carried out at the same temperature asthat used for the experiments on citropin 1.1 [9]. TheNMR sample of A4K14-citropin 1.1 had a pH of 4.1,compared to pH 2.3 for citropin 1.1. The difference in pHvalue was not expected to have an effect on the finalstructures as both peptides were fully protonated at theirrespective pH values.The 1H-NMR resonances were assigned using the

sequential assignment procedure of Wuthrich [58], whichinvolved the combined use of DQF-COSY, TOCSY andNOESY spectra. The a-13C resonances were assigned fromthe one-bond correlations to the assigned a-1H resonances,recorded in the HSQC spectrum. Similarly, an HMBCspectrum was employed to make the carbonyl-13C assign-ments from the two- and three-bond correlations to theassigned aH, bH and NH 1H resonances. Table 5 lists allthe assignments for the 1H and a-13C resonances.A qualitative indication of the peptide structure can be

obtained from an examination of the observed NOEs andchemical shifts. The NH region of the A4K14-citropin 1.1NOESY spectrum (mixing time¼ 250 ms), shown in Fig. 1,reveals a series of sequential NH–NH NOEs [dNN(i,i + 1)]that occur along the length of the peptide. A series of weakerdNN(i,i + 2)NOEs can also be observed at a lower contourlevel in this region. The various types of NOEs observed forA4K14-citropin 1.1 are summarized in Fig. 2. Here it canbe seen that, in addition to the NOEs mentioned above, anumber of weak sequential daN(i,i + 1)NOEs occur as wellas a series of NOEs from residues three and four aminoacids apart [daN(i,i + 3), dab(i,i + 3) and daN(i,i + 4)].Taken together, the observed NOEs and their intensities areconsistent with A4K14-citropin 1.1 having a helical struc-ture along the majority of its sequence. The pattern of NOEconnectivity is also similar to that found for the parentpeptide, citropin 1.1 [9]. However, the patterns extend overmore residues for A4K14-citropin 1.1. This is particularly

noticeable for the daN(i,i + 1) NOEs that cease at residue10 in citropin 1.1, but continue over the length of thepeptide for A4K14-citropin 1.1. Similarly, the daN(i,i + 3)NOEs extend right up to residue 16 in A4K14-citropin 1.1but stop at residue 14 for the parent peptide. Thus, from anexamination of the NOE data, it would seem the modifiedcitropin peptide has the greater a-helical character beyondresidue 10.A helical structure for A4K14-citropin 1.1 is also indica-

ted from an examination of the deviation from random coilchemical shift values of the a-1H and a-13C resonancesdetermined in water [58,59]. Smoothed over a window ofn ¼ ± 2 residues [60], the plot for the a-CH 1H resonancesshows a distinct upfield shift (Fig. 3A), while those for the13C resonances show a distinct downfield shift (Fig. 3B).The directions of these deviations from random coilchemical shift values are consistent with the peptide havinga helical structure along its length, with maximal helicity inits central region and less well-defined structure at its N- andC-termini [61–63]. For comparison, Fig. 3A,B also show thedeviations from random coil chemical shift for the 1H and13C a-CH resonances of citropin 1.1 [9]. Both peptides havevery similar plots over the central region of the peptide(from residues 4–10), i.e., where there is no difference inamino acid sequence between the two peptides and theyboth have the greatest helicity. However, from approxi-mately residue Ala10 onwards, the 1H and 13C chemicalshifts of A4K14-citropin 1.1 are consistently upfield anddownfield, respectively, of those of the parent peptide. Thesedifferences suggest that A4K14-citropin 1.1 forms a morestable a-helix than citropin 1.1 in the C-terminal region. Thesmall differences at the extreme N-terminal region (firstthree residues) for the plots of the 1H and 13C a-CHresonances are opposite in directional trend for structuralconclusions to be drawn. This may reflect the poorly definednature of the first turn of the a-helix due to the lack ofhydrogen bonds to their NH protons.Comparison of the observed NH chemical shifts of

A4K14-citropin 1.1 with the corresponding random coilNH chemical shifts [59] revealed a periodic distribution suchthat those from hydrophobic residues were shifted down-field with respect to the random coil values and those fromhydrophilic residues were shifted upfield (Fig. 3C). Thisbehaviour is characteristic of amphipathic a-helices [64,65]and is due to differences in backbone hydrogen bond lengthon either face of the peptide, which lead to slight curvatureof the helix. The curvature may not be significant forA4K14-citropin 1.1, as it consists of only 16 residues,however, the periodic distribution of NH shifts is consistentwith A4K14-citropin 1.1 forming an amphipathic a-helix.Furthermore, Fig. 3C also shows that the periodicity of theNH chemical shifts is very similar between the parent andmodified peptides.

Structural analysis

The conclusions derived from an examination of theNMR data were confirmed when the NOE data wereused as input for structural calculations. Sixty structureswere generated by restrained molecular dynamics (RMD)and dynamical SA calculations and the 20 structures oflowest potential energy were selected for close examination.

� FEBS 2003 Solution structure of a modified citropin 1.1 (Eur. J. Biochem. 270) 1147

Table 6. Structural statistics of A4K14-citropin following RMD/SA calculations.<SA> is the ensemble of the 20 final structures (SA) is the mean

structure obtained by best-fitting and averaging the coordinates of backbone N, a-C and carbonyl-C atoms of the 20 final structures. (SA)r is therepresentative structure obtained after restrained energy minimization of the mean structure. Well-defined residues are those with angular order

parameters (S) > 0.9. For A4K14-citropin 1.1, residues Leu2 to Gly15 are well-defined.

<SA> (SA)r

RMSD from mean geometry (A)

All heavy atoms 0.74 ± 0.10 –

All backbone atoms (N, a-C, carbonyl-C) 0.34 ± 0.09 –

Heavy atoms of well-defined residues 0.72 ± 0.11 –

Backbone atoms (N, a-C, carbonyl-C) of well-defined residues 0.21 ± 0.08 –

X-PLOR energies (kcalÆmol)1)Etot 75.34 ± 1.66 70.12

Ebond 6.76 ± 0.15 6.45

Eangle 23.39 ± 1.06 21.39

Eimproper 4.25 ± 0.56 3.29

Erepel 4.39 ± 0.34 4.80

ENOE 36.55 ± 1.31 34.19

Ecdih 0.00 ± 0.00 0.00

Table 5. 1H and 13C NMR chemical shifts for A4K14-citropin in 50% trifluoroethanol in water (by volume), at a measured pH of 4.12 at 25 �C.Data

are shown in p.p.m. Assignments for all the 1H NMR resonances are shown whereas only the a-13C and carbonyl-13C resonances are presented;NO, not observed.

Residue

Chemical shift of

13CONH a-CH b-CH Others a-13CH

Gly1 NO 3.93, 3.83 42.4 169.5

Leu2 8.45 4.15 1.64 c-CH 1.59 56.4 176.2

d-CH3 0.99, 0.92Phe3 8.12 4.25 3.17 H2,6 7.20 59.5 175.2

H3,5 7.31

H4 7.26

Ala4 7.71 4.02 1.57 53.8 178.4

Val5 7.38 3.70 2.35 c-CH3 1.09, 1.00 65.1 176.0

Ile6 7.96 3.67 1.94 c-CH2 1.72, 1.23 63.8 176.1

c-CH3 0.96d-CH3 0.88

Lys7 8.01 3.90 1.79 c-CH2 1.49, 1.39 58.5 177.1

d-CH2 1.68e-CH2 2.94NH3

+ n.o.

Lys8 7.71 4.10 2.21, 2.07 c-CH2 1.56 58.1 177.8

d-CH2 1.81, 1.73e-CH2 2.96NH3

+ n.o.

Val9 8.60 3.61 2.22 c-CH3 1.10, 1.00 66.0 176.7

Ala10 8.83 4.02 1.53 54.1 178.6

Ser11 7.83 4.17 4.12, 4.03 60.7 174.7

Val12 7.91 3.85 2.41 c-CH3 1.13, 1.02 64.8 176.9

Ile13 8.28 3.81 2.01 c-CH2 1.73, 1.34 63.5 177.2

c-CH3 0.99d-CH3 0.88

Lys14 8.18 4.13 1.99, 1.76 c-CH2 1.57 57.0 177.1

d-CH2 1.62e-CH2 3.04NH3

+ n.o.

Gly15 7.85 4.01, 3.93 44.7 173.5

Leu16 7.90 4.24 1.89 c-CH 1.65 54.5 179.2

d-CH3 0.95CONH2 7.24, 6.77

1148 J. Doyle et al. (Eur. J. Biochem. 270) � FEBS 2003

Some statistics for the 20 final structures are given inTable 6.The superimposition of the 20 structures over the

backbone N, aC and carbonyl-C atoms shows thatA4K14-citropin 1.1 forms a regular a-helix along itsentire length (Fig. 4A). Analysis of the angular orderparameters (S, / and w) [66] of these structures indicatedthat, except for the N- and C-terminal residues (Gly1and Leu16), all residues were well defined (S > 0.9 forboth their / and w angles). A Ramachandran plot [67]of the average / and w angles of the well-definedresidues reveals these angles are distributed within thefavoured region for a-helical structure (not shown). Themost energetically stable of the 20 final structures isdisplayed in Fig. 4B and from this representation it isapparent that A4K14-citropin 1.1 forms an amphipathica-helical structure with well-defined hydrophobic andhydrophilic faces.

Discussion

Citropin 1.1 is the major wide-spectrum antibiotic peptidein the secretion of the skin glands of L. citropa [9]. It is oneof the most potent membrane-active antibiotic peptidesisolated from amphibians and is particularly effectiveagainst Gram-positive organisms [8]. Citropin 1.1 is a 16residue peptide and is one of a number of amphibianantibiotic peptides containing the characteristic Lys7-Lys8pattern: a group which includes lesueurin (from L. lesueuri)[17], the aureins (from L. aurea and L. raniformis) [11] andthe uperins (from toadlets of the genus Uperoleia) [68].Citropin 1.1 does not cause lysis of red blood cells at aconcentration of 100 lgÆmL)1, but lysis is complete at1 mgÆmL)1 (B. C. S. Chia & J. H. Bowie, unpublishedresults). Citropin 1.1 is thought to be stored in an inactiveform (spacer peptide – citropin 1.1) in the skin glands, butwhen the frog is stressed, sick or attacked, an endoproteasecleaves off the spacer peptide and the active citropin 1.1 isreleased onto the skin. Citropin 1.1 must be cytotoxic to thefrog as after about 10 min of exposure on the skin a furtherendoprotease removes the first two residues of the peptidedestroying the antibiotic (and anticancer) activity [9].The solution structure of citropin 1.1 is shown in Fig. 5;

this should be compared with that of the syntheticallymodified A4K14-citropin 1.1 depicted in Fig. 4B. TheNMR studies reported here indicate that both peptidesadopt amphipathic a-helices, but that the helicity is morepronounced for A4K14-citropin 1.1. Each peptide has welldefined hydrophobic and hydrophilic regions. However,chemical shift and NOE connectivity data suggest that theC-terminal region of the a-helix may be more stable in themodified citropin. This is due probably to the replacementof Gly14 with Lys14. Gly is more conformationally mobilethan other residues, due to its lack of a side chain, and is awell-known breaker of helical structure [69]. The Lys residuewould therefore be expected to stabilize a helical structure inthis region. In addition, the positively charged side-chain ofLys would stabilize a C-terminal helix due to its inter-action with the negative end of the helix dipole [69]. Thereplacement of Asp4 with Ala4 does not have a significanteffect on the structure of the peptide. This may be becauseremoval of the negatively charged Asp4, which would

stabilize the N–terminal helix by interaction with thepositive end of the helix dipole [69], is compensated by theintroduction of Ala, which has a high helical propensity.Finally, we believe it is likely that all of the peptides listed inTables 1 and 4 adopt such structures when interacting witheither bacterial or cancer cell membranes.The antibiotic and anticancer activities of peptides of

this type are due to the disruption of the cell membraneby the peptide. In order to span the lipid bilayer ofbacterial and cancer cells, the peptide needs to have atleast 20 amino acid residues [4,14,70]. Citropin 1.1 hasonly 16 residues and thus is unable to fully span the lipidbilayer. Amphipathic peptides of this type are thoughtto operate via the �carpet� mechanism, which involvesaggregation of the helical peptides on the surface of themembrane by interaction of the positively charged sites ofthe peptide with negatively charged sites on the membranesurface. The peptides then insert into the lipid membrane,weakening the bilayer and making it susceptible toosmotic lysis [4,24,70]. From the work reported herein, thegreated helicity of A4K14-citropin 1.1 in its C-terminalregion may be responsible for its enhanced antimicrobialactivity.

Antibacterial and anticancer activity

Synthetic modifications of citropin 1.1, shown in Table 1,were made to investigate the relationship between activityand sequence. The first point to be made is that the naturalL-citropin 1.1 has, within experimental error (± 1 dilutionfactor), the same spectrum of antibiotic activities as thesynthetic all D-citropin 1.1. This is a feature of membraneactive peptides [4,13]. Other synthetic modifications weremade to the following plan: (a) to successively replace thehydrophilic residues (to ascertain the effect of a particularhydrophilic residue on the bioactivity), and some hydro-phobic residues (certain hydrophobic residues, particularlyterminal residues are often vital for good activity) with Ala,and (b) to change Gly and some hydrophilic residues to Lys(to determine the effect on activity of an increase in thepositive charge of the peptide). The spectrum of antibioticactivities for each synthetic modification is recorded inTable 2. The following observations can be made. Replace-ment of the following residues with Ala show (a) littlechange in activity for Asp4 and Ser11, and (b) significantreduction in the activity for Phe3, Lys7, Lys8 and Leu16;replacement of the following residues with Lys show (a)reduction in activity for Gly1 and Val12 and (b) significantincreases in activity against Gram-negative organisms forGly14 and Gly15. The conclusions from this study are that(a) modification of either of the terminal residues reducesthe activity, and (b) the activity against Gram-positiveorganisms is not significantly improved (in comparison withcitropin 1.1) by synthetic modification, but increasing thenumber of basic Lys residues in the hydrophilic zone of theamphipathic peptide markedly increases the activity againstGram-negative organisms like E. coli.Apart from particular detail, the trends in anticancer

activities of the modified citropins 1.1 mirror those outlinedabove for the antibiotic activities (Table 3). The citropin 1peptides are generally cytotoxic toward the majority of the60 cancers tested in the NCI regime: IC50 values are

� FEBS 2003 Solution structure of a modified citropin 1.1 (Eur. J. Biochem. 270) 1149

generally in the moderate 10)5 M range, with syntheticmodification 3 (Asp4 to Ala4) showing the strongestcytotoxicity (in the 10)6 M range). As was the case withantibiotic activity, L- and D-citropins 1.1 show almostidentical activity.The trends observed for antibiotic activity are more

marked when considering anticancer activity. For example,some synthetic modifications which decrease antibioticactivity, often destroy the anticancer activity, e.g., themodifications Gly1 to Lys1, Phe3 to Ala3, Lys7 to Ala7,Val12 to Lys12, and Leu16 to Ala16. The conclusions fromthis study are, that for best anticancer activity of citro-pin 1.1 type molecules, (a) the residues Gly1, Phe3, Ala4,Lys7 and Leu16 are essential, and (b) the charge needs to beP+2. The close correlation between the broad-spectrum

anticancer and antibacterial activity of membrane activepeptides, suggests that the anticancer activity is also due topenetration and disruption of the membranes of the cancercells. The selectivity of these peptides for cancer overnormal cells may be due to the significantly higher levels ofanionic phospholipids present in the outer leaflet of cancercells [71–74].

nNOS activity

We have already reported that citropin 1.1 causes the inhi-bition of nNOS by forming a complex with the regulatoryprotein Ca2+-calmodulin, thus impeding the attachment ofthis enzyme at the calmodulin binding site on nNOS [17].The actual nature of the complex is not known, but NMR

GLY1

LEU16

H3N+

+NH3

H3N+

OH

ONH2

+NH3

A

B

Fig. 4. Most stable structures of A4K14-citroprin 1.1. (A) Superimposition of the 20 most stable structures of A4K14-citropin 1.1 along the

backbone atoms (N, a-C and carbonyl C) (prepared with the program MOLMOL [52]) and (B) the most stable calculated structure of A4K14-

citropin 1.1. A ribbon is drawn along the peptide backbone in (B).

H3N+

+NH3

OH

O

H2N

H3N+

-OO

Fig. 5. Themost stable calculated structure of citropin 1.1.This figure was originally published byWegener et al. [9] inEur. J. Biochem. 265, 627–635.

1150 J. Doyle et al. (Eur. J. Biochem. 270) � FEBS 2003

studies on other peptide Ca2+-calmodulin complexes showthat the dumb-bell shaped calmodulin wraps itself aroundand then partially or fully encloses the a-helical peptide,completely changing the shape of the calmodulin system[75–79].The nNOS inhibition data for the various citropins and

related systems are collated in Table 4. The activity/sequen-cing relationship for effective nNOS inhibition is quitedifferent from that described above for antibiotic/anticanceractivity. The following observationsmay bemade: (a) L- andD-citropin 1.1 show quite different activities. Not only is theIC50 value for D-citropin 1.1 significantly less than that forL-citropin 1.1, but the Hill slope of 1.0 (2.0 for L-citro-pin 1.1), may indicate that the inhibition of nNOS byD-citropin 1.1 involves the Arg substrate site rather thaninteraction with Ca2+-calmodulin [36]. (b) Loss of residuesfrom the N-terminal end of the citropin system destroys theactivity against nNOS, whereas loss of activity is not somarkedwhen residues are removed from the C-terminal endof the peptide. For example, lesueurin (13 residues, charge+3) and citropin 1.2.3 (11 residues, charge+2) showmod-erate activity with IC50 values of 21.9 lgÆmL)1 (16.2 lm)and 24.4 lg/mL (20.5 lm), respectively. (c) A change in thenature of the end groups and some other residues ofcitropin 1.1 is not as important as it is for antibiotic oranticancer activity. For example, compare the data forchanges in Gly1, Phe3, Ser11 and Leu16 (see Table 4, forcitropin 1.1 and citropin modifications 2, 3, 7, 8 and 21). (d)The extent of positive charge on the peptide is important.For example, note the change in IC50 in the three lesueurinmodifications, i.e., lesueurin 1 [Lys11 (charge+3)], 2 [Gln11(+2)] and 3 [Glu11 (+1)] give IC50 values of 17.8, 49.0 and>100 lgÆmL)1, respectively, and also that in citropin 6(charge 0), the IC50 value is reduced to 30–40 lgÆmL)1

(Table 4). Maximum nNOS inhibition by a citropin occurswhen the charge is +3 or greater [e.g., citropin 14, charge+3, IC50 1.6 lgÆmL)1, and citropin 15 (A4K14–citro-pin 1.1, charge +4, IC50 1.6 lgÆmL)1]. As long as there isat least oneLys at residue 7 or 8, it does not seemparticularlyimportant where the other positive charges reside (e.g.,citropins 12, 14, 15, 20 and 21). Even retro citropin (charge+2) shows moderate activity.The prerequisites for maximum nNOS inhibition by a

citropin type peptide are (a) an a-helix; (b) preferably 16amino acid residues (c); Lys at either residue 7 or 8 and (d)an overall charge of +3.

Conclusions

Citropin 1.1, the major peptide in the skin secretion ofL. citropa, exhibits multifaceted biological activity withinthe 10)6 M concentration range, including widespectrumantimicrobial and anticancer activity, together with inhibi-tion of nNOS. This concentration is significantly less thanthat required to cause lysis of red blood cells. Syntheticmodification of citropin 1.1 can achieve a 10-fold increase inthese activities. Both citropin 1.1 and the more activesynthetic modification, A4K14-citropin 1.1, have beenshown to adopt amphipathic a-helical structures in aqueoustrifluoroethanol.As antibiotic and anticancer activity are thesame for L- and D-citropin 1.1, modified D-citropins couldbe useful as pharmaceutical agents, especially as the

citropins 1.1 are active against a number of pathogens thatshow resistance towards currently used antibiotics [80,81].The amphibian uses citropin 1.1 as a primary host-defencecompound against both small and large predators. It is notclear whether the animal utilizes the anticancer activity ofcitropin 1.1, orwhether this activity is simply a serendipitousbonus arising from the membrane activity of this peptide.

References

1. Bevins, C.L. & Zasloff, M. (1990) Peptides from frog skin. Ann.

Rev. Biochem. 59, 395–414.

2. Lazarus, L.H. & Attila, M. (1993) The toad, ugly and venomous,

wears yet a precious jewel in his skin.Prog. Neurobiol. 41, 473–507.

3. Erspamer, V. (1994) Bioactive secretions of the amphibian

integument. In Amphibian Biology. The Integument. (Heatwole,

H., ed.), Vol 1, 178–350.

4. Barra, D. & Simmaco, M. (1995) Amphibian skin: a pro-

mising resource for antimicrobial peptides. Trends. Biochem. 13,

205–209.

5. Anastasi, A., Erspamer, V. & Endean, R. (1968) The structure of

caerulein. Arch. Biochem. Biophys. 125, 57–63.

6. Stone, D.J.M., Waugh, R.J., Bowie, J.H., Wallace, J.C. & Tyler,

M.J. (1992) The structures of the caerins and caeridin 1 from

Litoria splendida. J. Chem. Soc. Perkin Trans. 1, 3173–3179.

7. Wabnitz, P.A., Bowie, J.H. & Tyler, M.J. (1999) Caerulein like

peptides from the skin glands of the Australian Blue Mountains

Tree Frog Litoria citropa. Part 1. Sequence determination using

electrospray mass spectrometry. Rapid Commun. Mass Spectrom.

13, 2498–2502.

8. Bowie, J.H., Wegener, K.L., Chia, B.C.S., Wabnitz, P.A., Carver,

J.A., Tyler, M.J. &Wallace, J.C. (1999) Host defence antibacterial

peptides from the skin secretions of Australian amphibians. Pro-

tein Peptide Lett. 6, 259–269.

9. Wegener, K.L.,Wabnitz, P.A., Bowie, J.H., Carver, J.A.,Wallace,

J.C. & Tyler, M.J. (1999) Host defence peptides from the skin

glands of the Australian Blue Mountains Tree Frog Litoria

citropa. The antibacterial citropin 1 peptides. The solution struc-

ture of citropin 1.1. Eur. J. Biochem. 265, 627–635.

10. Wabnitz, P.A., Bowie, J.H., Wallace, J.C. & Tyler, M.J. (1999)

The antibiotic citropin peptides from the Australian Tree Frog

Litoria citropa. Structure determination using electrospray

mass spectrometry. Rapid Commun. Mass Spectrom. 13, 1724–

1732.

11. Rozek, T., Wegener, K.L., Bowie, J.H., Olver, I.N., Carver, J.A.,

Wallace, J.C. & Tyler, M.J. (2000) The antibiotic and anticancer

active aurein peptides from the Australian Bell FrogsLitoria aurea

and Litoria raniformis. The solution structure of aurein 1.2. Eur. J.

Biochem. 267, 5330–5341.

12. Monks, A., Scudiero, D., Skehan, P., Shoemaker, R., Paull, K.,

Vistica, D., Hose, C., Langley, J., Cronise, P. & Vaigro-Wolff, A.

(1991) Feasibility of a high-flux anticancer drug screen using a

diverse panel of of cultured human tumor cell lines. J. Natl Cancer

Inst 83, 757–766 (see also http://dtp.nci.nih.gov)

13. Wong, H., Bowie, J.H. & Carver, J.A. (1997) The solution struc-

ture and activity of caerin 1.1, an antibiotic peptide from the

Australian Tree Frog Litoria splendida. Eur. J. Biochem. 247,

545–557.

14. Shai, Y.C. (1995) Molecular recognition between membrane-

scanning peptides. Trends Biochem. Sci. 20, 460–464.

15. Bechinger, B. (1997) Structure and function of channel form-

ing peptides; magainins, cecropins, melittin and alamethicin.

J. Membr. Biol. 156, 197–211.

16. Matsuzaki, K. (1998)Magainins as paradigm for themode of pore

forming polypeptides. Biochim. Biophys. Acta 1376, 391–400.

� FEBS 2003 Solution structure of a modified citropin 1.1 (Eur. J. Biochem. 270) 1151

17. Doyle, J., Llewellyn, L.E., Brinkworth, C.S., Bowie, J.H.,

Wegener, K.L., Rozek, T., Wabnitz, P.A., Wallace, J.C. & Tyler,

M.J. (2002) Amphibian peptides that inhibit neuronal nitric oxide

synthase: the isolation of lesueurin from the skin secretion of the

Australian Stony Creek Frog Litoria lesueuri. Eur. J. Biochem.

269, 100–109.

18. Waugh, R.J., Raftery, M.J., Bowie, J.H., Tyler, M.J. & Wallace,

J.C. (1996) The structures of the frenatin peptides from the skin

secretion of the Giant Tree Frog Litoria infrafrenata. J. Peptide

Sci. 2, 117–1124.

19. Bredt, D.S., Hwang, P.M., Glatt, C.L., Lowenstein, C., Reed,

R.R. & Snyder, S.H. (1991) Cloned and expressed nitric oxide

synthase structurally resembles cytochrome P-450 reductase.

Nature 351, 714–718.

20. Marletta, M.A. (1993) Nitric oxide synthase and mechanism.

J. Biol. Chem. 268, 12231–12234.

21. Marletta, M.A. (1994) Nitric oxide synthase: aspects concerning

structure and catalysis. Cell 78, 927–930.

22. Pfeiffer, S., Mayer, B. & Hemmens, B. (1999) Nitric oxide: a

protective or pathogenicmolecule.Angew. Chem. Int. Ed. Engl. 38,

1715–1731.

23. Forsen, S. (1995) Calcium induced structural changes and domain

autonomy in calmodulin. Nature Struct. Biol. 2, 777–783.

24. Venema, R.C., Sayegh, H.S., Kent, J.D. & Harrison, D.G. (1996)

Identification, characterisation and comparison of the calmodulin-

binding domains of nitric oxide synthases. J. Biol. Chem. 271,

6435–6440.

25. Lee, S.J. & Stull, J.T. (1998) Calmodulin-dependent regulation of

inducible and neuronal nitric oxide synthase. J. Biol. Chem. 273,

27430–27437.

26. Matsuda, H. & Iyanagi, T. (1999) Calmodulin activated

intramolecular transfer beteen the two flavins of neuronal nitric

oxide synthase. Biochim. Biophys. Acta 1473, 345–355.

27. Sautebin, L., Rombola, L., Di Rosa, M., Caliendo, G., Perissutti,

E., Grieco, P., Severino, B. & Santagada, V. (2000) Synthesis and

structure-activity of antisense peptides corresponding to the region

for CaM-binding domain of the inducible nitric oxide synthase.

Eur. J. Med. Chem. 35, 727–732.

28. Watanabe, Y., Hu, Y. & Hidaka, H. (1997) Identification of a

specific amino acid cluster in the calmodulin binding domain of

nNOS. FEBS Lett. 403, 75–78.

29. Choi, W.S., Chang, M.S., Han, J.W., Hong, S.Y. & Lee, H.W.

(1997) Identification of NOS in Staphyococcus aureus. Biochem.

Biophys. Res. Commun. 237, 554–558.

30. Morita, H., Yoshikawa, H., Sakata, R., Nagata, Y. & Tanaka, H.

(1997) Synthesis of nitric oxide from the two equivalent guanidino

nitrogens of 1-arginine by Lactobacillus fermentum. J. Bacteriol.

179, 7812–7815.

31. Choi, W.S., Seo, D.W., Chang, M.S., Han, J.W., Paik, W.K. &

Lee, H.W. (1998) Methylestersof 1-arginine and N-nitro-L-argi-

nine induce nitric oxide synthase in Staphyloccoccus aureus.

Biochem. Biophys. Res. Commun. 246, 431–435.

32. Er, H., Turkoz, Y., Ozerol, I.H. & Uzmez, E. (1998) Effect of

NOS inhibition in experimental Pseudomonas keratitis in rabbits.

Eur. J. Opthalmol. 8, 137–141.

33. Maeji, N.J., Bray, A.M., Valerio, R.M.&Wang,W. (1995) Larger

scale multi-pin peptide synthesis. Pept. Res. 8, 33–38.

34. Jorgensen, J.H., Cleeland, W.A., Craig, G., Doern, M., Ferraro,

J., Finegold, C.M., Hansen, S.L., Jenkins, S.G., Novick, W.J.,

Pfaller, M.A., Preston, D.A., Reller, L.B. & Swenson, J.M. (1993)

Methods for dilution antimicrobial susceptibility tests for bacteria

that grow aerobically, 3rd approved standard. Natl Committee

Clin. Laboratory Stand. 33, 1–12. (Document M7–A3).

35. Doyle, D.D., Gua, Y., Lustig, S.L., Satin, J., Rogart, R.B. &

Fozzard, H.A. (1993) Divalent cation competition with [3H]saxi-

toxin binding to tetrodotoxin-resistant and – sensitive sodium

channels. A two-site structural model of ion/toxin interaction.

J. General Physiol. 101, 153–182.

36. Fersht, A. Enzyme Structure and Mechanism. W.H. Freeman,

New York, NY, USA

37. Rance, M., Sørrenson, O.W., Bodenhausen, G., Wagner, G.,

Ernst, R.R. & Wuthrich, K. (1983) Improved spectral resolution

in COSY 1H-NMR spectra of proteins via double quantum fil-

tering. Biochem. Biophys. Res. Commun. 117, 479–485.

38. Davis, D.G. & Bax, A. (1985) Assignment of complex 1H NMR

spectra via two-dimensional homonuclear Hartmann-Hahn spec-

troscopy. J. Am. Chem. Soc. 107, 2820–2821.

39. Jeener, J., Meier, B.H., Bachman, P. & Ernst, R.R. (1979)

Investigation of exchange processes by two- dimensional NMR

spectroscopy. J. Chem. Phys. 71, 4546–4553.

40. Marion, D. & Wuthrich, K. (1983) Application of phase sensitive

two-dimensional correlated spectroscopy (COSY) for measure-

ments of 1H-1H spin-spin coupling constants in proteins. Biochem.

Biophys. Res. Commun. 113, 967–974.

41. John, B.K., Plant, D., Webb, P. & Hurd, R.E. (1992) Effective

combination of gradients and crafted RF pulses for water sup-

pression in biological samples. J. Magn. Reson. 98, 200–206.

42. Griesinger, C., Otting, G., Wuthrich, K. & Ernst, R.R. (1988)

Clean TOCSY for 1H spin system identification in macro-

molecules. J. Am. Chem. Soc. 110, 7870–7872.

43. Kay, L.E., Keifer, P. & Saaringen, T. (1992) Pure absorption

gradient enhanced heteronuclear single quantum correlation

spectroscopy with improved sensitivity. J. Am. Chem. Soc. 114,

10663–10665.

44. Summers,M.F.,Marzilli, L.G. & Bax, A. (1986) Complete 1H and13C assignments of coenzyme B12 through the use of new two-

dimensional NMR experiments. J. Am. Chem. Soc. 108, 4285–

4294.

45. Goddard, T.D. & Kneller, D.G. SPARKY 3. University of

California, San Francisco.

46. Xu, R.X., Word, J.M., Davis, D.G., Rink, M.J., Willard, D.H. &

Gampe, R.T. (1995) Solution structure of the human pp60c-src

SH2 domain complexed with a phosphorylated tyrosine penta-

peptide. Biochemistry 34, 2107–2121.

47. Brunger, A.T. (1992) A system for X-ray crystallography and

NMR. X-PLOR Version 3.851. Yale University, New Haven, CT.

48. Brunger, A.T. & Nilges, M. (1993) Computational challenges

for macromolecular structure determination by X-ray crystallo-

graphy and solution NMR-spectroscopy. Q. Rev. Biophys. 26,

49–125.

49. Nilges, M., Kuszewski, J. & Brunger, A.T. (1992) Sampling

Properties of Simulated Annealing. In Proceedings of the NATO

Advanced Research Workshop on Computational Aspects of the

Study of Biological Macromolecules (N.M.R.), Il Ciocco, Italy.

50. Folmer, R.H.A., Hilbers, C.W., Konings, R.N.H. & Nilges, M.

(1997) Floating stereospecific assignment revisited – application to

an 18 kDa protein and comparison with J-coupling data. J. Bio-

mol. NMR 9, 245–258.

51. Engh, R.A. & Huber, R. (1991) Accurate bond and angle para-

meters for x-ray protein structure refinement. Acta Crystallog. A

47, 392–400.

52. Koradi, R., Billeter, M. & Wuthrich, K. (1996) MOLMOL: a pro-

gram for display and analysis of macromolecular structures.

J. Mol. Graphics 14, 51–55.

53. Wegener, K.L., Brinkworth, C.S., Bowie, J.H., Wallace, J.C. &

Tyler, M.J. (2001) Bioactive dahlein peptides from the Australian

frog Litoria dahlii. Sequence determination by electrospray mass

spectrometry. Rapid Commun. Mass Spectrom. 15, 1726–1734.

54. Sonnichsen, F.D., Van Eyk, J.A., Hodges, R.S. & Sykes, B.D.

(1992) Effect of trifluoroethanol on protein secondary structure:

an NMR and CD study using a synthetic actin peptide. Bio-

chemistry 31, 8790–8798.

1152 J. Doyle et al. (Eur. J. Biochem. 270) � FEBS 2003

55. Ragan, R. & Balaram, P. (1996) A model for the interaction of

trifluoroethanol with peptides and proteins. Int. J. Peptide Protein

Res. 48, 328–336.

56. Schonbrunner, N.,Wey, J., Engels, J., George, H. &Kiefhaber, T.

(1996) Native like b-structure in a trifluoroethanol-induced par-tially folded state of the all b-sheet protein tendamistat. J. Mol.

Biol. 260, 432–445.

57. Dong, A., Matsura, J., Manning, M.C. & Carpenter, J.F. (1998)

Intermolecular b-sheet results from trifluoroethanol-induced

nonnative a-helical structure in b-sheet predominant proteins –infrared and circular dichroism spectroscopic study. Arch. Bio-

chem. Biophys. 355, 275–281.

58. Wuthrich, K. (1986) NMR of Proteins and Nucleic Acids, Wiley,

New York.

59. Wishart, D.S., Bigam, C.G., Holm, A., Hodges, R.S. & Sykes,

B.D. (1995) 1H, 13C and 15N random coil NMR chemical shifts of

the common amino acids. Investigations of nearest neighbour

effects. J. Biomol. NMR 5, 67–81.

60. Pastore, A. & Saudek, V. (1990) The relationship between che-

mical shift and secondary structure. J. Magn. Reson. 90, 165–176.

61. Wishart, D.S., Richards, F.M. & Sykes, B.D. (1991) Relationship

between NMR chemical shift and protein secondary structure.

J. Mol. Biol. 222, 311–333.

62. Wishart, D.S., Richards, F.M. & Sykes, B.D. (1992) The chemical

shift index: a fast and simple method for the assignment of protein

secondary structure throughNMR spectroscopy.Biochemistry 31,

1647–1651.

63. Wishart, D.S. & Sykes, B.D. (1994) The 13C chemical-shift index: a

simple method for the identification of protein secondary structure

using 13C chemical-shift data. J. Biomol. NMR 4, 171–180.

64. Zhou, N.E., Zhu, B.-Y., Sykes, B.D. & Hodges, R.S. (1992)

Relationship between amide proton chemical shifts and hydrogen

bonding in amphipathic a-helical peptides. J. Am. Chem. Soc. 114,

4320–4326.

65. Kuntz, I.D., Kosen, P.A. & Craig, E.C. (1991) Amide chemical

shifts in many helices in peptides and proteins are periodic. J. Am.

Chem. Soc. 113, 1406–1408.

66. Pallaghy, P.K., Duggan, B.M., Pennington,M.W. &Norton, R.S.

(1993) 3-Dimensional structure in solution of the calcium channel

blocker x-conotoxin. J. Mol. Biol. 234, 405–420.

67. Morris, A.L., MacArthur, M.W., Hutchinson, E.G. & Thornton,

J.M. (1992) Stereochemical quality of protein structure

coordinates. Proteins 12, 345–364.

68. Bradford, A.M., Raftery, M.J., Bowie, J.H., Tyler, M.J., Wallace,

J.C., Adams, G.W. & Severini, C. (1996) Novel uperin peptides

from the dorsal glands of the Australian Flood Plain Toadlet

Uperoleia inundata. Aust. J. Chem. 49, 475–484.

69. Richardson, J.S. & Richardson, D.C. (1988) Amino acid pre-

ferences for specific locations at the ends of a-helices. Science 240,

1648–1652.

70. Epand, R.M., Shai, Y.C., Segrest, J.P. & Anantharamaiah, G.M.

(1995) Mechanisms for the modulation of membrane bilayer

properties by amphipathic helical peptides. Biopolymers 37, 319–

338.

71. Zubay, G. (1986) Biochemistry, Addison-Wesley Publishers Co,

Massachussetts, pp. 507–509.

72. Conner, J., Bucana, C., Fidler, I.J. & Schroit, A.J. (1989) Differ-

entiation dependent expression of phosphatidyl serine in

mammalian plasma membranes: quantitative assessment of outer-

leaflet lipid by prothrombin complex formation. Proc. Natl Acad.

Sci. USA 86, 3184–3188.

73. Utsugi, T., Schroit, A.J., Conner, J., Bucana, C. & Fidler, I.J.

(1991) Elevated expression of phosphatidyl serine in the outer

membrane leaflet of human tumor cells and recognition of acti-

vated human blood monocycles. Cancer Res. 51, 3062–3066.

74. Tytler, E.M., Anantharamaiah, G.M., Walker, D.E., Mishra,

V.K., Palgunachari, M.N. & Segrest, J.P. (1995) Molecular basis

for prokaryotic specificity of magainin-induced lysis. Biochemistry

34, 4393–4401.

75. Malencik, D.A. & Anderson, S.R. (1982) Binding of simple pep-

tides, hormones and neurotransmitters by calmodulin. Biochem-

istry 21, 3480–3486.

76. Ikura, M., Kay, L.E., Krinks, M. & Bax, A. (1991) Triple

resonance multidimensional NMR study of calmodulin com-

plexed with the binding domain of skeletal muscle myosin light-

chain kinase: indication of a conformational change in the central

helix. Biochemistry 30, 5498–5504.

77. Ikura, M., Clore, G.M., Gronenborn, A.M., Zhu, G., Klere, C.B.

& Bax, A. (1992) Solution structure of a calmodulin-target peptide

complex by multidimensional NMR. Science 256, 632–638.

78. Scaloni, A., Miraglia, N., Orru, S., Amodeo, P., Motta, A.,

Marino, G. & Pucci, P. (1998) Topology of the calmodulin-

melittin complex. J. Mol. Biol. 277, 945–958.

79. Elshorst, B., Hennig, M., Forsterling, H., Diener, A., Maurer, M.,

Schultz, P., Schwalbe, H., Griesinger, C., Krebs, J., Schmid, H.,

Vorherr, T. & Carafoli, E. (1999) NMR solution structure of a

complex of calmodulin with a binding peptide of the Ca2+ pump.

Biochemistry 38, 12320–12332.

80. Hiramatsu, K., Aritaka, N., Hanaki, H., Kawasaki, S., Hosoda,

Y., Hori, S., Fukuchi, Y. &Kobayashi, I. (1997) Dissemination in

Japanese hospitals of strains of Staphylococcus aureus hetero-

geneously resistant to vancomycin. Lancet 350, 1670–1673.

81. Hancock, R.E.W. (1997) Peptide antibiotics. Lancet 349, 418–422.

� FEBS 2003 Solution structure of a modified citropin 1.1 (Eur. J. Biochem. 270) 1153