Surajit BhattacharjyaMolecular Biophysics UnitIndian Institute of Science

Bangalore—560012, India

Sunil A. David

V. L MathanThe Wellcome Trust

Research LaboratoryDepartment of

Gastrointestinal SciencesChristian Medical

College HospitalVellore—632 004, India

Polymyxin B Nonapeptide:Conformations in Water and inthe Lipopolysaccharide-BoundState Determined by Two-Dimensional NMR andMolecular Dynamics

P. Balaram’Molecular Biophysics UnitIndian Institute of Science

Bangalore—560012, India

Po/~tn~;vin B nonapep[ide (P.!4N) i,~u derivative (?fpolym~xin B, an a, y-diaminohut~’ric ucid-rich decapep:ide from Bacillus pcdymyxa that displayv antimicrobial and lip[~p[~l~~.raccharide(LPS) -antagonistic activities, The corrformation.s of Ph4N in aqueous solution us well as in theLPS-bound slate have been studied bj ‘H two-dimensional nmr methods in conjunction nilhmolecular d~namics techniques. The aqueous s[ructure oj’lhe,jiee peptide is chara(ltvized h~’at~’pe11’/3-turn cen[ered around [>-Phe5and Leu h, and an inverse y-turn at ‘Thrv. The I.PS-bound <twfiu-mation ofPMN was studied by [ransfirrcd nuclear Overhauser effict aperimen[s.The essential feaiures ~?fthe tyclic portion q{fiee aqueous PMN are tHOSf])s prewrved nhtw [hepeptidc is bound to LPS; however, the linear dipeptid(’,jragment as well as the side chuirts (?f[heheplatpep(ide ring .dunt a cortjiwmational change and a reduction in mohili:~. The LPS-h(ntndPh4N structure ~,as used 10 con.struc( a model of [he lipid.4 -pol~vny.vin B (dc(’apept ide) com-ple.r, which allow.y[he ralionalizatimr c?fseveral t’.rperimenlal ob.rervalion.rconcerning fhe hirrd-ing ofpol~’rny.vin to, and consequent neutralization l?l the [o.~icit?%of I.PS, and ma?’ be of vahwin the rational therapeutic targeting ofendotoxin. @ 1997 John ~ ‘iley & Sons, Inc.

INTRODUCTION riety of organisms, with humans among the mostsusceptible species. The release of LPS into sys-

Lipopolysaccharides ( LPS ), structural compo- temic circulation, which occurs commonly as anents of gram-negative bacterial outer mem- consequence of major gram-negative bacterial in-branes, 1z elicit a wide range of toxic effects in a va- fections, leads to a constellation of symptoms

Received AugusI 7. 1995; accepted May 29, 1996.* To whom correspondence should he addressed

Biopolymers. Vol. 41,25 I-265( 1997)[c!1997 John Wiley & Sons, [nc. CCC 0006-3525/97/03025 1-15

251

252 Bhaltacharj~)a et al.

termed “endotoxic shock,” characterized by he-modynamic abnormalities, coagulopathy, andmultiple system organ failure.3 No therapeutic mo-dality of documented value is available at the pres-ent time and mortality due to this syndrome hasremained essentially unchanged over the past de-cade at around 60’%0.4Our understanding of thestructural aspects of LPS and of the cellular mech-anisms underlying host responses to this toxin hasnow advanced to such a level as to render the ra-tional therapy of endotoxin-related disease states atractable goal. Indeed, several experimental ap-proaches, targeting almost every mechanism andprocess known or thought to be operational in en-dotoxicity, are being evaluated for possible clinicalbenefits.5-8

The structurally highly conserved amphiphiliclipid moiety of LPS, termed “lipid A“ has beenshown, by total organic synthesis, to represent thetoxic center of endotoxin.g A possible therapeuticoption, therefore, is the sequestration, or removal,of LPS, which can be accomplished with agentsthat bind lipid A, preventing its recognition by en-dotoxin-specific receptor molecules. Polymyxin B(PMB ), a cationic amphiphilic decapeptide antibi-otic obtained from Bacillus polymyxa, 10binds tothe lipid A portion of whole LPS 11with an appar-ent dissociation constant of 0.4w141 ~and the resul-tant complex is virtually devoid of toxicity. Is poly-

myxin B, however, is toxic, which, while proscrib-ing its parenteral use, has stimulated the search fornontoxic polymyxin anaiogues as candidate LPSantagonists. ]4 An understanding of the structuralaspects of polymyxin and its complexes with lipidA would serve as a useful paradigm for the rationaldevelopment of lipid A binding agents. The crystalstructure of polymyxin B has not been reportedand few studies exist in the literature on the solu-tion conformation of the peptide, 1‘lb none ofwhich are of sufficient detail to permit considera-tions of the interaction of the antibiotic with LPS.

This paper deseribes the confirmational charac-terization of polymyxin B nonapeptide ( PMN ) inwater in its free and LPS-bound state by two-di-mensional (2D ) nmr and molecular dynamics pro-cedures. The nonapeptide derivative lacks a singleN-methyloctanoy l-diaminobutyric acid residue 17of the parent polymyxin B molecule (Figure 1).We elected to characterize the nonapeptide sincepolymyxin B decapeptide aggregates in solutionwith consequent loss of spectral resolution. Fur-thermore, the decapeptide, but not the nonapep-tide, precipitates in the presence of such concentra-

mThr’-Dab’DJ6I Thrs Dab7

\ D~bti

FIGURE 1 The primary structure of PMN with thenumbering scheme of the amino acids shown. The Poly-myxin B decapeptide has an additional N-3-methylocta-noyldiaminobutyric acid shown in the box.

tions of LPS as are necessary for nmr experiments.The LPS-polymyxin B complex, modeled usingthe LPS-bound conformation of PMN as a tem-plate, permits the rationalization of several exper-imental observations concerning the binding of thepolymyxins, and subsequent neutralization of theactivity of lipid A and LPS, and may be of value inthe design ofendotoxin antagonists.

MATERIALS AND METHODS

PMN was obtained from Boehringer Mannheim as thedihydrochloride salt. The purity of PMN was found to beabout 90% by high performance liquid chromatographyon a reverse-phase C18column using a methanol/waterbinary gradient with O.I‘% trifluoroacetic acid( methanol: 40-90%; retention time: 11.82 min ). Sevenmilligrams of the peptide were dissolved in 0.5 mL of907. H20/ 107. D20 (ea. 3.5 mkf). All experiments wereperformed at pH to 3.0 (adjusted with HCI ) since atacidic pH, all the Dab y-amino groups are protonated,which helped alleviate the potential problem of peptideaggregation. LPS from Escherichia coli 01 I 1:B4 andDJO (99.9’%) were purchased from Sigma Chemicals,Inc. Since the polysaccharide moiety of LPS is consider-ably heterogeneous,’ concentrations of LPS are given asmass per unit volume.

NMR Experiments

1H-nmr spectra were acquired on a Bruker AMX-400 spectrometer (400 MHz ) equipped with an As-pect 3000 computer. A low power irradiation (55dB ) of the water signal was applied during the re-laxation delay to suppress the solvent resonance.

Double quantum-filtered correlated spectros-copy ( DQF-COSY )‘8 and total COSY (TOCSY ) 19experiments were performed in the phase-sensitivemode to assign the spin systems. The mixing timein the TOCSY experiments was 60 ms. TOCSY andDQF-COSY spectra were acquired with a maximumof512 experiments of 40 scans each. Spectral width

Polj,tn~\rin B Nonapeptide 253

(4504 Hz) was always identical in both dimensionswith the time domain in tz set to 1 K data points.

Two-dimensional nuclear Overhauser effectspectroscopy ( NOESY )~“ and rotating frameNOESY (ROESY )~‘ spectra were acquired in thepure absorption mode using time-proportionalphase increments along t,. NOESY and ROESYspectra were collected with mixing times of 300and 400 ms respectively, with 512 experiments of72-88 scans each. For all the experiments the recy-

cle delay was set to 1. 5–2 s. All 2D data were zero

filled to 1024 points in both dimensions and

multiplied by a m/~-shifted square sine-bell func-tion prior to Fourier transformation. The probetemperature was maintained at 298 K. In tempera-ture-dependence experiments performed to obtainthe temperature coefficients of the amide protons,sets of one-dimensional ( 1D) experiments wereperformed at various temperatures.

In order to examine the binding of PMN to LPS,line-broadening experiments were performed bysuccessively adding small aliquots of a concen-trated stock solution of LPS ( 10 mg/mL in DIO)to PMN (3.5 m.tl; in 90% HzO/ 10ToDZO). Two-dimensional transferred NOE (TRNOE ) exper-imentsz~-~s were carried out using a mixture ofPMN and LPS that yielded moderately broadenedPMN resonances with a mixing time of 200 ms at298 K. This mixture corresponds to a 20:1 w/wratio of PMN ( 3.5 m Al) and LPS (the considerableheterogeneity of the polysaccharide portion of LPSprecludes precise concentration estimates of LPS).

Computational Methods

A crude molecular model of PMN was built usingINSIGHT-II ( B1OSYM Technologies, CA) on aSilicon Graphics Iris workstation. The diaminobu-tync acid residue was constructed from lysine: allother residues were available in the INSIGHTamino acid library. Cyclization of the cyclic por-tion of the peptide was achieved by creating a pep-tide bond between the T-NH of Dab ~and the car-boxyl group of Thr’. All energy minimization pro-cedures were performed using the DISCOVERmodule in INSIGHT. The Consistent ValenceForce Field ~’ was used with Morse harmonic po-tential energy terms for bond lengths, and bondand dihedral angles. with cross terms for charge–charge interactions. A dielectric constant of 1.0 wasused. The T-amino functions of the Dab residueswere not protonated to avoid distortions due tocharge repulsive effects that are to be expected in

the absence of adequate solvent screening. A fewcycles of steepest descent minimization relaxedshort contacts: this was followed by minimization

using the conjugate gradients algorithm~’ with theconvergence tolerance set to 0.00 I of the averageabsolute energy derivative.

lnterresidue NOE-derived constraints, as well asH-bond constraints derived from the temperaturecoellicients, were considered in generating accept-able models. Since all the interresidue NOES weremore or less equally intense, the upper limit of theNOE distance cutoff was empirically set to 3 ~, areasonable approximation for relatively small mol-ecules.zXMain-chain to side-chain NOES were fewand very weak, and therefore were not consideredas constraints. For H bonds, the lower limit of theN ~ O distance was 2.5 ~. A force constant of 50kcal /mol was applied on all distance constraints asa penalty function for violation of distance criteria.All generic distance constraints are shown in Table 1.

Molecular dynamics ( MD) simulations wereperformed in an effort to search for alternate con-formations that are consistent with the experimen-tally observed distance-geometry constraints andto examine the confirmational flexibility of themolecule. zy~(iMD trajectories were computed at300 K using the crude starting model without con-straints employing the Verlet Leapfrog algorithm] 1with the same energy terms as those used in the en-ergy minimization procedures. The molecule wasallowed to equilibrate at 300 K in vacuo and in theabsence of solvent molecules until the temperaturedrifts stabilized completely ( usually less than I ps).Trajectories were then obtained for 10 ps with anintegration time step of 2 fs: atomic coordinateswere recorded every 1 ps, resulting in an ensembleof 10 structures. The distance constraints were thenimposed on these structures and were energy mini-mized to complete convergence. Alternatively, tra-jectories were also computed with NOE and H-bond distance constraints imposed. The formerprocedure of unconstrained dynamics simulationfollowed by restrained energy minimization waspreferred to the latter since this method resulted ina greater exploration of confirmational space asjudged by inspecting o,+ dihedral trajectory plots,and all MD results discussed in this paper are thosederived from the former procedure.

Distance constraints derived from TRNOE ex-periments were used to model the LPS-bound con-formation of PMN. In these procedures, all ob-served NOES (within the backbone protons, back-bone to side-chain and side-chain-sidechain protons )

2S4 Bhattacharjya et at.

Table 1 NOE and H-Bond Dktance Constraints of PMN in WateF

ObservedLower Bound Upper Bound Mean k SD

Constraint Atom 1 Atom 2 (A) (A) (A)

NOE Thr’ C“HThrl CBHDab2(YHDab3G’HDab4C“HD-Phe5(YHLetsbC“HLeu6 NHDab’ C*HDabBC“HThr9 C*HDPhe5 Ring

H bond Dab’ NDab37N

Dab2 NHDab2NHDab3NHDab4NHt3-Phe5 NHLeub NHDab’ NHDab’ NHDab*NHThr9 NHDab3N’HLets’MethylDab’ ODabaO

2.02.02.02.02.02.02.02.02.02.02.02.02.52.5

3.03.03.03.03.03.03.03.03.03.03.03.03.53.5

2.48 %0.052.50 t O.O42.75 t0.012.85 k 0.022.88 f 0.012.50 + 0.022.80 f 0.032.72 t0.012.57 k 0.032.55 ?0.042.58 t0.023.00 f 0.013.01 f 0.023.02 f 0.01

a Distances obtained from 10 structures generated by MD.

were used as constraints in molecular modeling per-formed as described above. The PMB deeapeptidewas then constructed from the LPS-bound PMNstructure by adding a N-3-methyloctanoyl-Dab resi-due to Dab 1.This was then docked onto a moleeularmodel of lipid A.3ZThe docking was performed inter-actively such that Coulombic interactions betweenthe lipid A phosphates and the yamino groups of theDab residues were maximal in view of the primarilyelectrostatic mode of interaction of polymyxin withlipid A as well as other anionic phospholipids (seeDkeussion ). The resultant complex was then sub-jected to a few hundred cycles of steepest descent en-ergy minimization to relieve short contacts.

RESULTS

Assignments of Amino Acid Spin Systems

Assignment of the proton resonances were doneusing 2D-TOCSY, DQF-COSY, ROESY, andNOESY experiments.33-3” All spin systems couldbe assigned unambiguously using TOCSY andCOSY spectra in water. The resonances of D-Phe 5,Leu6, and Thr9 were assigned by their characteris-tic chemical shifts of the CPH, C6H, and C7H, re-spectively (Table 11); the large downfield chemicalshifts of the Cd protons of the Thr residues helpedassigning both of them. The NH resonances of the

Table 11 1H Chemical Shifts (in ppm) of PMN in H20/D20 (9:1) at 298 K, pH 3.0, and Temperature Coefficientsof Amide Protons

db/dT X 10-3Residue NH C“H CtiH C7H C$H Others (ppm. K-’)

Thr’ — 4.01 4.25 1.32Dab2 8.99 4.57 2.15,2.25 3.2 4.6Dab’ 8.61 4.34 1.90; 1.98 3.13 NTH:7.74 4.4; N7H:4.0Dab4 8.50 4.55 2.01; 2.06 3.13 7.0D-Phe5 8.75 4.60 3.10,3.09 — Ring. lH: 7.2; 7.4 7.6Leu’ 8,58 4.22 1.51; 1.42 0.85 0.70; 0.75 6.6Dab’ 8.23 4.32 2.28 3.22 0.9Daba 8.70 4.29 2.24 3.13 6.2Thr9 7.91 4.21 4.28 1.22 4.8

X2

D-Fs &

4

Polymysin B Nonupeptide

TT9

I X3N’ H

~

X7

‘4AG!S==

—. ——..—

..-—r - -. ~–-.-.T—7 —~

-7. -,—.. .

85 8.0

FIGURE 2 The 400 MHz TOCSY spectrum of PMN in 90% H~O/ 10% D*O at 298 K. pH3.0, (60 ms mixing time) indicating the spin systems of the amino acids. Single-letter aminoacid codes are used. X denotes cr,~-diaminobuty ric acid ( Dab ). Chemical shiftsare referencedto the ‘H line of water at 4.8 ppm.

five Dab residues were fairly well dispersed in 1D enabling the assignment of the Dab~ spin systemspectra and their spin systems were clearly distin-guished in the TOCSY spectra (Figure 2); se-quence-specific assignments for the Dab residueswere performed on the basis of the observed NOESto DPhe5, Leu6, Thry, and Thr } resonances inROESY spectra (Figure 3a and b ). The amino ter-minus Thr 1C(’H appears at high field (4.01 ppm)and does not yield any NH/C ‘“Hcross peak in theTOCSY ( Figure 2 ) or COSY spectra ( not shown)

due to strong NOES from Thr 1C“H and CtiH pro-tons (Figure 3a). Dab3 was assigned from sequen-tial NOES from Dabz and Dab4 C’rH, and NH, re-spectively. The side-chain N ‘H group of Dab3 thatis bonded with the carbonyl of Thr9 could be dis-tinguished from its characteristic NOES with ThrqC’”H and CPH resonances. Dab4, Dab 7, and Dab’were assigned by the sequential NOES from D-Phe 5, Leu’, and Thr9 residues. The TOCSY spec-

256

I

Bhattacharj~a C(al

0

T’/X2

!b

T’CpH/X2

IL_.

‘o

rl

~9

+

X8/T9

_—

‘+2

B+T9/X N H

[ T9CPHi X3N+H

— t Ppm

,, ,!1 ,,, ,,!( ,,,

ppm 9!0 8:5 8!0 7!5

FIGURE 3 (a) PartiaI 400 MHz ROESY spectrum of PMN in 90% HzO/ 10%D*Oat 298K, pH 3.0 (400 ms mixing time). Sequential C“H /NH, intraresidues, and D-Phe 5ring protons

to Leu’ &methyl protons NOES are indicated. X denotes a,-y-diaminobuty ric acid ( Dab ). ( b)Partial 400 MHz ROESY spectrum of PMN in 90% HzO/ 10% DzO at 298 K, pH 3.0 (400 msmixing time ), showing the NH/NH cross peak between Leub and Dab’.

tra (Figure 2) also reveal conformation-dependentchemical shift perturbations of Leu 6, Dab 3, andDab4 side-chain protons. The two nonequivalentmethyl resonances of Leub are moved upfield (0.2ppm) from the position reported for unstructuredpolypeptides, 33presumably a result of ring currenteffixts due to the proximal ~Phe 5residue. Likewise,the chemical nonequivalence of the CSHZS andC7H2Sresonances of Dab4 are also found at relatively

high field positions. The chemical shifts perturba-tions may be indicative of specific side-chain interac-tions involving the residues BPhe5 and Leub andDab4, which are not conforrnationally averaged. TheCfiH2 protons of Dab3, also nondegenerate, appearat the highest field among the Dab residues, probablya consequence of the incorporation of its side chaininto the cyclic backbone. The vicinal proton couplingconstants (3JH~ceH) for aqueous PMN obtained

P(d~m~.vin B Nonapeptid(” 257

84

90

Yj~(

3I

ppm 90 88 8.6 84

ppm

82

FIGURE 3 ( Continaed,/rom [ht’pr~’viou$page)

from 1D spectra which provide dihedral angle infor- gree of solvent shielding (Table 111). It is note-mation [O]~’are listed in Table 111. worthy that in an early study ofpolymyxin B, two

unassigned NH resonances were shown to be

Temperature Dependencesolvent shielded. ‘bAll other NH resonances hadhigh temperature coefficients characteristic of

Temperature dependence of the chemical shifts of solvent-exposed protons.the amide protons were measured in order to iden-tify the NH protons involved in intramolecular hy-drogen bonding. The temperature coefficient of

Analysis of ROESY Spectra of PMN

Dab’ NH is very low (0.88 ppb K-l) indicativein Water

of its buried nature. The NYH of Dabs (4.0 ppb In NOESY spectra of aqueous PMN, NOE crossK 1) also has a relatively low temperature co- peaks for the protons of the cyclic portion do notefficient in water, suggestive of a moderate de- appear, whereas NOES were observed between

258 Bha[tacharjya et al

Table 111 3J},~CYHCoupling Constants and Corresponding Dihedral Angles for PMN in Water

3JHNC”HResidue (Hz) d%ala #modelb

ThrlDab2Dab3Dab’D-Phe5Leu6Dab’DabgThr9

—7.07.96.75.17.65.54.97.6

—+60; –78; –155–95; -150+40; +70; –80; – 160+20; +100; –75: –175-85; –150+30; +82; –70; – 170+25; –65; +100; –170–95; –160

—

–82.6 f 8.3– 120.6 * 24.4–101.6 *23.8

67.4 k 4.8–95.0 ? 12.2

–102.4 *24.5–70.8 ? 4.5–76.4 * 4.0

a Theo values obtained using the expression 3JH#UH = 6,4 COS20– 1.4 cos 0 + 1.9 Hz, where@ = –0 + 60, and 0 + 60,3’b Mean and SD of@ measured for 10structures obtained by MD simulations.

C“’flHof Thr 1and Dab2 NH (data not shown), in-dicating differential mobility of the linear and cy-clic components of PMN. The paucity of NOEcross peaks in the NOESY spectra, a likely conse-quence of the correlation time of the cyclic portionof PMN being in the zero NOE region, necessitatedthe ROESY experiments. All intra- and interresi-due cross peaks were present in the ROESY spec-trum along with a NH/NH NOE between Leu band Dab 7(Figure 3a and b). Since PMN has a cy-clic array of seven amino acid residues, the possi-bility of the existence of a @turn as proposed forpolymyxin B decapeptide by earlier studies in theliterature, ]516or a ~-turn, 38were examined. Par-ticular attention was paid to D-Phe 5 in view of itspropensity to adopt positive@ angles and thereforeto appear in turns. 39The characteristic diagnosticNOES between D-Phe 5 C“H / Leu6 NH and Leu6NH/ Dab’ NH as well as the solvent inaccessibilityof Dab’ NH proton indeed suggest a type II’ ( D,L)@-turn centered at D-Phe5/ Leu6. The relativelylow d6/dt value for the Dab3 N’H and the NOEbetween Thr9 C “H and Dab’ N 7H would be sug-gestive of a ~-turn centered around Thr9. The onlyobservable interside-chain NOE was that betweenthe D-Phe 5 ring protons and the LeuG &methylgroups. The chemical shifts of the two sets of&methyl protons of Leu G were nonequivalent asmentioned earlier, and the upfield-shifted protonsshow a more intense NOE with the D-Phe 5 ringprotons (Figure 3a).

Studies on LPS-Bound PMN. LineBroadening and TRNOE Experiments

The addition of LPS to aqueous PMN results in aconcentrationdependent line broadening of al-

most all PMN resonances, confirming that ex-change between lipid-bound and free peptide mol-ecule is fast on the nmr time scale. The side-chainresonances exhibit greater line broadening as com-pared to the backbone C“H and NH resonances(Figure 4). A mixture of LPS and PMN yieldingmoderately broadened PMN resonances was usedfor acquiring 2D TRNOE spectra.

In the TRNOE spectrum (Figure 5a and b), thesign of all cross peaks are negative, verifying thatthe observed cross peaks correspond to the LPS-bound state of the peptide. All backbone NOES ofthe cyclic portion of free aqueous PMN (Figure 3aand b) are preserved in the LPS-bound state aswell, with an additional NOE between Dab8 NH toThr9 NH (Figure 5b ). However, the intensity aswell as the number of backbone-to-side-chain andinterside-chain NOES are enhanced remarkably(Figure 5a), suggesting that while the cyclic por-tion of the peptide undergoes no major backboneconfirmational change, there is greater motionalrestriction of the side chains upon binding to LPS.It maybe noted that there is a steep dependence ofthe NOE magnitude on the rotational correlationtime in the negative NOE regime.40

New NOE cross peaks that were not observed inthe free molecule appear around the turns (Figure5a and b ). In the LPS-bound state, anew backboneNOE between Thr9 NH/Dabs NH and a side-chain NOE between Thr9-methyl protons/Dab 3N ‘H are observed. New NOES are also observedbetween the side-chain to backbone protonsaround the o-turn region. The pair of&methyl pro-tons of Leu6 gives NOES with its NH as well as withDab7 NH, the two sets of NOES being of differingintensities. The specific enhancement of these

—1

1I

I

Pma.

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07,5

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1.5

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Bhattacharjya cl al.

‘b) D-#Rjng -O O~D-#R’mg ~,.lD-#’NH

,p~~

X8/T9

I ,!, , I ,,, ,,! ,,( ,,

Dpm 85 8.0 7!5

(a) –-.- —..

a /=D #Ring

—w)/~C$HSfC6t+JX7NH

0 o_T9CfH3 / X’NYH

7,5

0.0

1,-8.5

1

1

9.0

ppm

-1

–2

,

–3

-4

- ~pm

FIGURE 5 (a) 2D TRNOE spectrum of LPS-bound PMN in 90%H20/ 10%DZOat 298 K,pH 3.0, 200 ms mixing time. PMN and LPS concentrations are 3.5 mM and 0.6 mg/mL.Sequential assignments of cross peaks between the NH protons to C“ and side-chain protonsare shown. X denotes a,~diaminobutyric acid ( Dab). (b) 2D TRNOE spectrum of LPS-bound PMN in 90%H20/ 10’%DZOat 298 K. Assignmentsof NH / NH cross peaks are shown.Also marked are NOES between ~Phe5 ring protons to its NH and to Leu b NH.

Po[) ‘tm’.rIrI B IVonapep[ide 261

Dab8

180

R(J+‘4+

120 7 -42

980

yp

-80P 8 5-120

i

●

II

-180 ~

-180 -120 -80 0 80 120 180

4

FIGURE 6 ( Left) SuDerrmsition of 10 remesentative MD structures of PMN in water. Onlv

backbone and ~U atoms’ ar~ shown. H atom-s are not shown. ( Right ) Backbone dihedral angle~of the MD structures indicated on a Ramachandran map. Numerals correspond to the residuenumbers as shown in Figure 1. Error bars represent SD, Sterically allowed regions are indicatedby contour lines.

NOES and the appearance of new NOES would beconsistent with a localized confirmational stabili-zation of the two turns upon complexation withLPS. It may also be noted that the NOES from the&methyl protons to D-Phe 53,4,5 ring protons arestronger than those to D-Phes 2.6 ring protons, in-dicating a specific orientation of the Leu ~ sidechain to the phen yl ring of D-Phe f.

Enhanced 7, value for the peptide side chainswill result in more intense NOES in the complexedstate. The Thr 1CijH /Dab2 NH and Thr 1methyl/C[’~H cross peaks that are present in the free mole-cule are absent in the LPS-bound form. This obser-vation is consistent with a confirmational changeinvolving the Thr 1 side chain upon interactionwith LPS.

Molecular Dynamics

Molecular dynamics simulations were performedwith NOE-derived constraints and H-bond criteriato obtain an ensemble of structures. Examinationof the 10 model structures of PMN in water gener-ated by MD superimpose closely ( Figure 6). with

an rms deviation of 0.30 ~ for all the C<’atomssuggesting a relative ngidit y of the backbone of thecyclic portion. The type II’ /l?-turn around D-Phefand Leu h were preserved in all conformers and the-y-turn centered around Thry was also present in allstructures: the @dihedral angle of Thrg was invari-ably around –76°, corresponding to an inverse T-turn.~~ The linear moiety was mobile and un-derwent marked excursions during MD simula-tion, Dabz exhibiting maximal mobility (C” rms:0.4 ~). The side-chain dihedral ( x ) angles are quitevariable, as expected. The “stereochernical good-ness” of the models were evaluated by plotting the@,Ydihedral angles on a Ramachandran map.4’The dihedral angles of all the eight residues fall inthe conformationally allowed region in the Rama-chandran plot ( Figure 6). D-Phe5 also lies in thesterically allowed region as the o,+ map would beinverted upon changing residue cbirality. The di-hedral angles about the C[’-C” (x,), and C“-C’(x2 ) of DabJ, are approximately 180°. Other Dabresidues fall into the extended region of the Rama-chandran map whereas Dab* adopts a right-handed helical conformation. The two hydrogen

262 Bhattacharjya et al.

FIGURE 7 A model of the polymyxin B-lipid A com-plex. Lipid A is shown as a ball and stick representationand polymyxin B is depicted by a stick representationwith van der Waals surface.

bonds modeled in the crude starting structure(Dab’: NH - Dab’: O; Dab3 N7H s Daba: O)were preserved and are of nearly ideal parameters:the N s O distances are 3.01 and 3.02 ~ and theH-N-O angles are 20.9° and 22.5”, respectively.

Figure 7 represents a model of the lipid A-poly-myxin B complex that maximizes favorable in-teractions between the two components. Poly-myxin B was generated by the addition of N-meth-yloctanoyl-Dab residue to the TRNOE-derivedPMN model. Docking was achieved interactivelywith a few cycles of energy minimization to relieveshort contacts. The salient features of the complexare the following: The peptide carbonyl groups ofthe cyclic portion of PMB lie on one face of thepeptide, and this surface overlies the disaccharideheadgroup of lipid A. The ~-amino groups ofDab 3/ Dab4 and Dab’/ Dab8 are within H-bonddistances of the phosphate groups at positions C-1and C-4’ on lipid A, respectively. The orientation

of the linear portion of PMB bearing the acyl resi-due is normal to the plane of the cyclic portion andis coaxial with the acyl chains of lipid A.

DISCUSSION

The conformation of free aqueous PMN has beenstudied by 2D-nmr methods, and in the LPS-bound state using transferred-NOE techniques inconjunction with molecular modeling methods.While considerable progress has been achieved inunderstanding the confirmational features of cy-clic hexa-, ‘z octa-, 43.44and nonapeptides, 45 rela-tively little has been reported on cyclic heptapep-tides. For this reason, and because the cyclic moi-ety of PMN is unusual in that it is composed of 23atoms (due to side-chain-to-main-chain cycliza-tion rather than the 21 in the case of main-chain-to-main-chain linkage ), PMN is interesting fromthe confirmational point of view. PMN is also dis-tinguished by the presence of the unusual Dab res-idue, a feature shared by other B. polymyxa derivedantibiotics such as the circulins and octapeptins. 10

The aqueous conformation of PMN is charac-terized by a type 11’~-turn with ~Phe5 at the i + 1and Leu 6 at the i + 2 position identified by the di-agnostic NOE pattern and H bonding, and an in-verse -y-turn centered at Thr9 as evidenced bysolvent shielding of Dab 3N ‘H and the presence ofNOES from the neighboring residues. The remain-ing portion of the ring is extended without strongtransannular H bonds, which is consistent with re-sults obtained by tritium-hydrogen exchange ex-periments.46

The LPS-bound conformation of PMN as ex-amined by TRNOE experiments indicate no majorconfirmational change of the backbone of the cy-clic portion, and both turns are preserved. This isconsistent with earlier reports 47 showing that thebinding of polymyxin to phospholipids do not re-sult in any change in the optical rotatory dispersionspectrum or the amide bond stretching frequenciesin the ir spectrum of the peptide. The segregationof the hydrophobic side chains of PPhe 5and Leuband the disposition of the carbonyl groups arrayedon one face of the peptide renders the mohm,de am-phiphilic. A model of the PMB-lipid A complexconstructed by doeking a model of PMB deducedfrom the TRNOEderived PMN structure on lipidA suggests that the polar face of PMB interacts withthe disaccharide headgroup of lipid A, the hy-drophobic side chains of mPhe5/Leu6 overhang-

Pol~my.rin B Nonapep~ide 263

ing the surface. The 7-NH2 pairs of the Dab resi-dues Dab~/Dab4 and Dab’/Dabx form ionic Hbonds with the lipid A phosphates. The linear par-tof PMB is so oriented as to facilitate hydrophobicinteractions with the acyl chains of lipid A.

Our investigations were motivated by the as-sumption that a detailed characterization of theconformation of polymyxin would enhance ourunderstanding of how the peptide functions as anantibiotic, and more importantly, as an LPS antag-onist. It would therefore be appropriate to attemptto rationalize polymyxin activity in terms of itsstructure. The antimicrobial action of polymyxinB is a manifestation of the binding of the peptideto, and subsequent disruption of, the outer mem-brane of gram-negative bacteria. 10 The outermonolayer of the outer membrane is comprised al-most exclusively of LPS~4Rand the initial event ofthe peptide action, therefore, is its binding to LPS.The presence of anionic constituents 2 ( lipid Aphosphates and anionic sugar residues ), and thehighly positively charged nature ofpolymyxin dueto the preponderance of the basic Dab residue, sug-gest that the binding is electrostatically driven. In-deed, the binding of polymyxin to LPS is inhibitedby divalent cations or under conditions of highionic strength.4’ LPS obtained from polymyxin-re-sistant bacterial strains whose lipid A phosphatesare esterified show decreased polymyxin bind-ing.~().fI Furthermore, acetylation of the Dab ~-amino groups of PMB result in loss of antimicro-bial activity.~z However, while the presence of thebasic amino functions are necessary, they are notsufficient for complete expression of activity.PMN, lacking a single terminal Dab residue cova-Iently linked to a fatty acyl residue, while bindinglipid A with a dissociation constant indistinguish-able from that of PMB (S. A. David, unpublishedresults ), shows negligible antimicrobial efficacy, 17and considerably less potent LPS-antagonistic be-havior.f~ These suggest that microbicidal and anti-LPS activities may have the same structural corre-lates, the cyclic portion conferring the ability tobind lipid A, and the linear fragment aiding the dis-ruption of LPS assemblies.

The strict structural requirements of the cyclicportion for biological activity were recognized dur-ing chemical synthesis of PMB.S4 These authors re-ported that the expansion of the cyclic peptide ringby one amino acid reduced antibiotic activity. Nomajor change in the backbone conformations ofPMN on LPS binding would therefore suggest thatthe cyclic ring backbone serves only as a scaffold to

appropriately position the Dab side chains, andthat the conformation of the ring per se may be rel-atively unimportant. This conjecture is supportedby our earlier findings that gramicidin S and tyro-cidin A, basic cyclic decapeptides with rigid @-pleated structures obtained from B. brevis, alsobind lipid A with binding constants comparable tothat of PMB 55; furthermore, subsequent studieshave shown that small, linear dibasic nonpeptidemolecules (aliphatic diamines and aromaticdiamidines) of appropriate dimensions also bindlipid A.56 The salt bridges observed in modeling thePMB-lipid A complex are likely to be responsiblefor the high affinity of the peptide for lipid A. Theformation of such ionic hydrogen bonds appears tobean obligatory requirement for the interaction ofPMB with anionic Iigands, since the peptide bindsto anionic phospholipids possessing a free phos-phate group. but not to analogues with electrostat-ically similar N-methylated phosphate groupss’and poorly to lipid A bearing esterified phos-phates.? 1Interestingly, a variety of cationic organiccompounds possessing free protonatable basicgroupss’ bind to lipid A, but not molecules withquarternized nitrogen atoms.

The penetration of the fatty acid tail into the hy-drophobic regions of lipid A observed in the modelis consistent with increased surface pressure of lipidA monolayer in film balance experiments.s” It islikely that the insertion and subsequent apposi-tioning of the hydrophobic component of the pep-tide destabilizes LPS assemblies on bacterial mem-branes. It also appears possible that the fatty acylmoiety may mask regions on the hydrophobic do-main of lipid A, thereby impeding the recognitionof lipid A by its receptors. It has recently been re-ported that the hydrophobic domain of lipid A iscrucial for cellular activation.4B

These results provide a first step toward an un-derstanding of the structure-activity relationshipsof polymyxin B, and also permits a greater appre-ciation of the desirable structural requirements ofpotential endotoxin antagonists and may be ofvalue in designing analogs of polymyxin withgreater LPS-antagonistic properties and lower tox-icity.

This work was supported by grants-in-aid from the In-dian Council of Medical Research, New Delhi, India. Wethank Dr. M. Kastowsk y, Institute for Crystallographyy,Berlin, for graciously providing the atomic coordinatesof the lipid A model. Computational facilities were pro-vided by the Supercomputer Education and Research

264 Bhatlacharjya e[ al.

Center and NMR spectra were recorded at the Sophisti-cated Instruments facility. We are most grateful to ananonymous reviewer for pointing out incorrect assign-ments in an earlier version of the manuscript.

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