Helical epitope of the group B meningococcal α(2-8)-linked sialic acid polysaccharide

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Helical epitope of the group B meningococcal α(2-8)-linked sialic acid

polysaccharide

Brisson, Jean-Robert; Baumann, Herbert; Imberty, Anne; Pérez, Serge;

Jennings, Harold J.

Biochemistry

Helical

_Epitope

of

_{the Group}

B

_{Meningococcal}

_{a(2-8)-Linked}

Sialic

Acid

Polysaccharide*

Jean-Robert Brisson,1Herbert Baumann,1 Anne Imberty,****8

SergePérez,8 and

Harold

J. _{Jennings*’1} Institute

_for

_BiologicalSciences, NationalResearch Council

_of

Canada,Ottawa, CanadaK1A OR6, andLaboratoire de

PhysicochimiedesMacromolécules, Instituí Nationalde_{la Recherche Agronomique, BP}527, 44026NantesCédex, France Received December28, 1991; RevisedManuscriptReceivedMarch 11, 1992

abstract: The _{immunologicalproperties}

of

the _group B_{meningococcal}

_{a(2-8)-linked}

sialicacid poly-saccharide have been rationalized interms

of

a model where the randomcoil nature

of

the _polymer can

bedescribed by thepresence

of

local helices. Theconformational versatility

of

the

aNeuAc(2-8)aNeuAc

linkagehasbeen_exploredby

NMR

studiesat 600

MHz

_{in conjunction}

with

_{potentialenergy}calculations

for colominicacid,an

_a(2-8)NeuAc

_polymer,andthetrisaccharide

_{aNeuAc(2-8)aNeuAc(2-8)j8NeuAc.}

Potential_{energy calculations}were usedto estimate the_{energetically}favorable conformers andtodescribe thewide range

of

heliceswhich_{the polymer}can _adopt. No_{unique conformer}was _{found to satisfy}all

NMR

constraints, andonly ensemble averaged nuclear Overhauser enhancements could _{correctly simulate} the

experimentaldata. Conformationaldifferences between thepolymerandthe trisaccharide couldbebest

explainedinterms

of

slightchangesintherelative

distribution

of

conformersin solution.

Similar

helical parametersforthe

a(2-8)NeuAc

polymerand

poly(A)

were _proposedasthebasisfor their _{cross-reactivity}

toa monoclonal_{antibodyIgMNOV. The unusual length dependencyfor binding}

of

oligosaccharide to group B_{specific antibodies}was _{postulated to}arisefrom_{the recognition}

of

a_{high-order local helix}withan extended

conformation whichwas not _{highly populated}in solution.

(jTroup

BNeisseriameningitidisandEscherichiacoliK1

are _majorcauses ofmeningitis. Both bacteriahaveidentical capsular polysaccharides composedofa(2-8)-linkedsialic acid residues. _{Althoughvaccine development}hasbeen_hampered

by thepoorimmunogenicity ofthe_group B meningococcal polysaccharide inhumans,byN-propionylation ofits sialic acid _residues, antibodies are _{produced which} are both

bac-tericidal and _{protective [for} reviews, see _Jennings (1989, 1990)].

The_{a(2-8)-linkedsialic acid homopolymer}has_many in-terestingimmunological propertieswhichhave been_extensively

studied. The_presenceofa conformationalepitopewas

hy-pothesizedby Jennings et al. (1984) to explain theinability of_{oligosaccharides}ofup tofiveresiduestoinhibitthe_binding

ofthe_{group B polysaccharide to}a_homologoushorse_antibody. Later,

it

was established that an _{unusually large} deca-saccharide was _required to bind or inhibit _group B poly-saccharide specific antibodies (Jennings et al., 1985;Finne

&

Makela, 1985;Hayrinenet al., 1989). Thisisincontrastwith

aconventional_{sequentialoligomer}where_onlyamaximum6 or 7 residues are _necessary for _{binding (Rabat,} 1960). Cross-reactivityofahuman_{monoclonal antibody(IgMNOV)}

withthe _{a(2-8)NeuAc‘ polymer, poly(A),} and _other

poly-nucleotideshas also been observed _{(Rabat et al., 1988).} Various attemptshavebeenmade to relatethese immuno-logical properties to the three-dimensional structure ofthe antigen. Lindon et al. _(1984), from

NMR

studies at 360

MHz,determined differencesintheorientationof_{the exocyclic}

chainsofthe_a(2-8)NeuAcanda(2-9)NeuAcpolysaccharides andin_{the segmental}motionof_{the polysaccharides.} Michon etal.(1987) determinedfrom

NMR

studiesat 500MHzthat the conformation ofthe _linkagesofthe polysaccharideand fThisisNationalResearchCouncilofCanadaPublicationNo.32011.

Part ofthisworkwas _{made possiblethrough the France-Canada} ex-changeprogram grantedtoJ.R.B.andfinancial support from INRA.

1_{NationalResearchCouncilofCanada.} 8_{InstituíNationalde laRecherche}

Agronomique.

short_{oligosaccharides}were different. Yamasakiand Bacon (1991),froma_{quantitativeanalysis}of2DNOE’sat 500MHz

usingrigidmodels, suggestedthatthe_{polysaccharide adopts} orderedhelical structures in solution. Inthe present paper, the_{conformational properties}ofcolominicacid,an

a(2-8)-NeuAc _polymer, and the trisaccharide

aNeuAc(2-8)-aNeuAc(2-8)/3NeuAcwere _{evaluated by}

NMR

studiesat 600

MHz,in conjunctionwith_{potentialenergy calculations.} The interpretationoftheNOE’swas based onamodelwhichallows

fortheinherent

_flexibility

about the_linkages. The confor-mational versatilityofthemoleculewas _{estimated bypotential}

energy calculations. The biological implications were

ra-tionalized in termsofamodel wheretherandom coil nature

ofa_{polysaccharide}can bedescribedbythe presenceoflocal helices (Perez

&

_{Vergelati, 1985).}

Experimental

Procedures

Nuclear MagneticResonance_Experiments. Thecolominic acid polysaccharideand the trisaccharidewere _preparedas

describedbefore(Michonetal., 1987),at concentrations

of

10-20mgin0.5mLof95%_{D20, 5% acetone-1/6,pH7,}Na+ counterion. Samples were _purgedwith helium _{gas. Upon} storage atacoldtemperature,samplesinsolutionwere found to bestableformonths, withno _signs of_{degradation. The}

H3a and H3e resonances of the _{reducing terminus} were

partiallydeuterated_{(>50%) after}severalmonthsinsolution.

All experiments were _performed on a Bruker AMX 600 spectrometer at 288 R. Foradditional temperature stability, thedeuteriumlockwas establishedon internalacetone-</6. No

significantchangesin chemicalshiftwere _{observed upon the}

addition

of

_acetone-d6. Forcolominicacid,pureabsorption

phasesensitive2DNOEexperimentswere doneinTPPImode

(Bodenhausen et al., 1984) withsix mixing timesof25, 50, 75, 100, 150, and 200 ms. The _{relaxation delay} between

1

Abbreviations: COSY, correlation spectroscopy; NeuAc,

N-acetylneuraminicacid;NOE,nuclear Overhauser enhancement;NMR,

nuclear magneticresonance;TPPI, time-proportionalphase increments.

ConformationofMeningococcalB _{Polysaccharide Biochemistry,} Vol. 31, No. 21, 1992 4997

TableI: MM2-MinimizedCoordinatesofaNeuAc-O-Me

atom X Y Z Cl 2.00350 -0.00013 1.42684 C2 1.42467 0.00407 0.00253 C3 1.85264 1.28886 -0.71271 C4 3.33580 1.26891 -1.05815 C5 3.63666 0.02645 -1.89345 C6 3.16028 -1.22252 -1.14587 C7 3.36974 -2.51478 -1.94809 C8 2.79059 -3.75974 -1.27483 C9 3.14742 -5.05286 -1.99458 CIO 5.59251 -0.31857 -3.37196 Cll 7.10639 -0.36067 -3.44988 Ola 1.35286 0.85734 2.23587 Olb 2.93208 -0.64255 1.83271 02 0.01649 -0.00037 0.00817 04 3.67387 2.43642 -1.80927 06 1.78881 -1.12261 -0.78919 07 2.77959 -2.38247 -3.24501 08 3.28173 -3.86537 0.06502 09 2.61127 -6.11483 -1.24381 010 4.90479 -0.55473 -4.34865 N5 5.06865 -0.02067 -2.13024 H3a 1.26995 1.39780 -1.65856 H3e 1.61658 2.18718 -0.09476 H4 3.95711 1.27566 -0.13132 H5 3.07491 0.12311 -2.85219 H6 3.76751 -1.31926 -0.21869 H7 4.46117 -2.67404 -2.11132 H8 1.67985 -3.67829 -1.20742 H9R 4.25246 -5.18468 -2.05049 H9S 2.71761 -5.10088 -3.02114 Hila 7.50134 -1.14014 -2.75920 Hllb 7.53118 0.62782 -3.16186 HI lc 7.44975 -0.60184 -4.48183 HN5 5.72929 0.09162 -1.36044 HOla 1.79198 0.79277 3.09998 H04 4.54535 2.33014 -2.15201 H07 3.35196 -1.87683 -3.79615 H08 4.20238 -3.66611 0.05735 H09 2.75650 -5.93429 -0.33581 CM2 -0.56945 -1.16138 0.57428 HM2a -0.19855 -1.33684 1.60824 HM2b -0.37743 -2.05165 -0.06578 HM2c -1.66970 -0.99793 0.61710

acquisitions was 6 s, and the sweep width was 2400 Hz. Matricesof512_{by 2048 points}were recordedwith_eightscans

pertxincrement. Transformationwasdoneusinga _/2-shifted

sine-squaredbell

filter

in both dimensionand zero-fillingto

a _square matrix

of

1024 X 1024 points for a final digital

resolutionof2.4_{Hz/point. Afirst-order polynomial}baseline correlationwas _{applied in the}

fl

dimension_prior to volume integrationofthe cross-peaks. The_averageofthe_equivalent volumes aboveandbelow_{the diagonal}was usedfor_analysis.

All

data processingwas _{done using}uxnmr _{software provided}

by Bruker. Steady-state ID NOE experimentson the

tri-saccharidewere doneasdescribed before_{(Michonet al., 1987).} The spectrum

of

afew_{milligrams of}colominic acidwas

taken_{at various temperatures up to}343_K,inorder to observe the proton coupling constants. _{As described previously}

(Michonet al., 1987),protonassignmentswere obtainedfrom

a COSY _{experiment. Spin simulations} were _{done using}

LAOCN5_{(QCPE) with}ftnmr _(HareResearch_Inc.). Theerror on the _coupling constantswas ± 0.3 Hz.

Calculations. The starting geometryof aNeuAcwas built

fromthe_{crystal structure}oftheßsialicacid_methylglycoside (Flippen, 1973) afterproper settingofthe anomeric config-uration.

It

was thensubmitted to a_{complete refinement}

of

bond lengths,valence, and torsion angles byusing the

mo-lecular mechanics _{program MM2(87) (QCPE) (Burket}

&

Allinger, 1982).

All

calculationswere _{done using}the

mini-mized coordinatesforthe_{methylglycoside}ofaNeuAc (Table

I).

Fora_{givendisaccharide, the conformationalanalysis}was

evaluated usingthePFOS_{approachwhich included the} par-titioned contributions arisingfromvan derWaals interactions andtorsionaland exo-anomeric_{potential (Tvaroska}

&

Perez, 1986). Rotational barriersof1.0and0.5_kcal/molwere used for and

_,

_{respectively.} Noelectrostaticcontributionwas

considered. _{The ring geometry}was treatedasinvariant,and

hydroxylichydrogen atoms were not takenintoaccount. Four angles determine the linkage conformation for

aNeuAc(2-8)aNeuAc(h-a): =

(¿>06-¿>C2-¿>02-aC8),

= _{(¿>C2-¿02-tiC8-tiC7),}

a>8 =

(07-C7-C8-08),

and 7 =

(06-C6-C7-07).

Theorientationofthe pendant groups for

Cl

andC9was defined_by =

(Q6-C2-C1

-01

A) ando>9 =

(08-C8-C9-09). All

anglesare _expressedindegrees. The

angles<vb 7, 8, and ₉ were _alwaysset the same forboth

residues. Thecv7anglewas setat60°,and separate mapswere

generatedfor 8 = _60°,

_-60°,

_{and 180°}

(g+,g",t). The

( , )

mapswere calculated in the_{followingway.} Fora_givenset of _7, w8values, theglycosidic torsionalangles and were

stepped in increments of 5° over _{the angular range} which containedlow_{energyminima,}-120°to+240°for and40° to 220° for

.

At

each_pointofthis_{grid search, the wj and}

9 angleswere _stepped in increments

of

20° over the whole angular range, andtheminimum_energy conformerwas

se-lected. Sucha_procedureallowedsevere steric constraintsto

berelievedbetweenthe_{hydroxymethyl group at}C9 and the carboxyl group at

Cl

across _{the glycosidic linkage.}

All

the low energy conformers derivedfrom_{the analysis}ofsuch_“rigid

potentialenergysurfaces”were _{refined using theMM2(87)}

software. From alltheseminimizedconformers, theaverages

ofthe and<v8anglesforthethree staggeredtu8conformers

were takenand the_rigidmapswere _{calculated againusing}

thesemean values(Table

II).

The_{averageglycosidicbridge}

angleforbC2-b02-aCS of 117° was also used.

Helicesare _customarilydescribedintermsofasetofhelical parameters, (n,h),withn_{being the number}ofresiduesperturn

of

the helix and h _being the translation

of

_{corresponding}

residues along thehelixaxis. _{The parameter}nisderivedfrom the rotationangle (µ) about the helix axis per repeatunit,

wheren =

2 /µ.

Withsetvaluesofglycosidic valence angles andresidue geometry, these parameterscan becalculatedfor

TableII: MinimumEnergy Conformersfor<*NeuAc(2-8)a:NeuAc

E“ _^ OJg (Ug "1 <oi>6 n hc _h/L

Gl+ 3.2 -45 130 55 70 138 -40 9.1 2.3 4.8 0.8 G2+ 0.0 75 100 55 70 138 -20 6.5 3.6 -3.2 -0.5 G3+ 2.9 -180 105 55 70 138 -40 6.7 11.3 5.7 0.9 Gl- 3.6 -50 120 60 -62 -162 -40 6.3 2.5 2.9 0.7 G2- 0.3 60 115 60 -62 78 -20 7.4 2.2 -4.2 -1.0 G3- 2.3 170 135 60 -62 -162 -40 6.9 2.8 -1.9 -0.5 T1 5.2 15 155 67 179 138 180 3.8 2.9 1.2 0.2 T2 1.0 80 160 67 179 98 180 4.7 3.0 '4.0 0.8 °_Relative

Biochemistry, 31,

figure 1: Structureof aNeuAc(2-8)aNeuAcshowingthefour torsion

angleswhich determine thelinkage conformation with

(06-C6-C7-07) = ₇ = _{60° and(07-C7-C8-Q8)} =

8 = _{60°. The}

ori-entation of_{the pendant groups} at Cl and C9 is_{defined by} =

(06-C2-C1-01A)and 9 = _{(08-C8-C9-09),respectively.}

any given value of the set of torsional _angles (Sugeta

&

Miyazama,1967). Thelengthofthevirtualbond(L) between two adjacentlinking atoms,namely,

02

and

08,

represents themaximum ofthe _{possible extension. The quotient}

_h/L

yields the percentageoftotalextension. The chiralityisdefined by the signofh,positivefor right-handedhelices and negative

forleft-handedhelices.

The NOE was calculated using the complete relaxation

matrix, under the assumption of _isotropic tumbling. The

steady-stateNOEwascalculatedasdescribedbefore (Brisson

&

Carver, 1983). The 2DNOEwas _{calculated according}to Borgias and James(1988). Determinationofdistancesfrom 2D NOE data was done using the distance extrapolation method (Balejaet al., 1990). The ensemble averaged

NOE

was calculatedas_{described byGumming}andCarver (1987). For better_{Boltzman weighting}oftheNOE,a_{potentialenergy}

mapwith Io increments in

( , )

was _generatedfromthe 5° interval map using cubic spline interpolation. Only (r™6)

motional averaging was used. _Averagingwas onlydonefor

( , ),

theo>7, 8, and 9 anglesbeingfixed. Foragiveno>7

and ₈value, the

( , )

potentialenergy surfaceforwhich , and _(ti9adopted minimum_energyconformers was used _(see above) andtheaveragedNOEwas _{calculated separately}for

(ti9 = 60°,

-60°,

and 180°. This couldbe done becauseno

major interglycosidic NOE was observed on the H9

reso-nances. Therelative_energyofconformerswithinawellwere

modifiedbyfirstdefiningacontourlevelwhichencompassed

alocalminimaandthen changing the energyoftheconformers at the_gridpointswithinthewellbythesame amount. The relative population within a well was calculated using the Boltzmanndistributionon a 10 X 10 _{gridinterpolated}from a 5o X 5° _grid.

Inorder to calculate theNOE,therotationalcorrelation timeofthe molecule mustbedetermined. It was estimated fromabest

fit

ofthecalculatedNOEtoH3a-H5and

H3e-H5 forvarious _rigid conformers. Correlation timesof8 ns

and0.5nswere usedfor simulationoftheNOEfor colominic acid and _{the trisaccharide, respectively.} The “standard deviation”,Sdev,fortheNOEwas calculated_{using7V(Sdev)2}

=

( „

)2,

where

N

isthenumber

of

points. The n - h

plots for poly(A) were _generated ina similar manner as described before (Yathindra & Sundaralingam, 1976) usingX-raycoordinates _{(Saenger et}al., 1975). Ball andstickmodelswere _generatedwiththeschakal _program.

Calculationswere _performedon Micro VAX 3200 and 3500.

Results

In order to understand the conformational behavior

of

«NeuAc(2-8)«NeuAc(Figure 1), thelinkage conformation

,

,

7, and 8, andtheorientation ofthe pendant groups mustbedetermined. The_{pyranoseringadopts}awell-defined

figure 2: Potential_{energy contourmap}for aNeuAc(2-8)«NeuAc with( 7, 8)= _{(55°,70°)(A), (60°,-62°) (B),and(67°,179°) (C)}

and(tijand 9 adoptingminimumenergy conformers. Withrespect

tothe globalminimumofeach section,the_8,6,and10_kcal/mollevels are drawnfor_(A),(B),and_{(C),respectively.} For_(A),fromleftto right,three seperate wells,WG1+, WG2+,andWG3+,arethusdefined.

Similarly for (B),three wellsWG1",WG2™,andWG3'are defined. Localenergyminima (TableII)areindicated. Thehelical parameters

n _{(solid lines) and}h(dotted line) are also_{displayed. For poly(A)}

(D),then- _h

map and the energetically favoredhelices (dashed area)

are shown. The heliceswith (n,h) = _{(9,2.8Á) and (9,5.5Á)are}

marked.

2C5conformation,andthe

NAc

groupat C5 adoptsa_planar

configuration withthe

H5-C5-N5-HN5

inthe trans con-figuration (Christian

&

Schultz, 1987).

Potential_{Energy Calculations. Potential}energy calcula-tionswere _{performed in order}toassess the

_{( , )}

conforma-tional spaceforthestaggeredrotamers

of

8, with 7 fixed to theg+rotamer (Figure2). Thehelical parameters, ob-tained upon propagation of the molecule, are also_plotted. Three

_{( , )}

mapswere _{represented wherethe ,} and ₉

ro-tamers were allowed _{to adopt}minimum _energyconformers

foreach_settingofthe_{linkage conformation. The 7} and ₈ angleswere _optimizedbymolecularmechanicscalculations. For the

( 7, 8)

=

(55°,70°) map(Figure2A), three major wells,WG1+, WG2+,andWG3+, whichencompassminima

Gl+,G2+, and G3+(Table

II)

are definedbythe8 _kcal/mol

levelwith_respecttothe_{global minimum. Forthe ( 7, >8)} =

(60°,-62°) map (Figure 2B), three separate wells, WG1", WG2~, andWG3“, couldbedefined_{by the}6 _kcal/mollevel abovetheG2~_{conformer (Table}

_II).

The

_{( , )}

_{energy map} for((ti7,(ti8) = (67°,179°) mapswas more restricted,and_only the 10_kcal/mollevelwith_{respect to}minimum T2 (Table

II)

isshown_{in Figure}2C. _Althoughother localminima could befoundwithinthese wells,onlytheareas _{defined by}these wellswere _{useful in interpreting the}NOEresults. Relevant parameters forvarious low-energy conformers are _givenin

TableII. Molecularmechanics calculationswere _performed

forvariousconformerswithinthesewells _{(data not} shown).

It was foundthattheseconformerswere stable,the linkage

anglesnot changingsignificantlyuponcompleteminimization.

Multipleorientationsofo>9were alsofavorable. The ,

ori-entationwas _{mostly restricted}to conformers around -170°

and-40°.

In orderto estimate the role

of

the charge, the _average distance betweenthefour_{oxygen atoms}forthecarboxylgroup

Conformationof_{Meningococcal}B Polysaccharide Biochemistry, Vol. 31, No. 21, 1992 4999

Table III: _{Experimental 2D}NOEVolumes‘ _{with75msof MixingTimeforColominicAcidandCalculated Relative NOE6}

protons exp average' minima <G+> <G-> <T> Gl+ G2+ G3+ H3a-H3e 100 100 100 100 100 100 100 H3a-H8 9 8-11 8 2 32-41 2 28 H3e-H8 5 5-6 5 2 17-23 2 47 H3a-H7 3 3 2 2 4-6 2 74 H3e-H7 3 3 1 1 3 3 60 H5-H8 4 2-4 3 2 12-16 2 5 H3a-H5 24 22 22 22 24 22 30 H3e-H5 12 12 11 11 12-14 11 16 Sdev1' 1-2 2-3 4 10-15 4 34 H8-H6 28 26-33 5-6 7 30-42 26-30 40 H8-H7 30 20-24 27 9 22-31 19-22 31 H3a-H9“ 2 2-19 1-6 1 7-80 1 3 H3e-H9' 1 1-11 1-3 1 4-57 1 4 H7-H9' 11 3-4 6-18 4-19 5 3 4

“_{Estimatederror} = _{±25%. 6TheH3a-H3eNOEissetto 100. ( 7, 8) forG+conformers} = _{(55°,70°), for(G}

> = (60°,-62°),andfor<T) = (67°,179°).

( , )

forGl+= _{(-45°,130°), forG2+}= _{(75°,100°),}for_G3+= _{(180°, 105°). Significant}

changesfor 9 = _{60°,-60°,and 180°} are shown. '

_{( , )}

ensemble averagedNOE. Relativeenergies(in kilocaloriespermole)forwells WG1+, WG2+,andWG3+are 1.2:0:3andforwells

WG1", WG2“ andWG3"are 0.5:0:2. ‘'Standard deviationofthe fitfor six_{mixing times}andforthefirst8volumes_{only. The calculated}NOE

rangeforH9RandH9Sisalsoincluded.

5

figure 3: Partial NMRspectraof colominicacid at 600MHz and343K: (a)spinsimulated spectrum and (b) resolutionenhanced

spectrum.

less favored in solution due to the close _{proximity of} the charged groups betweenadjacentresidues,whileforall the gauche conformers the carboxyl groups are further _apart.

Colominic Acid. Colominic acid, which has a _proton

spectrum identical tothat

of

thegroupBmeningococcal po-lysaccharide (data not shown), was used since

it

was more

readily available. Thechoiceofcounterion, thepH,andthe molecular weightfractionofthe polymerare _importantfactors whichwere consideredin order to_geta _sampleas

homoge-neous as_{possible and}a _{stable sample}which did not degrade

with time. The high molecular fraction of commercially available colominic acid (Na+ salt) was _{selected by gel}

fil-tration. ThepH was _{adjusted to} 7. Different_sampleswere

preparedwhichalways gave thesame _{spectra(Michon}et al., 1987). Especially, the H8andH9resonances were resolved which permittedthe detectionof

NMR

parameterswhichwere

crucialinassessingthesolutionconformationofcolominic acid. In order to determine more _{accurately the} vicinal _proton

couplingconstants forcolominicacid, the spectrumwas

obtained at343

K

(Figure3).

All

multipletscouldberesolved and_{correctlyspin}simulatedto _{obtain accurate parameters.} The proton coupling constants did not changewithtemperature (Lindonet al., 1984). Proton couplingconstantswere found to bethesame asthoseforresidue b

of

thetrisaccharideat 300K(Michonet al., 1987), exceptfor

_/(H8,H9')

whichwas

5.5 Hzinstead of6.9 Hz.

The 2DNOEexperimentwitha_{mixing time}of75_{ms, at}

600MHz,and 288K, forcolominic acidisshownin Figure

4. Thevolumesthatcouldbe_accuratelymeasuredare _given

7

figure 4: 2DNOE_{spectrum at 600}MHzand 288K ofcolominic

acid. The_{mixing time}was 75ms. The IDspectrumisshownon

top along the

fl

axis.

in Table

III,

and some are _{plotted in Figure} 5.

All

NOE

calculations were done _{using the coordinates} for the di-saccharide(b-a). Adimercan be usedtosimulate theNOE’s

becausethe mostfavorable conformers formextended helices

when_{propagated (Figure}2)with pitches(nh) greater than

5 Á. Hence, long-range interactions between nonadjacent residues

will

notbe_{important. For colominic}acid, theNOE betweentwo protonswas taken asthesum

of

theinter-and intraresidue NOE ofthe equivalent pair. Forexample, the

H3a-H7 NOEwas thesum ofthe ¿H3a-¿>H7NOEandthe

¿>H3a-aH7 NOE. Forsome conformers, theinter- and in-traresiduecontributions were _comparable.

TheNOE’s for

H3a-H5

and H3e-H5are dominated by

theintraresidueNOEonly. Fromthe2DNOEbuilduprates, using the distance extrapolation approximation (Table

IV),

theH3a-H5 andH3e-H5distanceswere foundto be2.5and

Biochemistry,

figure 5: 2DNOE_{buildup rate for colominic}acid. _{The experimental}

volumesfor_(H3a-H3e)/2

(·),

H3a-H5

_{( ),}

H3e-H5

_{( ),}

H3a-H8

(A), H3e-H8

( ),

H5-H8 (O),H3a-H7 (X),andH3e-H7(+)are shown. The

_{( , )}

ensembleaveraged theoreticalNOE builduprates (solid lines) using thepotentialenergymapfor 7 =

55°, 8 = _70°

(Figure 2A) withan_{altered relative energy}forwells WG1+, WG2+, andWG3+of1.2:0:3_kcal/moland the 9valuewhichgavethebest

fitare also_plotted.

TableIV: DistancesforaNeuAcObtained from Minimized

Coordinates, from Distance Extrapolation0ofCalculated 2DNOE

Using ConformerGl+,andfromthe_{Experimental 2D NOE6 buildup}

RateforColominicAcid

NOE

protons coordinates caled exptl

H3a-H3e 1.79

H3a-H5 2.51 2.5 2.5

H3e-H5 3.74 3.3 3.3

°a0 fromfit of y =

a0+ axx+ a2x2,where (y/r0)6 =

V/VQ, V0 =

H3a-H3evolume,r0= _{1.79A, V}=

cross-peakvolume, andx = mix-ing timesof25, 50, 75, 100, 150, and 200ms. ‘Estimatederror = _0.2

in_agreementwiththe distanceof2.51

Á

calculatedfromthe coordinates ofTable I,adistanceof3.74

Á

is_expected for H3e-H5. However,

if

the_buildupratesare calculated_using thecoordinatesofconformer

Gl+

andtheH3e-H5distance extrapolated from thecalculated volumes, avalue

of

3.3

Á

isobtained. Thereason for this discrepancy is that_higher

order effectsare _neglectedinthe distance extrapolation method (Landy

&

Rao, 1989). Hence,inorder to accountforcorrect summation of the inter- and intraresidue NOE’s and the complete spin geometry,afullmatrixanalysiswas _alwaysused.

TheNOEalso depends on allpairson _interprotondistances

and thuson

,

,

_{7, 8,}and 9. For motionalaveraging, the

NOEmustbe_averagedina_{certain way depending}ontherates ofinternal motion (Kessler etal., 1988). Inthiscase_only

_(r6)

averagingwas considered, since the modeofaveraging

will

notaffect anyoftheconclusions reachedin this study. The r"6 terms for a conformer were _{weighted according to the}

Boltzmann distribution function.

TheNOE’swere _{analyzed using}amodel wherethe molecule

was allowed to_{sampleenergetically favored conformers}over

( , )

with the ₈ and 9 rotamers _{adopting staggered}

con-formers_{only. The 7 angle}was _kept fixedtothe_g+rotamer. Since,ingeneral,NOE’s thatwere _{highlydependent}of and

were _{not greatly affected by the}valueof 9, thepotential energy map where 9 isallowed tofreelyrotate couldbeused

to calculatethe

_{( , )}

ensemble averagedNOE for different

values

of

<u9. TheNOE’s for H3a-H8, H3e-H8, H3a-H7,

H3e-H7,andH5-H8 satisfied this conditionandthuscould

be usedtoassessthe

_{( , )}

linkage conformation. The NOE’s

between

H3a-H9

andH3e-H9were notused because_they

were _{highlydependent}on 9, and theH9assignment toeither

figure 6: NOEconstraints mapofcolominic acidforamixing time of75ms. Thecalculatedrelative NOE’susing thefullrelaxation matrix, whichsatisfy theexperimental NOE’s (Table

III)

withan error boundof±25%are _plottedas afunctionof

_{( , ),}

with 7 =

55°, 8 = _{70°. The}NOE_{constraintsfor}H3a-H8 _(-),H3e-H8

(|), H3a-H7_(/),H3e-H7_(\),andH5-H8(O) relative toH3a-H3eare

displayed. The mapsfor 9 = _{60°,-60°,and 180°areoverlaid. The}

8_kcal/mollevelswhichdefine the threemajorenergy wellsWG1+, WG2 ,and WG3+(lefttoright) around theGl+,G2+, and G3+ confomers(Table

II)

are also shown.

H9Ror H9Swas _{ambiguous. Still,}thesecalculatedNOE’s for differentvalues of<o9 were within the range of the

ex-perimentalones. _{Conversely, the}NOE’s_{for H8-H6, H8-H7,} and

H7-H9

couldbeusedto determinetheorientationofthe exocyclic chainsince_theywere not_{highlydependent}on

( , ).

Theconformationofthe exocyclic chainwas determined from_{the proton}vicinal_{coupling constants}whichare_dependent on thetorsionalangle

H-C-C-H

andfromNOE’s. Froma

spinsimulationofthe spectrum at343 _K,

_/(H6,H7)

and

/(H7,H8)

were found to be 1.0 and 2.3 _{Hz, respectively}

(Figure 3). Also, the strongNOE for

H8-H6

and

H5-H7

(Figure4), indicatedthatthe ₇ = _60°and

8 = _{60° rotamers}

were _{predominant in solution. For}the_{hydroxymethyl group} at C9,

/(H8,H9)

and

_/(H8,H9')

were 4.5and 5.5Hz,

indi-catingthatthestaggeredrotamersfor 9 were allaccessible. These resultsare similarto theones obtainedforthe

corre-spondinglinkage inthetrisaccharide (Michonetal., 1987). However, an NOE between the H7 and the H9 resonance

indicated thatthe other rotamers for 8 were _{also present.}

Most probably,the_{g" rotamer} was _{present due}to the small

/(H8,H7)

= _2.3Hz_{andthe large}

H8-H7

_NOE,whichcan

arisefrom both_{gauche rotamers (Table}

_III).

FiveNOE’s couldbe used to evaluate the

_{( , )}

_linkage conformation,namely thoseforH3a-H8, H3e-H8, H3a-H7,

H3e-H7, and

H5-H8.

AnNOE_{constraints map}was

cal-culated to determine

if

allsetsofNOE’scan _satisfya_unique

conformer. TheNOEwas calculatedforeach_gridpointand

if

itsvalue fallswithinerror boundsofthe experimentalNOE, it was indicatedon themap. Forg+rotamers of 7 and 8, three_mapswere calculatedfor_{staggered rotamers} of 9 and overlaid. No_{unique energetically favorable conformer satisfied}

the_{five experimental}NOEconstraints (Figure6). Similar resultswere obtainedfortheotherstaggered rotamer of 8. The experimental NOE’swere _successfullysimulatedusing motionalaveragingover

( , )

_(Table

III,

_Figure5). Forthe

Conformation of_{Meningococcal} B _{Polysaccharide Biochemistry, Vol. 31,} No. 21, 1992 5001

Table V: Experimental ID_{NOE" for aNeuAc(2-8)«NeuAc(2-8)/3NeuAc (c-b-a)}andCalculated Relative NOE4

protonssat.obsd exptl

average' minima (G+> <G> <T> Gl+ G2+ G3+ cH3a-cH3e 100 100 100 100 100 100 100 cH3a-6H8 16 10-17 14 3 39-44 1 19-21 cH3a-6H6 3 2 1 4 6 1 12 cH3a-cH4 31 28 27 28 28 27 33 cH3a-cH5 53 55 57 59 51-54 56 78 cH3a-Z>H7 _d _{3 2 2 5 2 80} cH3a-cH6 6 6 4-7 8 6 6 8 cH3a-cH7 3 4 3 3 4 4 6 cH3e-cH3a 100 100 100 100 100 100 100 cH3e-6H8 7 4-7 8 5 13-16 1 58-65 cH3e-6H6 4 1 1 6 2 1 22 cH3e-cH4 46 55-64 54 52 63-102 51 66 cH3e-cH5 21 22 21 22 20-26 21 23 cH3e-6H7 ud ₄ 1 2 3 5 55 cH3e-cH6 7 6-8 4-8 8 7-12 6 8 cH3e-cH7 -e _{2 2 2 4 2 3} SdeV 4-6 4 5 9-19 6 40 6H8-6H7 100 100 100 100 100 100 100 6H8-6H6 114 102-106 12-23 71-87 108-122 104 240 6H8-6H3a 21 16-19 12-14 6 33-64 2 43 ¿>H8-6H3e 14 7 6-8 8 12-21 2 130 6H8-6H5 12 8 5 37-48 3 13

“Estimatederror = _{±25%. 4ThereferenceNOE}issetto 100. _{( 7, 8) for}G+conformers = _{(55°,70°), for<G)} =

(60°,-62°),andfor (T) =

(67°,179°).

( , )

forGl+= _{(-45°,130ß), forG2+}= _{(75°,100°),andforG3+}= _{(180°,105°). Significant}

changesfor , = _{60°,-60°,and 180»}

areshown. '

_{( , )}

ensemble averagedNOE. Relativeenergies(in kilocaloriespermole) forwellsWG1+, WG2+,andWG3+are 0.4:0:3andforwells

WG1", WG2",are 0.5:0:2. ‘'ResonancescH5and6H7overlap. ‘AbsoluteNOE<1 ± 1. ^ForcH3aandcH3e_{NOE’s only.}

7 = _{55°, 8} = _{70° energy surface, threemajorwells,WG1+,}

WG2+,andWG3+, whichencompassconformers

Gl+,

G2+, and G3+can be_{defined by the}8_kcal/mollevelwithrespect tothe_globalminimumG2+ _(Figure2A).

With

therelative energy for minima

Gl+,

G2+, and G3+ of 3:0:3 _kcal/mol obtainedfrom_{the potential energy calculations, the relative}

populationofwellWG1+was _99%,whiletheotherwells had

lessthan 1%_occupancy. The

_{( , )}

_{ensemble averaged}NOE’s calculated using this distribution were the same as those calculated using the singleminimum conformerG2+, sincefor allconformerswithinwell WG2+, interglycosidic NOE’swere

small, compared tothoseforconformer

Gl+

or G3+_(Table

III).

Hence, poor agreementwas obtainedwith_the

experi-mental values. _However,

it

was observed that the NOE’s

showed_very

little

_dependenceon andthatinterglycosidic

NOE’s had maxima near conformers

Gl+

and G3+. For

conformer

Gl+,

H3a interglycosidicNOE’sare _{bigger than}

those

of

H3e, whilefor conformerG3+ the reserve is true. Hence,a_slightincreasein_{the population}ofwellWG1+,would leadto_{goodagreement between}the_averagedNOE’sand the experimental data. The occupancyofwellWG3+mustremain low,orthe_{relative magnitude}oftheH3a-H3eNOE’s would change.

A

relativeenergy between wellsWG1+,WG2+,and WG3+ of 1.2:0:3 _{kcal/mol, equivalent to} a _population of

13:86:1, was found togive the best

fit.

Only the ratio for

WG1+andWG2+was _{altered, the}one for WG2+andWG3+ being kept fixed as that found from _{the potential energy}

calculations. The

_{( , )}

ensemble averaged NOE’s were

calculatedforthe three_{staggered 9 rotamers}andexhibited

no _{majordependency}on _{9, except those}involvingH9(Table

III).

Sinceother rotamersfor ₈ can be_present,

( , )

ensemble averagedNOEcalculationswere alsodonefor allstaggered 8 rotamers. Forthe g"rotamer,energy wellssimilar tothose fortheg+rotamerwere observed, anda_good

fit

ofall NOE’s, exceptfor

H8-H6,

couldbe_{obtained (Table}

_III).

Distinct

wells WG1-, WG2-, and WG3“ could be _{defined by the} 6

kcal/mollevelwithrespecttoconformerG2" (Figure2B). The relativeenergyofwellWG1"hadtobemodifiedfora _good

figure 7: ID NOEdifference spectraof

aNeuAc(2-8)aNeuAc(2-8)/3NeuAc(c-b-a)for saturationofH3aofresidueaandc(a),of

H3eofresiduec,andofH8 andH9ofresidueb_(c)are _{shown along}

withthe ID spectrum(d).

fit.

The relative energy

of

well WG3" was not altered. Relative energiesforwellsWG1",WG2“,andWG3™of0.5:0:2

kcal/molequivalent toa _populationof8:90:2 gavethe best agreement between the averagedNOE’sandthe_experimental data. _{Poor agreement}was foundforthe trans_{rotamer (Figure} 2C),sinceno _{energetically}favored conformersnear = -50°

were _{present where stronginterglycosidic NOE’s}withthetwo

H3 _geminalresonances occurred.

Trisaccharide.

NMR

experimentswere _performedon the trisaccharide in order to compare the linkage conformation

of_{the polymer to the}oneof_{the linkage}c-bofthetrisaccharide

aNeuAc_{(2-8) aN}eu_Ac(2-8)0Neu_Ac,(c-b-a). The

ID

NOE

spectra for the trisaccharide at 600 MHz and 288 K are

plotted inFigure7,and theNOEdataare _givenin Table V. Byperforming the experimentsat a_{lower temperature,} the correlation time

of

the molecule was _decreased, leading to bigger negative NOE’s, which couldbemore _accurately

in-tegrated. The chemicalshiftassignments andcoupling

con-stants were similartothose_{reported before} (Michonet al., 1987).

Asfor colominicacid,a_strongNOEbetween¿>H8andZ>H6,

Biochemistry,

the 7 = _{60° and 8} = _{60° rotamerswere predominant in}

solution. Due to_partialoverlapof6H9and6H8resonances,

the6H9resonance could notbe_selectivelysaturatedand the

smallNOEbetween H9 andH7, which might indicate the presence of other 8 rotamers, could not be evaluated, as observed forcolominic acid.

Four sets of NOE’s could be used to evaluate the

_{( , )}

linkage conformation distribution, namely interglycosidic

NOE’s between cH3a-6H8, cH3e-6H8,

cH3a-iH6,

and

cH3e-6H6. AlthoughthecH3a-6H7andcH3e-6H7 NOE’s couldbe observed,the overlapofthe 6H7resonance withthe

6H5resonance _{preventedtheir quantification. The}¿>H8-cH5

NOEwas detected, but its magnitude

(<1)

and the _noisy baseline prevented its accurate determination.

All

NOEcalculationswere _{done using}the coordinates

of

the disaccharideo¡NeuAc(2-8)aNeuAc, whichcorrespondto residuesc-b ofthe_{trisaccharide (Table}V). TheNOE

con-straints_mapswere similar totheones for colominicacid, and they did not showthe_presenceofa_{preferred conformer in}

solution (data notshown). However, the

( , )

ensemble

av-eragedNOE’s,usingmodifiedenergy surfacesforthe gauche rotamers of 8, were able _{to reproduce adequately the}

ex-perimental NOE’s. Relativeenergiesfor WG1+, WG2+, and WG3+ of 0.4:0:3 _{kcal/mol, equivalent} to a _population of

20:79:1, were found toincrease_{the magnitude}ofthe

inter-glycosidicNOE’scomparable tothose

of

the observedones.

The

_{( , )}

ensemble averagedNOEcalculationsfor

_g'

andthe transrotamers_{of 8}were performedusing thesame _potential

energy surfaces asforcolominicacid.

Discussion

The conformation

of

colominic acid has been _previously discussed by a number

of

authors, and all have relied on

interpretation

of

NMR

results. Lindonet al. (1984) deter-mined,from

NMR

at 360_MHz,thatH7andH8were_gauche

andmolecularmechanicscalculationswere usedtoassessthe

rotational barriers about the C7-C8 bond.

All

staggered rotamerswere found tobeaccessiblewithbarriersofabout

10_{kcal/mol. The}13C

NMR

_{spin-lattice relaxation times,}were

interpreted as _suggesting that the

_a(2-8)NeuAc

poly-saccharidetumbled isotropically in solutionasa_{rigidspecies.}

Michonetal.(1987) studied differencesin NOE’sat 500MHz between _{the polysaccharide} and trisaccharide and from a

qualitative interpretationoftheNOE’sonly,

it

was _proposed

that_{the polysaccharide adopted}adifferent conformation from the _{trisaccharide. Recently, Yamasaki} and Bacon _(1991), froma_{quantitativeanalysis}of2DNOE’sat 500

MHz

using

rigid conformers, suggested that the polysaccharide adopts orderedhelical structures in solution.

Inour _study,boththe results and_{their interpretation}differ

fromthoseofYamasaki and Bacon (1991). Differentchemical

shiftsforthe_polymerwere observed,whichcould possiblybe

attributed to sample preparation. While the H8 and the

downfieldH9resonance _overlappedfor theirsample,theywere

resolvedinthis study. Thus, the overlapoftheH8 andH9

resonances _{prevented these}authorsfromdetermining theC8 stereochemistry. Usingrigidmodels and steric constraints, they proposed ordered helical structuresforthe_following

_{( , )}

ranges:

(-60°

to 0°, 115° to _175°) for 8 = _{60°and (90°}

to 120°, 55° to _{175°) for 8} =

-60°.

_Their

( , )

ranges correspond to thetwoareasintheNOEmap(Figure6), where

some NOEconstraints intersect the most. Frommodels,they estimated the numberofturns _perhelix,n, tobe from 3 to

4 and _{the pitch (nh) to} be from 9 to 11 Á. Their helical parameters were overestimated_by a factor of2, since it is difficultfrommolecular models alone to determine therotation

angle(µ) along thehelixaxis betweentworesidues,from which

=

_{2 /µ.}

_Valuesof_{ngreater than2}for ₈ = _{60° and also}

n_{greater than}2_{for 8} = -60° _{correspond totheir}

( , )

_ranges

(Figure 2).

Inthis study,NOEdataat600

MHz

for_{the polysaccharide} andthe trisaccharidewere _{interpretedbyinvokingflexibility}

about the exocyclic linkages. Thebest

fit

oftheNOEdata

forcolominic acid (Table

III,

Figure 5)was obtainedfrom

a

( , )

_{ensemble average} over the whole _{energy surface.} Proton couplingconstants andNOEdataindicatedthatthe

g+ rotamers for o>7 and 8 were predominant in solution.

However,flexibilityhad tobeinvoked about theC7-C8bond due toNOE’s _{which could only}arisefrom other rotamers, most probably the g"one. From the determination of the proton couplingconstantsfortheH9resonances,allstaggered conformersforthehydroxymethylgroup at C9were_postulated

tobe accessible. Hence, dueto the widerange

of

conforma-tionswhichcan be_{sampled,the polysaccharide}is_suggested

to exist predominantly as a random coilin solution. As Perez and_{Vergelati (1985)}notedintheirstudyof po-lysaccharidehelicalstructures, the spatialorganizationofthe polymerchaincan be_{defined by consecutivefragments which}

havewell-definedhelicalparameters. Theselocalhelicescan

alsobereferredtoa_{pseudohelices. Compared to other} hom-opolysaccharides,a(2-8)-linkedsialicacid ranksfirstinterms

of

_displaying awide range

of

helical structures (Figure 2). Obviously, thepresenceoffourbondsinthe linkage junction amplifiesthenumberofallowedconformationalstates,aswell

asthe numberofhelicaltypeswhichcan be_generated. The helical_parametersfora_{few low-energy conformers}are _given

inTable

II.

Threehelicesforthe_g+rotamers

of

a>7and 8

are shown_{in Figure}8. Wheneverthevalues h = _{0 or} =

2 are crossed,thechirality ofthehelix_changes. The = ₂

helix_correspondstoa_{zigzagplanarconfiguration, while}h =

0 correspondsto cyclic oligomers. Conformers withsmall values

of

h would foldbackon themselves after nresidues. For the 8 =

g+ rotamer, most _{energetically favored}

con-formerscouldformlocalhelices, since the h= ₀line_crosses

a _regionwhere values

of

nwere _{greater than}4 _(Figure2A).

Also, high-orderhelicescan form with > _6,while forthe other ₈ rotamersnisfrom2to3 _{only. For theg+}rotamer

ofo>8inthe high-order helical region,asmall changeinlinkage

conformationleadstoa_largechangein helical_parameters. Hence, the topological featuresofthe polymercanchangewith

little

changein energy. Suchproperties wouldbefavorable to topological rearrangements fromdisordered randomcoils tomore ordered_{conformations which promote}theformation

ofthehelical_epitope.

Fora_{polysaccharide,}theNOE’s

will

be_averagedover all

the conformerssampledin solution.

If

a _predominant

con-formeror a_{pseudohelix existsin highenoughproportion, this}

shouldbereflectedintheNOE’s,andaNOE_{constraints map}

could detectthe presence

of

a_{preferred conformer.}

If

more

thanone _{conformer satisfied the constraints,}or no_preferred

conformerwas _{found, then thepresence}ofmolecular_flexibility mustbeinvoked_{to explain}the observedNOE’s. Even

if

the NOE’s indicatethepresenceofa_preferredconformerandthe motionalaveragingcalculationsstillsatisfytheexperimental conditions, motional

flexibility

cannot beruled out.

For thecase ofcolominic acidandthe trisaccharide, the

NOE’sare bestexplained by motional averaging. A small proportionofconformerswas found to_{greatly influence the} averaged NOE’s, since their NOE’swere of_{much greater}

magnitude than themajority

of

conformers,as can beseen

con-Conformation of_{Meningococcal}B Polysaccharide Biochemistry, Vol. 31,No. 21, 1992 5003

figure 8: _{Side and top view}with_{respect to the}helixaxisforhelicesofa(2-8)NeuAc10 which

G2+, and G3+. The linkage conformationhavebeen_{slightlyaltered to giveintegral}helicesofr

for poly(A)isshownon _top. The_{charged groups}inthe moleculesare _representedas _spheres.

formersGl+and G2+ (Tables

III

andV). Therelative_energy

of

_{the potential} energy wells had to be altered in order to reproducethe observedNOE’s. Empiricalpotentials, which neglect molecularflexibility,electronic interactions, and solvent effect, are used _{only tocrudely estimate}theallowable

con-formationalspace. Therigid potential wouldalsooverestimate barrierstointerconversion. TheNOEdatacan thusbeused

totestdifferent population distributions calculatedfrom

em-pirical potentialenergy functions(Gumming

&

Carver, 1987). For colominic acid a _{relative population} between wells WG1+ and WG2+ of 15:100 was _{found to reproduce} the NOE’s correctly. Forthetrisaccharide,a ratioof25:100for

thesame wellswas needed. The_{relative population}

of

well WG3+ to well WG2+ must remainlow_(1-2%) in bothcases

or theHe3-H8wouldbecome_{larger than}theH3a-H8 NOE.

Hence, differenceinlinkage conformation between thepolymer and_{short oligosaccharides}can be_explainedas_arisingfrom

slightchangesintherelative populationdistributionof

con-formersinsolution,as_{opposedto different rigid}conformations

(Michonet al., 1987).

Theabovemodelfortheconformational behaviorof

colo-minicacid in solutionhas_{important biological implications.} The cross-reactivityofIgMNOVwithboth thea(2-8)NeuAc

polymerand _poly(A)can now berationalized(Kabatet al., 1988), sincethey bothcan adoptsimilarhelices _(Figure2). From the crystal structure of _{trinucleoside diphosphate} of

adenosine,amodelforthe_poly(A) helixwas _proposedwith

= _9andh = _2.82

Á

(Saenger et al., 1975). Energetically preferredconformers due tofavorablebasestackingcan form

helicesfrom = _{8to12(Yathindra} &_{Sundaralingam, 1976).}

Therefore, helicesforthe_a(2-8)NeuAcpolymerandpoly(A)

can _easilybe_generatedwhichhavesimilar helical_parameters. The = _9helixfor_{both polymersisshow}in Figure_8. There

isnot _onlya _similarityin the spatialdistributionofcharges betweenthe_{carboxylgroups}of a(2-8)-linkedsialicacid and the phosphate groupof poly(A) butalsoa_strikingresemblance

betweenthe_{helical parameters} of_{both polymers (Figure2).}

Thisobservationwouldtendtosuggestthattheconformational

epitopeoccurs withinone turn

of

thehelix

of

a(2-8)-linked

sialic acidor_poly(A). Since_{the energetically preferred helices}

of poly(A) have more than 8 _{residues per}helical_turn,

it

is

proposedthattheconformationalepitopeforthe<x(2-8)NeuAc

polymerprobablyoccurs withina_{high-order helix which} has an extended_{conformation (Figure8).} However, there isso

farno indicationoftheexactnumberofresidueswithinthis helix _{that actually bind to group} B polysaccharide specific antibodies.

Theunusual_{length requirementof a(2-8)-linked}sialicacid oligosaccharides to bind to group B _{meningococcal} poly-saccharide_{specific antibodies}can alsoberationalized. Jen-nings et al. (1985) and Finne andMakela(1985)observedthat at leastadecasaccharidewas neededtoinhibitor bind tothese

antibodies. Also,for_{large oligomers}

of

up to 17sialic acid

residues,theinhibitorypropertiesdidnotmaximizebutsteadily

progressedwithincreasingsize. _Hayrinenetal._{(1989) showed}

thatthecriticalchain lengthforbindingwas 10units which contributed90%ofthe_{bindingenergy. Thebinding forlonger} oligomersalso_{steadily progressed}with_increasingsizebutwith minor contributions tothe_{bindingenergy}fromtheadditional

residues. Becauseofthelimitationsofthesizeofan _antibody

site(maximum6 or 7_{residues) (Kabat, 1960), these}

obser-vations do_{not imply that}the_antibodywas _bindingtoas_many as 10 or more consecutive residues, rather that at least 10

residuesare _requiredfor_{the recognition}site toform.

If

the conformational_{epitope corresponds to}a_{high-order helix with} an extendedhelical conformation, as _suggested from

cross-reactivityofIgMNOVtopoly(A),large oligosaccharideswould

be _{required in order} for the epitope to form. Since poly-saccharides have_{to undergo}topologicalrearrangementsfrom disordered randomcoils tomore ordered conformations fa-vorable to theformation

of

the_{helical epitope}and because

Biochemistry,

unlikely that the _epitope would occur _{in any short}

oligo-saccharidesequence. Inadditionforthe decasaccharide, the

conformational behaviors

of

_{the reducing}and_nonreducing residuesare _expected tobedifferent from that ofthe inner residues,asobservedforthetrisaccharideandcolominicacid (Michonet al., 1987). Thecontinued increasein bindingabove 10units couldbeattributed tothe_{length stabilization}ofthe epitope, i.e.,theincreasedprobability

of

the presenceofthe epitope.

The poorimmunogenicityofthe groupBpolysaccharide probably arises from the recognition ofa helical structure

whichisalsoan_{integralcomponent}oftheneural cell adhesion molecule

N-CAM

(Finneet al., 1983). Despitestructural

mimicry withmammaliantissue,antibodies specificforthe

a(2-8)-linkedsialic acid homopolymercan be_producedand from_{the examples wherebinding}studies havebeen_reported

(Jennings et al., 1985; Finne

&

Makela, 1985;Hayrinen et· al., 1989;Kabatet al., 1988) allare _specificforthe extended helicalepitope. Becauseour conformationalstudiesindicate thatthis epitopeis_onlyaminor contributorto thetotalnumber

of _epitopes available, we must therefore presume that the dominanceof_{this epitope}intheimmune_responsemustbethe resultofimmunologicalselection. Thefailureoftheimmune systemtoproduceantibodies specificforthemore _populous

lower_{order helices present}in the_{polysaccharide}randomcoil probably occurs because these shorthelices are

conforma-tionallysimilarto theones_{found in shorter sialyloligomers.}

Theseshort_{a(2-8)NeuAcoligosaccharides}are _{also present}

in humantissue_(Finneet al., 1983), andthe production of

specificantibodyto_{them appears to}beeven more _stringently

avoided.

Acknowledgments

We thank FrancisMichonforcriticalreview ofthe

man-uscriptandRobertPonforhelp insamplepreparations. We

are _grateful to _{Andy Byrd} and

Bill

_Eagan for access to a

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