1995 j exp med. sag binding to a tcrb chain of known three dimensional sturcture

8/2/2019 1995 J Exp Med. SAg Binding to a TCRb Chain of Known Three Dimensional Sturcture

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Superantigen Binding to aT Cell Receptor R Chain ofKnownThree-dimensional StructureBy Emilio L . Malchiodi,* Edward Eisenstein, * $ BarryA Fields,*DouglasHOhlendorf PatrickMSchlievert,11 Klaus Karjalainen,l andRoyA Mariuzza*

From t h e * CenterforAdvanced Research i n Biotechnology, University of Maryland BiotechnologyI n s t i t u t e , Rockville, Maryland 20850 $Department of Chemistry an d Biochemistry, University ofMaryland, Baltimore County, Maryland 21228 Department of Biochemistry and DDepartment ofMicrobiology, University of Minnesota Medical School, Minneapolis, Minnesota 55455 and'Basel I n s t i t u t e for Immunology, Postfach CH-4005, Basel, Sudtzerland

SummaryThe three-dimensional s t r u c t u r e of an unglycosylated T c e l l antigen receptor (TCR) R chainh as recently been determined t o 1 . 7 A resolution . To i n v e s t i g a t e whether t h i s s o l u b l e R chain(murine V(38 .2JR2 .IC(31) r e t a i n s superantigen (SAG)-binding a c t i v i t y , we measured i t s a f f i n i t yfor various b a c t e r i a l SAGS in the absence of MHC c l a s s I I molecules . Dissociation c o n s t a n t s(KD s ) were determined using two independent techniques : s u r f a c e plasmon resonance detec-t i o n an d sedimentation equilibrium. S p e c i f i c b i nd in g was demonstrated to staphylococcal en -terotoxins ( S E s ) B, C1 , C2 , and C3 and to streptococcal pyrogenic exotoxin A(SPEA), c o n s i s -tent with th e known p r o l i f e r a t i v e e f f e c t s of t h e s e SAGs onTc e l l s expressing V(38 . 2 . In c o n t r a s t ,SEA, which does not s t i m u l a t e V(38 .2-bearing c e l l s , does not bind th e recombinant R chain .Binding of th e R chain t o SAGs wa s characterized by extremely f a s t d i s s o c i a t i o n r a t e s (>0. 1s - 1 ) , s i m i l a r to those reported for c e r t a i n leukocyte adhesion molecules . Whereas th e R chainbound SEC1, 2 , an d 3 with KDs of 0 .9-2 . 5 [LM, th e corresponding value f o r SEB was ^140p,M . The much weaker binding t o SEB than t o SEC1, 2 , or 3 wa s s u r p r i s i n g , e s pe c ia l l y s i n c eSEB wa s found to a c t u a l l y be 3- to 10-fold more e f f e c t i v e , on a molar b a s i s , t han t he other tox-i n s in stimulating th e p a r e n t a l T c e l l hybridoma . We i n t e r p r e t these r e s u l t s i n terms of th e a b i l -i t y of SEC to a c t i v a t e T c e l l s independentl y ofMHC, in contrast to SEB. We ha v e a l s o mea-sured SE binding to th e glycosylated form of th e R chain an d found t h a t carbohydrate apparentlyd oe s n ot contribute to recognition, even thoug h th e N-linked glycosylation s i t e s a t V(38 . 2 r e s-idues Asn24 an d Asn74 a r e a t or near th e putative SAG-bindin g s i t e . This r e s u l t , along with th es t r u c t u r a l b a s i s for the V( 3 s p e c i f i c i t y of SEs, are discussed i n r e l a t i o n to th e c r y s t a l s t r u c t u r e ofth e unglycosylated R chain .

Atigen recognition by T lymphocytes i s mediated byhighly diverse c e l l s u r f a c e glycoproteins known a s T

c e l l receptors (TCRs)t . In addition t o recognizing peptideantigens complexed with products of th e MHC complex,TCRs i n t e r a c t with a c l a s s of molecules known a s superan-t i g e n s (SAGs), which stimulate T c e l l s bearing p a r t i c u l a r V( 3

' A b br e v ia t i on s u s e d in t h i s paper : APC, a n t i g e n - p r e s e n t i n g c e l l ; RCHO,g l y c o s y l a t e d 1 3 c h a i n ; ( 3 mu t , u n g l y co s y l a t ed 1 3 c h a i n ; HBS, Hepes-buffe r e d s a l i n e ; K, d i s s o c i a t i o n c o n s t a n t ; Ms, minor l y m p h o c y t e s t i m u l a t i n ga n t i g e n ; r , c o r r e l a t i o n c o e f f i c i e n t ; RU, r e s p o n s e u n i t ; SAG, s u p e r a n t i g e n ;SEA, s t a p h y l o c o c c a l e n t e r o t o x i n A SEB, s t a p h y l o c o c c a l e n t e r o t o x i n B;SEC, s t a p h y l o c o c c a l e n t e r o t o x i n C S EE, s t a p h y l o c o c c a l e n t e r o t o x i n E ;SPEA, s t r e p t o c o c o c c a l p y r o ge n ic e x o to x i n A ; TCR, T c e l l a n t i g e n r e -c e p t o r

r e g i o n s , l a r g e l y i r r e s p e c t i v e of th e peptide/MHC s p e c i f i c -i t y of th e TCR ( 1 - 3 ) . SAGs include s e l f a n t i g e n s , such a sth e minor lymphocyte stimulating antigens ( M l s ) encodedby endogenous murine r e t r o v i r u s e s , a s well a s foreign a n t i -g e n s , such a s staphylococcal an d streptococcal pyrogenictoxins .T c e l l stimulation by SAGs i s generally thought t o r e -q uire t he p a r t i c i p a t i o n of MHC c l a s s 1 1 molecules ( 4 - 8 ) ,which bind some b a c t e r i a l SAGs with a f f i n i t i e s in th e mi -cromolar range (9-12) . Most current models assume t h a tSAGs a c t i v a t e Tc e l l s b y simultaneously binding c l a s s l I mol-e c u l e s on APC an d th e VR element on T c e l l s (1-3, 1 3 ) .Recently, however, through th e use of c l a s s 1 1 knockoutmice, staphylococcal enterotoxin C (SEC) an d staphylococ-c a l enterotoxin E (SEE) h av e b e en shown t o a c t i v a t e Tc e l l s

1833 J . Exp . Med. C Th e R o c k e f e l l e r U n i v e r s i t y P r e s s " 0022-1007/95/12/1833/13 $2 .0 0Volume 182 December 1995 1833-1845


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independently ofMHC(14) . The response resembled theconventional c l a s s 11-associated one i n that T c e l l s express-ing th e same V( 3 gene segment were selectively stimulated .However, T e l l activation by SEC and SEE i n MHC-deficient mice was not followed by deletion ofclones bear-ing th e relevant V( 3 element . Thus, MHC-independent Tc e l l activation may produce certain physiological conse-quences whichare different from those associated withMHC-dependent activation .To investigate th e biochemical basis ofTe l l activation

by SAGS, we have measured th e binding of various bacte-r i a l toxins t o a soluble murineTCRR chain (V(38 . 2 J ( 3 2 . 1C(31) i n th e absence ofMHCl a s s 1 1 molecules As we haverecently determined the three-dimensional structure of t h i s( 3 chain to atomic resolution ( 1 5) , t h i s represents an excel-l e n t system for delineatingTCR-SAGinteractions . We showthat th e reactivity of t h i s R chain toward different toxins i sconsistent with their e f f e c t s on th e parentalTe l l hybridomaWe a l s o find that th e ability ofcertain bacterial SAGs to ac-t i v a t e Te l l s independently ofMHCcorrelates with th erelatively high a f f i n i t y of these particular toxins f or th e i s o -lated ( 3 chain. By measuring th e a f f i n i t y of glycosylated andunglycosylated forms of th e R chain fo r SEs, we have beenable t o directly a s s e s s the contribution of carbohydrate toSAG binding F i n a l l y , th e identification of amino acid r e s i -dues on th e putative SAG-binding surface ofth e VP8. 2 do-main which are conserved i n other V(3 domains with similaror different toxin r e a c t i v i t i e s provides i n s i g h t s into th e struc-t ur a l b a s i s ofTCRrecognition by SAGS .

Materials and MethodsSuperantigens . P u r i f i e d S EA, S EB , SEC1, SEC2 and SEC3 were

purchased from Toxin Technology, Inc . ( S a r a s o t a , FL ) . Strepto-c o c c a l pyrogenic exotoxin A(SPEA) was prepared a s described ( 1 6 )RecombinantTCRProteins . The s o l u b l e a( 3 TCRused i s a c h i -

meric molecule i n which the Vand e x t r a c e l l u l a r C regions of botha and ( 3 chains were fused with immunoglobulin C i c regions( 1 7 ) TheTCR (designated 14 . 3 . d ) i s s p e c i f i c f o r a hemagglutininpeptide of influenza virus (HA 110-120) i n the context of I - E dand u t i l i z e s the Va4.1Ja2134/V(38 . 2 J ( 3 2 . 1 gene combination ( 1 8 )The chimeric protein i s assembled and s e c r e t e d a s a f u n c t i o n a l ,glycosylated VaCUCic/V(3CRCic heterodimer by J558L my-eloma c e l l s t r a n s f e c t e d with t h e recombinant genes ( 1 7 ) . A f f i n i t yp u r i f i c a t i o n using the anti-mouse Cic monoclonal antibody 1 87 . 1( 1 9 ) was c a r r i e d ou t a s described ( 1 7 ) .

Glycosylated 14 . 3 . d V(3C(3 chain ((3CHO) was derived fromthe VaCaCic/V(3C(3Cic heterodimer by treatment with papain,which was found t o s e l e c t i v e l y degrade the a chain a s well a s bothCrc domains ( 2 0 ) . In a t yp ic a l preparation, o a / 3 heterodimer a t aconcentration of2 mg/ml i n 0 . 1 MKP04 , pH 7 . 2 , was digestedf o r 35 min a t 37Cwith papain (Worthington) a t an enzyme/sub-s t r a t e r a t i o of 1 :500 i n the presence of 1 .2 5mMEDTA and 1 . 5mM2-mercaptoethanol . The reaction was terminated by the ad-d i t i o n ofN-ethylmaleimide t o a f i n a l concentration of 1 0 MM

Unglycosylated VRCR chain ((3mut) was obtained by elimina-t i o n of four ou t of f i v e p o t e n t i a l N-linked glycosylation s i t e sthrough s i t e - d i r e c t e d mutagenesis and introduction of a termina-t i o n codon a t the end of the f i r s t C region exon of the ( 3 chain

1834 Superantigen Binding to TCR/3 Chain

( 1 5 ) . Asparagines a t positions 24 , 7 4 , and 12 1 (numbering accord-ing t o reference 21 ) were mutated t o glutamine, while the glyco-s y l a t i o n site a t position 236was eliminated by mutation of Ser238t o v a li n e . TheRmut chain was produced i n J558L c e l l s and a f f i n i t yp u r i f i e d using the anti-mouse C( 3 monoclonal antibody H57-597( 1 5 , 22 ) . The unmutated N-linked glycosylation s i t e a t positionC( 3 1 8 6 wa s used i n


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where c , i s t h e c o n c e n t r a t i o n of t h e p r o t ei n a t a g i ven r a d i a l p o s i -t i o n , c m i s the c o n c e n t r a t i o n of t h e p r o t e i n a t some r e f e r e n c e po -s i t i o n ( e . g . , t h e m e n i s c u s ) , Mi s t h e molecular w e i g h t , v i s t h e p a r -t i a l s p e c i f i c volume, p i s t h e s o l v e n t d e n s i t y , C o i s t he a ng ul a rv e l o c i t y , r i s t h e r a d i a l p o s i t i o n i n c e n t i m e t e r s from t h e c en t e r o fr o t a t i o n , r , i s t h e d i s t a n ce i n c e n t i m e t e r s from t h e c e n t e r of r o t a -t i o n t o t h e m e n i s c u s , R i s t he g as c o n s t a n t , Ti s t h e a b s ol u t e ( K e l v in )t e m p e r a t u r e , and B i s a c o r r e c t i o n term f o r a nonzero b a s e l i n e .

Equilibrium c o n s t a n t s f o r t h e a s s o c i a t i o n of t h e [ 3 c h a i n a n dSAGS were c a l c u l a t e d from t h e d a t a o b t a i n e d a t sedimentatione q u i l i b r i u m a c c o r d i n g t o

where c, , R i s t h e concentration of r e c e p t o r a t t h e r e f e r e n ce p o s i -t i o n , c . , T i s t h e concentration of t h e t o x i n a t t h e r e f e r e n c e p o s i -t i o n , M an dMa r e t h e molecular we ig hts o f t h e Pmut c h a i nan d SAGS a s determined i n s e p ar at e sedimentation e q u i l i b r i u me x p e r i m e n t s , Mc i s the molecular weight o f t h e complex, and KDi s t h e d i s s o c i a t i o n c o n s t a n t f o r t h e v a r i o u s [ 3 chain-SAG i n t e r a c -t i o n s . Parameters were e v a l u at e d u s i ng n o nl i n e ar l e a s t - s q u a r e s a n a l -y s i s ( 2 9 ) , or b y u s i n g a modified v e r s i o n of IGOR (Wavemetrics,Lake Oswego, OR) running o n a Macintosh computer ( 3 0 , 3 1 ) .Data f o r t wo d i f f e r e n t c o n c e n t r a t i o n s or t wo r o t o r s p e e d s werea n a l y z e d s i mu l t an e o us l y a s a t e s t f o r homogeneity a nd r e v e r s i b i l -i t y ( 3 2 ) a n d t o c o n s t r a i n confidence i nt e rv al s f or t h e parameterv a l u e s ( 3 3 ) . E r r o r s o n KD s were -20% of parameter v a l u e s .T C e l l S t i m u l a t i o n Assay . T c e l l s t i m u l a t i o n was monitored b yt h e r e l e a s e o f IL-3 a s p r e v i o u s l y d e s c r i b e d ( 3 4 ) . B r i e f l y , 5 X 10 4i r r a d i a t e d ( 1 0 , 0 0 0 r a d ) A20 APC were preincubated with d i f f e r -e n t c o n c e n t r a t i o n s of SAGS or p o si t iv e c o n tr o l p e p t i d e (HA 110-1 1 9 ) i n 100-N . 1 volumes f o r 2 h i n 96-well p l a t e s . T h e r e a f t e r , 2 X10 4 14 . 3 . d T c e l l hybridoma c e l l s were added i n 100-WI volumesan d s u p e r n a t a n t s were h a r v e s t e d 20 h l a t e r f or a s sa y of I L - 3 a c t i v -i t y . P r o l i f e r a t i o n of t h e IL-3-dependent c e l l l i n e DA-1 wa s mea-s u r e d 26 h l a t e r u s i n g [ 3 H]thymidine . A l l measurements were d o n ei n d u p l i c a t e i n t h e p r e s e n c e of 5% FCS i n IMDMc u l t u r e media .

Results

c T=B+c, R exp[MR ( 1 -vr)to (r2 -rmz )l2RT]+( c m, T) e x p [MT(1 - vr) o) (r2 - rm2) / 2 R T] + ( 2 )

(Cm R ) (CV1,T)/KD e x p [MC ( 1 - v p) co 2 ( r z - rm2) / 2 RT]

Th e B in di ng o f S o l u b l e TCR Components t o SAGS I s Char-a c t e r i z e d b y Rap i d A s s o c i a t i o n a n d D i s s o c i a t i o n Rates . To ana-lyze t he interaction of re c om bi n a n t TCR m ol ec u l es w i thSAGs by B IAcore, SAGS were c o u p le d d i r e c t l y t o t he dex-tran matrix through primary a m i n e g ro up s o f t he p ro t ei ns( 2 7 ) . I n j e c t i o n o f d i f f e r e n t concentrations o f 1 4 . 3 . d o t p TCR,RCHO, a n d Pmut o ver SEC1 g a v e c o n c en t r a ti o n - d epen -dent b i n d i n g ( F i g . 1 , A, B, a n d C, r e sp e ct i v e l y ) t h at c a n b edistinguished f r o m the p r o f i l e obtained when SEA wa s im-mobilized ( F i g . 1 E ) . Results s i m i l a r to those f o r SEC1were obtained when SEC2, SEC3, a n d SPEA were c o u -pled t o the matrix (not shown) . These data a r e in agree-ment with the a b i l i t y o f SEC a n d SPEA, b u t n ot SEA, tostimulate VR8.2-bearing T c e l l s ( 2 ) . S u r p r i s i n g l y , the b i n d -in g to S E B , which i s a l s o known to a c t i v a t e c e l l s expressingV(38 . 2 ( 2 ) , appeared markedly weaker : whereas i n j e c t i o n o f8 RIVI Pmut o ver 4 , 0 0 0 response u n i t s (RU) o f immobi-

1 83 5 Malchiodi e t a l .

l i z e d SEC1 p r o d u c e d a t o t a l response ( s p e c i f i c a n d n o n sp e-c i f i c ; s e e below) of-1,750 RU ( F i g . 1 C ) , i n j e c t i o n o f thesame concentration o f R chain o v er 4 , 00 0 RUo f immobi-l i z e d SEB r e s u l t e d in a response o f only a bo ut 4 00 RU( F i g . 1 F) . S i m i l a r l y , i n j e c t i o n o f 8 u,M otp TCR over SEBp r o d u c e d a response o f only -200 RU (Fig . 1 G) , i n d i c a t -i ng t h a t the weak interaction o f Rmut with t h i s t o xi n i s n o tdue to t h e a bse n ce o f carbohydrate or o f a chain . I n j e c t i o no f 8 N.M 1 9 3 4 . 4 Vo t o ver SEC1 p r o d u c e d a response ( F i g .1 D) s i m i l a r to t h a t observed a f t e r i n j e c t i o n o f Pmut o verSEA ( F i g . 1 E ) .

E f f o r t s to e x am i ne bin d i n g in t he reverse orientation( i . e . , w i t h TCR c o m p o n e n t s on the sensor s u r f a c e a n dSAGS in s o l u t i o n ) were n ot s u c c e s s f u l . In the c a s e o f a(3TCR, a l t ho u g h b i n d i n g o f SEC wa s d e t e c t a b l e , b a s e l i n e d r i f tprecluded q u a n t i t a t i v e a n a l y s i s ; s i m i l a r d i f f i c u l t i e s have beenreported f o r t he b in di ng o f SEB to an immobilized humanTCR ( 1 2 ) . In the case o f (3CHO and (3mut, no bin d i n g o fa n y SAG c o u l d b e detected, implying t h a t immobilizat i o no f the ( 3 chain t hr ou g h a mi n e groups l e d to i t s i n a c t i v a t i o n ,a s described in other systems ( 2 6 ) . These d i f f i c u l t i e s promptedus to measure ( 3 chain-SAG i n t e r a c t i o n s b y sedimentationequilibrium ( s e e below), which does n o t req uire ligand i m-mobilization .When 8 R1VI aR TCR were injected o ver SEC1, SEC2,SEC3, a n d SPEA, the response occurred in two d i s t i n c tphases : a f as t i ni ti al increase o f -800 RU, followed by amuch slower i n c r e a s e o f -300 RU ( F i g . 1 A, f o r the c a s eo f SEC1) . When buffer f l o w wa s r e e s t a b l i s h e d , the r e -sponse dr o p pe d in two phases : a f a s t i n i t i a l d ro p o f -800RU within 4 s , followed b y a slower decrease o ver the r e -m a i n i n g 300 RUWe a t t r i b u t e the slowly d i s s o c i a t i n g phaseto the presence o f multimeric aggregates o f upTCRwhichb in d wi th hi gh a v i d i t y , e ve n t ho u g h the protein ha d beenp u r i f i e d b y s i z e exclusion chromatography . I t ha s been shownt h a t even s m a l l am o u n ts o f aggregated m a t e r i a l ( G 2%) , whichc o u l d form during the concentration s t e p a f t er g el f i l t r a t i o n ,c a n p r o d u c e t h i s type o f biphasic d i s s o c i a t i o n ( 2 5 , 26) . Ont he o t he r h an d , when (3CHO o r Pmut were i n j e c t e d o verSEC1, SEC2, SEC3, a n d SPEA, equilibrium bin d i n g l e v e l swere rea che d w i thi n s ec o n ds ( F i g . 1 , B a n d C) . Upon com-pletion o f the i n j e c t i o n s , t he responses d r o p p e d r a p i d l y a swell (>90% decrease within the f i r s t 4 s i n t he c a s e o f Pmutd i s s o c i a t i n g f r o m SEC1 ; Fig . 1 C) . Because (3CHO a n dPmut displayed r e l a t i v e l y simple k i n e t i c behavior, and be-cause the r e a ct i v i ty p r o fi l e s o f i s o l a t e d R chain a n d up het-erodimer t o w a r d SEC, SPEA, a n d SEB had been f o u n d tob e q u a l i t a t i v e l y i n d i s t i n g u i s h a b l e , a l l subsequent measure-ments were c a r r i e d o ut with RCHO o r ( 3mu t . I t i s w o r t hn o t i n g t h a t t he monodisperse behavior o f the R chain ( s e ea l s o below) i s c o n s i s t e n t with i t s a b i l i t y to c r y s t a l l i z e : whereasb o t h RCHO a n d Pmut have yielded x-ray d i f f r a c t i o n qual-i t y c r ys ta l s , we were u n a b l e t o c r y s t a l l i z e t he otp het-erodimer ( 1 5 , 20) .The k i n e t i c s o f the bin d i n g o f soluble TCR c o m p o n e n t s

to SAGS were t oo f a s t to be accurately measured, a l t h o u g hthe association a n d d i s s o c i a t i o n r a t e constants may be e s t i -mated a t >100,000 M1 s - 1 a n d >0 . 1 s -1 , r e s p e c t i v e l y .


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hCOC.

2000,

1500

50 0

0-

1000-~

2000Time ( s )

E

Consequently, a f f i n i t i e s were determined under equilibriumbinding conditions a s described below .

G l y c o s y l a t e d an d U n g l y c o s y l a t e d TCR 0 Chains Bind SECand SPEA w i t h Micromolar Amities . Conditions f o r equi-librium binding a n a l y s i s were chosen on th e b a s i s of t he k i-n e t i c behavior of t h i s system ( F i g . 1 ) . Approximately 2,000RUo f e ac h SAG were immobilized a nd buffer flow r a t e swere s e t a t 5 Ntl/min to avoid diffusion-limited r e a c t i o n s( 2 8 ) . Each ( 3 chain wa s i n j e c t e d over th e s u r f a c e f o r 20 s

18 3 6 Superantigen Binding to TCR,6Chain

yCOGHd

0.yd

1600

20007

1500-,.

500

2000

1500

50 0

8

1000Time ( s )

1000Time ( s )

D

F

2000

2000

a nd report points f o r Scatchard a n a l y s i s were taken 10 s a f -t e r i n j e c t i o n (Fig . 2 ) . To estimate apparent Kn s , decreasingconcentrations ( 6 4 , 3 2 , 16 , 8 , 4 , 2 , and 1 [ 1 , M ) of RCHOor(3mut were injected over SEA, SEC1, SEC2, SEC3, andSPEA. To estimate the i n c r e a s e in RU r e s u l t i n g from th enonspecific e f f e c t of protein on th e bulk r e f r a c t i v e index,binding of (3CHO and (3mut t o a control s u r f a c e with noimmobilized ligand were a l s o measured ( F i g . 2 A) . (3CHOa nd (3mut showed th e same behavior over SEA a s over th e


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C0a

2000,

9 15 00dH 1000

0s

G

0 1000 2 000Time ( s )

Figure 1 . Concentration-dependent binding of soluble TCR compo-nents t o immobilized SAGS. B inding t o SECT by soluble a ( 3 TCR ( A ) ,PCHO ( B ) , (3mut (C), an d Va (D) was recorded a t the concentrationsindicated (N ,M). Similar p r o f i l e s were obtained when SEC2, SEC3 an dSPEA were immobilized . I n E an d F , Pmut was injected over SEA an dSEB, respectively . In (G), up TCR was injected over SEB For eachSAG, 3,0 0 0 -5,0 0 0 RUwere immobilized . The buffer flow r a t e was 2 N , l /min an d TCR components were injected f o r 1 5 min Dissociation wasc a r r i e d out f o r 15 min with HBS an d th e s u r f a c e s were regenerated by i n -jection of 1 0 mMCl between a s s a y s . Responses a r e gi ven as i n c r e a s e sover baseline values .

c o n t r o l s u r f a c e . As an a d d i ti o n al c o n tr o l f o r nonspecific bind-i n g , th e same concentrations of an i r r e l e v a n t protein, BSA,were injected over a s u r f a c e with no protein immobilized( F i g . 2 C) . As can b e s e e n , th e response to 32 N.M BSA i scomparable t o th e response to 64 p,M [3mut . Taking i n t o ac-count t h a t th e molecular mass ofBSA i s roughly twice t h a tof Pmut (66 . 2 versus 26 . 4 kD), an d t h a t there i s a l i n e a r r e -l a t i o n s h i p between th e mas s ofboundprotein and th e mea-sured RU ( 3 5 ) , i t i s apparent t h a t both proteins give s i m i l a rnonspecific e f f e c t s on th e bulk r e f r a c t i v e index . When BSAwa s i n j e c t e d over a s u r f a c e with S EC 1 , th e s i g n a l s were sim-i l a r t o those in Fi g . 2 C, an d much lower than th e responsesto equivalent masses of (3mut (Fig . 1 B ) .The s p e c i f i c binding of (3CHO an d [3mut to immobi-

l i z e d SAGS wa s calculated a s the difference between th e r e -sponses on th e SAG-derivatized an d control s u r f a c e s . Plotsof these data indicated t h a t th e b i nd in g of [ 3 chain t o SEC1,SEC2, SEC3, an d SPEA was s a t u r a b l e , a s shown in Fig . 3 ,Aand Cf o r the interaction of Pmut with SECl an d SPEA,respectively . A Scatchard plot f o r th e b i nd in g of [ sm ut t oSEC1 wa s l i n e a r ( co r r e l a ti o n c o e f f ic i e n t [ r ] = 0 . 9 9 ) an dgave a KD of 1 9 . 8 [,M . The predicted maximum s p e c i f i cbinding was 1 9 8 0 RU, while th e amount of immobilizedSEC1 was 2 0 5 0 RU Since SEC1 an d Pmut have s i m i l a rmolecular masses (2 7 . 5 an d 26 . 4 kD, r e s p e c t i v e l y ) , t h i s i n -d i c a t e s that nearly a l l th e SEC1 an d Pmut in th e a s s a y werea v a i l a b l e for binding . The r e s u l t s for SPEA are shown inFi g . 3 , Can d D a KD of 6 . 2 NLM wa s obtained . Similar a n a l -y s e s were c a r r i e d ou t for the binding of Pmut t o SEC2 andSEC3, an d for the binding of [3CHO to SEC1, SEC2, an dSEC3 .The r e s u l t s of t h e s e experiments are summarized in Ta-

b l e 1 . The apparent KD s f o r th e binding of Pmut to SEC1,1837 Malchiodi e t a l .

SEC2, SEC3, an d SPEA were 19 . 8 , 7 . 9 , 9 . 2 , an d 6 . 2 N,M,respectively . For th e binding of [3CHO to SEC1, SEC2,and SEC3, th e KD s were 1 8 . 2 , 5 . 4 , and 8 . 5 N.M . T h eser e s u l t s demonstrate t h a t ( a ) th e a f f i n i t y of recombinantV[38 . 2 J ( 3 2 .1C[31 chain f o r SEC or SPEA i s s i m i l a r t o thoseofsome other T c e l l s u r f a c e glycoproteins for t h e i r l i g a n d s ,such a s th e adhesion molecule CD2 f o r CD58 an d CD48( 2 5 , 2 6 ) , or of TCRs for peptide/MHC ( 1 2 , 1 7 , 37 , 38)and ( b ) glycosylation does not s i g n i f i c a n t l y contribute t oth e SAG-bindin g a c t i v i t y of a t l e a s t t h i s p a r t i c u l a r R chain .1 4 . 3 . d i 6 Chain Binds SEB with Much Lower A f f i n i t y thanSEC and SPEA . The markedly weaker binding of Pmutto SEB than t o SEC1 suggested in Fig . 1 , Can d F was con-firmed by equilibrium binding experiments ( F i g . 4 ) . BIA-core measurements were c a r r i e d ou t a s described above forSEC an d SPEA, except t h a t much higher concentrations of[ 3 chain (up t o 256 p,M) were required to approach s a t u r a -t i o n . The Scatchard plot was l i n e a r ( r = 0 . 9 7 ) an d gave aKD of 1 44 p,M with predicted maximum binding of 2460RU (Fig . 4 D) . Given t h a t about 2700 RU of SEB wereimmobilized, an d t h a t SEB and Pmut have s i m i l a r molecu-l a r masses ( 2 8 . 5 an d 26 . 4 kD , r e s p e c t i v e l y ) , t h i s i n d i c a t e st h a t >90% of th e coupled SAG retained binding a c t i v i t y .The weak b i nd in g of SEB to th e 14 . 3 . d [ 3 chain w as u n-

expected, p a r t i c u l a r l y in v iew of th e finding t h a t , on a mo-l a r b as i s, t h i s toxin wa s a c t u a l l y 3- to 10-fold more e f f e c t i v ethan SEC or SPEA i n stimulating th e p a r e n t a l 1 4 . 3 . d T c e l lh y bridoma ( F i g . 5 ) . We therefore decided t o check our r e -s u l t s by an independent technique, sedimentation equilib-rium . Amajor advantage of t h i s method i s t h a t both i n t e r -acting s p e c i e s a r e in s o l u t i o n , thereby avoiding an y p o s s i b l ea r t e f a c t s a r i s i n g from ligand immobilization .

Th e Much Weaker Binding of 0 c h a i n t o SEB t h a n t o SEC o rSPEA I s Confirmed by Sedimentation Equilibrium . Beforea n a l y s i s of the equilibrium c o n s t a n t s f o r th e [3mut chain withth e various b a c t e r i a l SAGS , th e behavior of the individualreceptor fragment an d toxins was evaluated in s e p a r a t e sed-imentation experiments . A l l of th e s p e c i e s under consider-ation were well behaved, showing v i r t u a l l y no tendency t oaggregate a t concentrations up to -25 ~LM . An estimate f o rth e mol ecu lar w ei g h t of th e [ 3 chain wa s 28,800, in goodagreement with t h a t expected from i t s amino acid compo-s i t i o n (26,400) . The molecular we ig h t of SEC3 wa s deter-mined to b e 28,300, compared with a r i expected value of28,900 . Similar agreement was obtained for SEC1, SEC2an d SEB. However, th e molecular weight of SPEA was e s -timated a t only 22,500, somewhat lower than th e expectedvalue of2 5,8 0 0 .

The equilibrium d i s so c i at i o n c o n s t a n ts determined by s e d i -mentation equilibrium f o r Pmut with various SAGS a r epresented in Table 1 ; a representative sedimentation p r o f i l ei s shown i n Fig . 6 . As can b e seen i n Table 1 , th e i n t e r a c -tion of th e ( 3 chain with v i r t u a l l y a l l t he tox in s occurs withKDs in th e low micromolar range ( 0 .9-2 . 5 p,M) . An excep-tion i s S E B , for which an estimate f o r th e KD ofonl y 70p,M cou ld b e made . However, t h i s estimate should be con-sidered only a lower l i m i t f o r th e a c t u a l KD s i n c e , under th ef e a s i b l e conditions f o r t h i s measurement i n th e u l t r a c e n t r i -


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f u g e , weaker binding would no t have been detected . At-tempts t o b e t t e r define th e KDusing 5- and 10-fold higherprotein concentrations (up t o 0 . 1 mM) to f avor c o m p l e xformation i n t h i s weakly interacting system were unsuc-c e s s f u l due to problems of nonspecific aggregation a t theseconcentrations . I t i s nevertheless apparent t h a t th e SEB bindsth e 1 4 . 3 . d ( 3 chain with much lowe r a f f i n i t y than SEC orSPEA, i n agreement with th e r e s u l t s f r o m BIAcore .A c o m pa r is o n o f apparent KDvalues obtained by se dimen-

t a t i o n equilibrium with those fromBIAcore r ev e a l s t h a t th el a t t e r a r e c o n s i s t e n t l y h i g h e r , by up t o a f a c t o r of 11 in th ec a s e ofSPEA (Table 1 ) . We believe t h a t the sedimentationequilibrium values more accurately r e f l e c t t he t ru e bindingc o n s t a n t s because t h i s method does no t involve chemicalcoupling of l i g a n d s to a s o l i d support . I n extreme c a s e s ( e . g . ,2 6 ) , immobilization can r e s u l t in complete l o s s of bindinga c t i v i t y , a s we observed f o r ( 3 chain coupled to the dextranmatrix ( s e e above) . Such experiences strongly suggest t h a tintermediate e f f e c t s on ligand a c t i v i t y ar e a l s o p o s s i b l e . In-deed, we a t t r i b u t e th e g e n e r a l l y weaker binding of ( 3 chainto SAGS measured by BIAcore than by sedimentation equi-librium t o s u b t l e e f f e c t s on SAG conformation/accessibilitya r i s i n g from th e immobilization procedure . These r e s u l t s i l -

1838 S u p e r a n t i g e n Binding t o TCR0 Chain

Discu ssion

Time ( s ) Time ( s )Figure 2 . Binding of 1 4 .3 . d 0 chain t o immobilized SECT an d SPEA (3mut was injected a t the indicated concentrations ( N M) over a control s u r f a c ewith no protein immobilized , ( A ) , or over ones t o which SEC1 (2,050 RU) ( B ) o r SPEA (1,350 RU) (D) ha d been coupled . The same concentrations ofBSA were a l s o injected over a control surface (C ) . Buffer f low r a t e s were 5 N,1/min . Equilibrium binding l e v e l s were reached within 4 s . After d i s s o c i a -t i o n with HBS, r e s i d u a l bound protein was e lute d us ing 10-s pulses of 1 0 mMHCI. Similar p r o f i l e s were obtained f o r SEC2 an d SEC3 .

l u s t r a t e th e i m po r t an c e o f determining binding c o n s t a n t s bya t l e a s t two independent techniques .

Our r e s u l t s demonstrate t h a t unglycosylated 1 4 . 3 . d Pc h a i n , whose three-dimensional s t r u c t u r e has been deter-mined t o high resolution ( 1 5) , r e t a i n s th e SAG-binding ac -t i v i t i e s ofth e assembled, glycosylated up heterodimer . Thea b i l i t y of th e recombinant ( 3 chain to s p e c i f i c a l l y bind SEB,SEC1, SEC2, SEC3, and SPEA i s consistent with th eknown p r o l i f e r a t i v e e f f e c t s of these toxins on V(38 .2-bear-in g T c e l l s ( 2 , 3 6 ) ; in c o n t r a s t , S E A, which d oes n ot s ti mu -l a t e c e l l s expressing VR8. 2 ( 2 ) , does no t bind the 1 4 . 3 . d ( 3chain with measurable a f f i n i t y . We therefore conclude t h a tth e s t r u c t u r e ofth e ( 3 chain, a s determined by x-ray c r y s t a l -lography, represents a b i o l o g i c a l l y a c t i v e c o nf o rm at io n o ft h i s molecule, a t l ea s t with r e s p e c t t o t ho se r egi on s respon-s i b l e f o r SAG recognition . However, we cannot formallyexclude th e p o s s i b i l i t y t h a t other regions of th e R chainmonomer migh t have a d i f f e r e n t conformation when a s s o -c i a t e d with c x chain in t he u (3 heterodimer .

Binding of th e 1 4 . 3 . d ( 3 chain t o a l l f i v e SAGS t e s t e d i s

20 0 0CONTROL

2 0 0 0 T 64SECT B

a 1400 - a 14004 32dNC y 160Gu 90 0- 0C9004 a

6 44

20 0 1 6 9 4 2 1 z o o 2~f~1f1 0 0 0 2 0 0 0 3000 400 0 0 1 0 0 0 2 0 0 0 3000 4000Time ( s ) Time ( s )

20007 2 0 0 0 TCONTROL C SPEA

a S1400 1 1400 + 32dy H 16O COGN $004 64 GH 8004 4

32 162 0 0 + 4 20 0

1 0 0 0 20 0 0 30 0 0 400 0 0 1 0 0 0 2 0 0 0 30 0 0 40 0 0


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20001

2000

10001

0 40 60 80R mut ( PM)

p mut ( E t M)

characterized by very f a s t association r a t e s (> l X 10 5M1 s - 1 ) a n d extremely f a s t d i s s o c i a t i o n r a t e s (>0. 1 s -1 ;Fi g . 1 ) ; the l a t t e r a c c o u n t f o r th e r e l a t i v e l y weak a f f i n i t i e so b s e r v e d ( i . e . , 0 . 9 RM, a t b e s t , i n th e c a s e ofSEC3) . Rapiddissociation k i n e t i c s h a v e a l s o been r ep o r t e d for th e i n t e r -action o f th e r at c e ll a d h e s i o n molecules CD2 a n d CD48( 2 5 ) , and f o r th e binding o f TCRs t o peptide/MHC com-plexes ( 3 7 , 38) . Fo r a d h e s i o n molecules, f a s t d i s s o c i a t i o nr a t e s may provide a mechanism t o f a c i l i t a t e deadhes io n , arequirement for c e l l m o t i l i t y ( 2 6) . Fo r TCRs, on th e o t h e rhand, rapid d i s s o c i a t i o n r a t e s would n o t appear to be com-p a t i b l e wi t h th e r e l at i ve l y s t a b l e interaction wi t h ligand be -lieved t o be a pr er equ is i t e fo r s i g n a l transduction t h r ou g hth e TCR/CD3 c o m p l e x ( 3 8 , 39) . This s u g g e s t s t h a t T c e l lactivation by SAGS r e q u i r e s the participation o f additionals u r f a c e molecules t o s ta bi li ze t h e t r a n s i e n t SAG-TCR in-t e r a c t i o n , i n a g r e e m e n t with other s t u d i e s (4-8) . Whilec l a s s 1 1 MHC pro ducts h av e be e n c l e a r l y implicated in t h i sr o l e (1-3, 1 3 ) , recent e v i d e n c e ( 1 4 ) i n d i c a t e s t h a t o t he r , y etunidentified, molecules may s u b s t i t u t e fo r MHC in pre-senting SAGS to T c e l l s ( s e e below) . Multivalent bindinga r i s i n g f r o m recepto r cross-linking would a l s o e f f e c t i v e l ydecrease the offr a t e (and i n c r e a s e t h e h alf - ti me ) o f theTCR-SAG i n t e r a c t i o n . A l t e r n a t i v e l y , rapid d i s s o c i a t i o n r a t e s

18 3 9 Malchiodi e t a l .

RU

Fi gur e 3 . Affinity of 14 . 3 . d Rchain fo r SECT a n d SPEA . Plotsa r e from data i n Fi g . 2 R e s p o n s e sr e s u l t i n g f r o m i n j e c t i o n o f th e i n -d ic a te d c o nc e nt r at i on s of (3muto v e r control surface (O , A, C) ,SEC1 (O, A ) a n d SPEA (O, C)ar e shown . Specific binding (A , A ,C) was c a l c u l a t e d as t h e differencebetween t h e responses f o r SECTa n d SPEA a n d t h e c on tr o l surfaceS c a t c h a r d a n a l y s i s o f t he s p e c i f i cbinding of (3mut t o SECT ( B ) a n dSPEA ( D ) gave l i n e a r p l o t s withc o r r e l a t i o n c o e f f i c i e n t s of 0 . 9 9 a n d0 . 9 8 , r e s p e c t i v e l y . The a ppa r e n tK s f o r th e (3mut -SEC1 and(3mut-SPEA reactions were 19 . 8a n d 6 . 2 N .M , r e s p e c t i v e l y .

may a c t u a l l y be c r i t i c a l f o r T c e l l triggering by S A G S , a s sug-gested by a r ec en t s t ud y in which a s i n g l e peptide/MHCc o m p l e x wa s f o u n d t o be able t o s e q u e n t i a l l y bind a n d t r i g -g er n e ar ly 20 0 TCRs ( 4 0 ) .The c o mp le xi t y o f TCR-SAG i n t e r a c t i o n s i s further i l -

l u s t r a t e d by the f inding t h a t a s oluble human TCR express-i ng t he VR3 . 1 ge n e s e g m e n t binds SAGS with quite d i f f e r -ent k i n e t i c s than t h e mouse VR8. 2 TCR described here ( 1 2 ) .Bind i ng o f the human VR3 . 1 TCR t o SEB i s characterizedby a s s o c i a t i o n and d i s s o c i a t i o n r a t e s which a r e only moder-a t e l y f a s t : 1 . 3 X 10 4 M1 s - 1 and 1 . 1 X 10 -2 s - 1 , respec-t i v e l y . The a f f i n i t y o f t h i s TCR fo r SEB, h ow ev e r, i s com-p ar ab le t o t h a t of the 14 . 3 . d R chain f o r SEC3 : 0. 8 p,Mv er sus 0 . 9 pLM, respectively . On th e other h a n d , SEB wasshown to bind HLA-DR1 with k i n e t i c s very s i m i l a r to t h o s ewe obs erve f o r the V08.2-SAG interaction ( 1 2 ) ; in b o t hc a s e s , th e association a n d d i s s o c i a t i o n r a t e s were t o o rapidt o be accurately m e a s u r e d . This implies t h a t bi n di n g o f hu-man V(33 . 1 to SEB h e l p s s t a b i l i z e th e t r a n s i e n t SEB-DR1 in-t e r a c t i o n and, consequently, th e TCR-SEB-DR1 c o m p l e x( 1 2 ) . We m i g h t therefore e xpe ct t he bi nd in g o f m u ri n eVR8. 2 t o SEB to be s t a b i l i z e d by a more k i n e t i c a l l y favorableinteraction o f SEB with c l a s s 1 1 molecules ( i . e . , o n e with aslower d i s s o c i a t i o n r a t e ) .


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3000aa

4000

d 2000 .eo -Gd 1000 -0-

25 6

4000-

3000

2000

12E 1000

0

CONTROL A

6432 E 4

t1000 2000 3000 4000

Time ( s )

16

P mut ( i1 M) RUFigure 4 . Binding of 14 . 3 . d ( 3 chain t o immobilized SEB . (3mut wa s injected a t the indicated concentrations (11M) over a control surface with no pro-t e i n immobilized ( A ) or on e with 2,700 RUSEB ( B ) . Buffer flow r a t e s were 5 NU/min ; equilibrium binding l e v e l s were reached within 4 s . After d i s s o -c i a t i o n , r e s i d u a l protein was eluted using 10-s pulses o f 10 mMHCI . Specific binding (A , C) wa s c a l c u l a t e d a s th e d i f f e r e n c e between th e t o t a l response(O ) and th e non-specific response (O ) . The Scatchard p l o t wa s l i n e a r (r = 0 . 9 7 ) and gave a K of 14 4 ltM .

The much weaker binding o f the 14 . 3 . d ( 3 chain, or ofaR heterodimer, t o SEB than to SEC1, SEC2, SEC3 orSPEA wa s s u r p r i s i n g , s i n c e a l l f i v e t o x i n s a r e known t o stim-u l a t e V(38 .2-bearing T c e l l s ( 2 , 36) . Furthermore, SEB wasconsiderably more potent than the other t o x i n s i n stimula-tion a s s a y s using the 14 . 3 . d hybridoma ( F i g . 5 ) . These r e -s u l t s may be interpreted in terms of a recent study d e s c r i b -ing functional d i f f e r e n c e s b etween SEB and SEC in MHCc l a s s I I kno c k out mic e ( 1 4 ) . Incubation of lymph node c e l l sf rom c l a s s I I - d e f i c i e n t mice with SEC, b ut no t SEB, r e s u l t e din th e c l o n a l expansion of VP8-positive T c e l l s a nd the gen-e r a t i o n of CTL Similar r e s u l t s were obtained with SEE,except t h a t V(311-positive c e l l s were s e l e c t i v e l y expanded .The responses t o SEC a nd SEA resembled conventionalMHC-associated responses i n t h a t T c e l l bearing the sameV( 3 elements were s p e c i f i c a l l y a f f e c t e d . Our data provide a

c0G

1840 Superantigen Binding to TCR13 Chain

12E

6432

16

1000 2000 3000 4000Time ( s )

straightforward explanation f o r the finding t h a t a c t i v a t i o nof V(38-positive T c e l l s by SEC c an occur in t he absence ofMHC, in c o n t r a s t to activation by SEB : whereas SEC1, 2 ,and 3 bind the 14 . 3 . d R chain with Kp s of 0.9-2 . 5 p,M, thecorresponding value fo r SEB i s b etween 70 and 140 1LM(Table 1 ) . Thus, SEs able to bind TCRs with micromolara f f i n i t i e s maynot a b s o lu t e l y r e q ui r e the p a r t i c i p a t i o n ofMHCc l a s s I I molecules to s t a b i l i z e t h e i r interaction with the Rchain . Even in the case of MHC-independent a c t i v a t i o n ,however, APC fromMHC c l a s s I I - d e f i c i e n t mice ( o r f rommice lacking both c l a s s I a nd c l a s s I I ) were required f o rstimulation ( 1 4 ) . This s u g g e s t s t h a t other s u r f a c e moleculesmay s u b s t i t ut e f or MHC i n binding c e r t a i n SEs, and per-haps a l s o th e TCR, thereby helping t o s t a b i l i z e TCR-SEi n t e r a c t i o n s .A f u r t h er d i s t i n c t i o n b etween SEB and SEC may l i e i n


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60

50-

20-

10 -I

0U (SEB)U (SECI)U (SEC2)

ng/mlU (SEC3)U(SPEA)

U(SEA)

Figure 5 . IL-3 production by the parental 14 . 3 .d hybridoma a f t e r stim-ulation with different SAGS in the presence ofA20APC TheSAG con-centrations used are as indicated with the different symbols.

t h e i r r e l a t i v e a f f i n i t i e s f o r MHC c l a s s I I . While SEB has beenshown to bind HLA-DR1 an d -DQ with a KD in th e mi -cromolar range ( 9- 11 ) , th e d at a on SEC i s l e s s c l e a r . In on estudy, SEC2 was found able to compete with SEB f o rTable 1 . Dissociation Constantsfor the Binding ofBacterialToxins to Glycosylated and Unglycosylated 14.3 .d /3 Chain

Affinity measurements by BIAcore and sedimentation equilibriumwere carried out as described in Materials and Methods . All experi-ments were performed at 25C using the molecular weight values pre-sented in the text and the specific extinction coefficients provided byToxin Technology, Inc . The value for the association of SEB withRmut obtained from sedimentation equilibrium is a lower limit for thedissociation constant . NB,no detectable binding; ND, notdone

1841 Malchiodi et al .

Ec 0 8 ,N

cW

1 .0-~

06

Q 0,2-

0.0- T __V ~_6.90 6.95 7.00 7.05 7.10

Radius, cmFigure 6 . Sedimentation equilibrium of an equimolar mixture ofthe14 . 3 .d R chain with SECT . Sedimentationwas performed at 22,000 rpmin 50mMris-HCI, pH 8. 0 , a t 25C . Starting concentrations of(3 chainandSEC1 were 7 . 5 p.M . (Bottom) Absorbance at 280 nm versus distancefrom the center of rotation in centimeters . (Top) The residuals (AZ80theoretical - A8o observed) for th e equilibrium between th e twocomponents, yielding aKof2. 51 p.M,are small andrandom . Similar re -sults were obtained for SEC2, SEC3, andSPEA

binding t o 1 3 1 1 , 1 - p o s i t i v e B c e l l s ( 4 1 ) , whereas i n anotherstudy no binding of SEC1 to c e l l s expressing DR3,5 couldbe demonstrated ( 4 2 ) . Indeed, th e d i f f e r e n t i a l a s s o c i a t i o n ofc e r t a i n SAGS with d i f f e r e n t c l a s s I I molecules has been putforward a s a p o s s i b l e explanation f o r the lack ofan absolutec o r r e l a t i o n between V( 3 expression an d SAG r e a c t i v i t y ( 1 3 ) ,

40-M

Figure 7. Space-filling model of the 14. 3 .d i 3 chaia showing the loca-tion of potential N-linked glycosylation s i t e s . The V(3 andCi3 domainsare labeled ; CDRl (residues 25-33) i s shown in green, CDR2 (residues48-56) in purple, andHV4 (residues 69-75) in light brown . TheN-linkedglycosylation s i t e s at VP positions 24 and 74 and a t C(3 position 236 arelabeled 1 , 2, and 3 , respectively . Those at C(3 positions 121 and 186 arenot v i si b le i n this orientation .

SAGo c

BIAcore

RCHOBIAcore

(X 10-6 M)

(3mutBIAcore

(X 10-6 M)

RmutCentrifuge(X 10-6M)

SEC1 NB 18.2 19.8 2.51SEC2 NB 5.4 7 .9 2.32SEC3 N13 8 .5 9 .2 0.86SPEA NB ND 6 .2 2.11SEB NB ND 144 >70SEA NB NB NB ND


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Figure 8 . Space-filling model of the putative SAG-binding face of the V(38.2 domain showing the location of conserved residues relative to Vpl, V(33,V(37, and V(311 . The amino acid sequences ofVP segments 1, 3, 7, and 11 were aligned with that ofV(38 .2 according to reference 21 ; no insertions ordeletions with respect to V(38 .2 were present. V(38 .2 residues which are conserved relative to each ofthe other V(3 segments are shown in red in the pan-els marked Vp1, V/33, V(37, and V(311 . CDR1 is in green, CDR2 in purple, and HV4 in light brown . The potential N-linked glycosylation sites at V(38 .2positions 24 and 74 are in gray; neither site is conserved in any of th e other V(3 segments . Single-letter abbreviations for amino acid residues are : E, Glu ;I, Ile ; K, Lys ; L, Leu ; N, Asn ; P, Pro ; Q, Gln ; R, Arg; S, Set ; T, Thr ; and V, Val .

While no affinities have been reported for the interactionof SAGs with mouse class 11 proteins, it tempting to specu-late that the very weak binding of SEB to VR8 .2 we ob-serve is compensated by a fairly strong association between

1842 Superantigen Binding to TCR,8 Chain

SEB and mouse class 11, hence accounting for the fact thatT cell activation by SEB requires MHC (6) . Conversely,SEC, even though it may lack appreciable affinity for mouseclass 11 , binds V(38 .2 sufficiently tightly to enable it to stim-


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u l a t e T c e l l s independently of MHC Yet a third scenario i ssuggested by SEB, which binds both human VR3 . 1 a ndDR with mic romola r a f f i n i t i e s ( 1 2 ) . Thus, th e same en-terotoxin may a c t i v at e d i f f e r e n t T c e l l s by d i f f e r e n t mecha-n i s m s , depending on th e p a r t i c u l a r VP element they expressa nd on th e s p e c i f i c MHCb ack gro un d of th e h os t .As shown in Table 1 , glycosylated 14 . 3 . d R c h ain bindsSEC1, 2 , and 3 with e s s e n t i a l l y th e same a f f i n i t y a s unglyc-

osylated R c h ain . This i n d i c a t es t ha t carbohydrate does notcontribute to SAG binding, a t l e a s t in t h i s p a r t i c u l a r case .This r e s u l t i s somewhat s u r p r i s i n g s i n c e th e p o t e n t i a l N-linkedglycosylation s i t e s a t VP r e s i d u e s Asn24 a nd Asn74, whichwere mutated t o glutamine ( 1 5 ) , a r e a t or near the putativeSAG binding s i t e in th e three-dimensional s t r u c t u r e of th eR c h ain ( F i g . 7 ) . In p a r t i c u l a r , Asn74 i s part of th e s o c a l l e dfou rth hyperv a riable region (HV4), which has been d i r e c t l yimplicated in SAG recognition ( 3 ) , while Asn24 would beexpected t o l i e adjacent t o Asn74 i n th e unmutated s t r u c t u r e .One explanation f o r th e lack of e f f e c t of mutations a t t h e s ep o s i t i o n s i s that neither Asn24 nor Asn74 i s a c t u a l l y glyco-s y l a t e d . We consider t h i s u n l i k e l y , a s both r e s i d u e s a r e lo-cated on th e s u r f a c e of the protein . A l t e r n a t i v e l y , these r e s i -dues may in f a c t l i e a t th e periphery of th e combining s i t ef o r b a c t e r i a l SAGs . I t i s worth noting in t h i s r e s p e c t t h atm ut a ti on o f Asn24 t o glutamate has been shown t o conferr e a c t i v i t y t o Us-1 ( 4 3 ) . This s u g g e s t s t h at v i ra l a nd b a c t e r i a lSAGs bind somewhat d i f f e r e n t regions of th e VP domain .To i n v e s t i g a t e th e s t r u c t u r a l b a s i s for th e SAG-binding

s p e c i f i c i t y of d i f f e r e n t R c h a i n s , pairwise comparisons werec a r r i e d ou t b e twee n mouse V08. 2 a nd Vol, V03, VP7,a nd VR11 based on th e c r y s t a l s t r u c t u r e of th e VR8. 2 do-main (Fig . 8 ) . While VR3, VR7, VR8. 2 , and VP11 a l l r e a c twith SEC (and VR3, VR7, a nd VR8. 2 r e a c t with SEB a sw e l l ) , Vol does no t r e a c t with e i t h e r SEB or SEC, butonly with SEA, which does not bind VR8. 2 ( 2 ) . As mightbe expected, a compa r i son of conserved r e s i d u e s on th e

We a r e g r at ef u l t o Michael Robinson (Pharmacia B i o s e n s o r ) f o r i n v a l u a b l e a d v i c e a nd d i s c u s s i o n s through-ou t t h e c o u r s e of t h i s work .T h i s r e s e a r c h wa s s u p p o r t e d by N a t i o n a l I n s t i t u t e s of H e a l t h (NIH) g r a n t AI36900 and a g r a n t from t h eR ic h a rd Lou ns b er y Foundation (R . A. M a r i u z z a ) , NIH g r a n t RR08937 (E . E i s e n s t e i n ) , NIH g r a n tHL36611 ( P . M S c h l i e v e r t ) , a nd g r a n t s from t h e Kimberly-Clark C o r p o r a t i o n , (Neenah, WI), t h e P e r s o n a lP r o d u c t s Co. (New Brunswick, N J ) , a nd Tambrands ( P a l m e r , MA) ( P .M . S c h l i e v e r t and D.H . Ohlendorf) .Support from t h e L u c i l l e P . M a rkey C h a r i t a b l e T r u s t i s a l s o g r a t e f u l l y acknowledged . Th e B a s e l I n s t i t u t e f o rImmunology wa s founded a nd i s s u p p o r t e d by F . Hoffmann-LaRoche Ltd . , B a s e l , S w i t z e r l a n d .E . L . Malchiodi i s a F e l l o w of CONICET, A r g e n t i n a .

putative SAG-binding face of th e VR8. 2 domain r e v e a l st h a t Vol indeed has fewer r e s i d u e s in common with VR8. 2than do VR3, VR7, or VR11 . Excluding r e s i d u e s unlikelyto be involved i n d i r e c t contacts with b a c t e r i a l SAGS be-c au se they l i e a t th e periphery of t h i s s u r f a c e (Thr5, Arg9,Va112, As n3 0, P ro110, Ar g113 , a nd Leu116a), VR8. 2s h a r e s f i v e r e s i d u e s with VR3 (Pro8, L y s1 8 , P r o7 0, Ser71,a nd Ser76), f i v e residues with VR7 (Va119, Ser68, Glu73,Ser76, a nd I l e u 7 8 ) , f i v e r e s i d u e s with VR11 (Pro8, Lys11,Thr20, Pro70, a nd Gln72), but only three r e s i d u e s withVol ( S e r 7 , Pro8, a nd Pro70) . What i s remarkable, how-e v e r , i s t h a t even between VR8 . 2 a nd VR3, VR7, or Vol 1 ,a l l of which r e a c t with a s i m i l a r s p ec t r um o f SAGS, th e num-ber of common r e s i d u e s i s r e l a t i v e l y small ; furthermore,t h es e r e si du es a r e not p a r t i c u l a r l y concentrated in HV4This i n d i c a t e s that c e r t a i n SAGs (SEC3, f o r example, whichr e a c t s with VR3, VR7, a nd VR8. 2 ) a r e a b l e to recognizevery d i f f e r e n t molecular s u r f a c e s present on d i f f e r e n t VPdomains . One way t h i s could oc c u r i s i f a s i n g l e SAG pos-s e s s e d s e v e r a l d i s t i n c t VR-binding s i t e s . However, a recentx-ray c r y s t a l l o g r a p h i c study of a n idiotope--anti-idiotopecomplex ( 4 4 ) s u g g e s t s t h a t i t i s not necessary to p o s t u l a t emultiple binding s i t e s t o explain th e recognition o f t op o-graphically d i f f er e n t s u r f a c e s by a s i n g l e molecular s p e c i e s .I n t h i s study, t h e c on ta c ts made between a n anti-lysozymeantibody (D1 . 3 ) a nd an a n t i - i d i o t o p i c a n ti body ( E 5 . 2 ) r a i s e da g a i n s t i t were compared with the c o n t a c t s made b e twee nD1 . 3 a n d l ys o zy me in the o r i g i n a l antigen-antibody com-plex ( 4 5 ) . S u r p r i s i n g l y , i t wa s found t h a t D1 . 3 employede s s e n t i a l l y th e same s e t ofcombining s i t e r e s i d u e s (and mostof th e same atoms) in binding lysozyme a s i n binding E5 . 2 ,d e s p i t e th e f a c t t h a t th e s i t e s on l ys o zy me a n d E5 . 2 recog-nized by 13 1 . 3 were s t r u c t u r a l l y unrelated . Whether s i m i l a rme chan i sm s operate t o permit individual SAGs to i n t e r a c twith a number of d i f f e r e n t VP f a m i l i e s must await d i r e c ts t r u c t u r a l s t u d i e s of R chain-SAG complexes .

A d d r e s s correspondence t o Dr. RoyA. M a r i u z z a , Center f o r Advanced Research i n B i o t e c h n o l o g y , 9600Gudelsky D r i v e , R o c k v i l l e , MD20850 .Receivedfor publication 11 May 1995 and i n r e v i s e d form 10July 1995 .Note added i n proof S i n c e t h i s manuscript was s u b m i t t e d , we have determined t h e c r y s t a l s t r u c t u r e of a com-p l e x between t h e 1 4 . 3 . d R c h a i n a nd SEC3 . S u p e r a n t i g e n c o n t a c t s a r e m a i n l y with CDR1, CDR2, andHV4, r a t h e r than with framework r e s i d u e s . The g l y c o s y l a t i o n s i t e s a t V ( 3 p o s i t i o n s 2 4 a nd 74 a r e no t d i r e c t l yi m p l i c a t e d i n t h e R chain-SEC3 i n t e r f a c e .18 4 3 Malchiodi e t a l .


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