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  • 8/2/2019 2)Brodsky Review

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    M at r ix B io lo g y Vo l . 1 5 /19 9 7 , p p . 5 4 5 -5 5 4 1997 by Gu stav Fischer Verlag

    The Col lagen Tr ip le -Hel ix StructureB A R B A R A B R O D S K Y * a n d J O H N A . M . R A M S H A W +""Departm ent o f Biochemistry, UM DNJ-R obert W ood Johnson Medical School, Piscataway, New Jersey,US A CSIRO , Division of B iomolecular Engineering, Parkville, Australia

    A b s t r a c tRecen t ad v an c es , p r i n c ip a l l y t h ro u g h t h e s t u d y o f p ep t i d e m o d e l s , h av e l ed t o an en h an ced

    u n d er s t an d in g o f t h e s t ru c tu re an d fu n c t i o n o f t h e co l l ag en t r i p l e h e li x . I n p a r t i cu la r , t h e f i r s tc ry s t a l s t ru c tu re h as c l ea r l y sh o wn th e h ig h ly o rd e red h y d ra t i o n n e two rk c r i t i ca l f o r s t ab i l i z i n gb o th t h e mo lecu l a r co n fo rmat io n an d t h e i n t e r ac t i o n s b e tween t r i p l e h e l i ces . Th e seq u en ce d e -p en d en t n a tu re o f t h e co n fo rmat io n a l f ea tu res i s a l so u n d er ac t i v e i n v es t i g a t i o n b y NM R an do th e r t ech n iq u es . Th e t r i p l e -h e l i x mo t i f h as n o w b een i d en t i fi ed i n p ro t e in s o th e r t h an co l l ag en s ,an d i t h as b een e s t ab l i sh ed as b e in g imp o r t an t i n man y sp ec i fi c b io lo g i ca l i n t e r ac t i o n s as we l l a sb e in g a s t ru c tu ra l e l emen t . T h e n a tu re o f r eco g n i t i o n an d t h e d eg ree o f sp ec i f ic i t y fo r i n t e r ac t i o n sin v o lv in g t r i p le h e l i ces may d i f f e r f ro m g lo b u l a r p ro t e in s . T r ip l e -h e l i x b in d in g d o ma in s co n s i s t o fl i n ea r seq u en ces a lo n g t he h e l i x , ma k in g t h em am en ab l e t o ch a ra c t e r i za t i o n b y s imp le mo d e l p ep -t i d es . Th e ap p l i ca t i o n o f s t ru c tu ra l t ech n iq u es t o su ch mo d e l p ep t i d es can se rv e t o c l a r i fy t h ein t e r ac t i o n s i n v o lv ed in t r ip l e -h e l i x r eco g n i t i o n an d b in d in g an d c an h e lp ex p l a in t h e v a ry in g im-p ac t o f d i f f e r en t s t ru c tu ra l a l t e r a t i o n s fo u n d i n m u tan t co l l ag en s i n d i seased s t a t e s .

    Key w o rd s : co ll ag en , tr i p l e -h e l i x , h y d ra t i o n , x - r ay c ry s t a l l o g rap h y , N M R

    In t roduct ionTh e co l l ag en f ami ly r ep resen t s a g ro u p o f d iv e r se ex t r a -

    ce l l u l a r ma t r i x mo lecu l es l i n k ed b y t h e p resen ce o f t h eco l l ag en t r i p l e -h e l i x s t ru c tu re a s a co mmo n s t ru c tu ra l e l e -men t (Bro d sk y an d S h ah , 1 9 9 5 ; Ba t eman e t a l . , 1 9 9 6 ) . I nh ig h er an imal s , a t l eas t 1 9 d i s t i n c t co l l ag en t y p es a r ek n o w n t o s h a r e t h i s c o m m o n m o t i f , b u t t h e y v a r y i n t h e i rh ig h er o rd e r s t ru c tu res an d fu n c t i o n s . Th e t r i p l e h e l i xco n s t i t u t es a ro d - l i k e s t ru c tu re imp o r t an t fo r f i b r i l f o rma-t i o n an d s t ru c tu ra l i n t eg r i t y , b u t i t i s n o w c l ea r t h a t th et r i p l e h e l i x a l so i n t e r ac t s wi th a wid e r an g e o f mo lecu l esimp o r t an t i n ex t r ace l l u l a r ma t r i x o rg an i za t i o n an d fu n c-t ion . In add i t ion , the co l lagen t r ip le hel ix has been foundas a d o main i n a v a r i e ty o f o th e r p ro t e in s , i n c lu d in g t h o sein v o lv ed i n ea r l y h o s t -d e fen se fu n c t i o n s (Tab l e I ) (Ho p p ean d Re id , 1 9 9 4 ) .

    i Abbreviations u sed: MS R, macrophage scavenger receptor,Hyp, hyanzyprotine.

    Table I. Examples of occurrences of triple-helix domains, forms ofself-association and binding of ligands (Kielty et al., 1993; H opp eand R eid, 1994; Brodsky and Sh ah, 1995).Protein Self-A ssociation Ligand BindingActivitiesFibri l lar col lagens:Type I collagen D-periodic fibrils

    Non- f i b r i l l a r co l l agens:Basem ent mem brane irregular networkcollagen (type IV)N o n - c o l l a g e n o u s p r o te i ns :C lq hexamer of trimers

    collagenase, deco-rin,integrins,phospholipids

    integrins, heparin,nidogen

    C1 r,Cls af teractivation;C 1q receptorMSR membrane OxLDL, tetraplexcompone nt nucleic acids

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    5 4 6 B . B ro d s k y a n d J . A . M. R a m s h a wT h e c o n f o r m a t i o n o f co l l a ge n w a s r e c o g n i z ed e a r ly t o b e

    d i s t in c t f ro m o th e r p ro te in s s tu d ie d . I t s a m in o a c id s e -quence show s two un ique fea tu res : (1 ) g lyc ine is eve ry th i rdre s id u e, g e n e ra t in g a r e p e a t in g (G ly -X-Y ) n p a t t e rn ; a n d (2 )a h ig h p ro p o r t io n o f re s id u e s ( a b o u t 2 0 % ) a re th e im in oa c i d s p r o l i n e a n d h y d r o x y p r o l i n e . A r o u n d 1 9 6 0 , t h e m o -l e c u la r s t ru c tu re w a s d e t e rm in e d to b e th re e s u p e rc o i l e dp o ly p ro l in e I I - l i k e c h a in s (R ic h a n d C r i c k , 1 9 6 1 ; R a m a -c h a n d ra n , 1 9 6 7 ) . I t is o n ly r e c e n t ly th a t a c ry s t a l s t ru c tu reo f a t r i p l e -h el i c a l m o le c u le h a s b e c o m e a v a i l a b le a n d a l -lo w e d a d e t ai l e d c o n s id e ra t io n o f it s f e a tu re s a n d th e d e f i -n i t i o n o f i t s e x t r a o rd in a r y h y d ra t io n n e tw o rk (B e ll a e t al . ,1 9 9 4 ; 1 9 9 5 ) . P e p t id e s h a v e p l a y e d a m a jo r ro l e in th e r e -c e n t a d v a n c e s in k n o w le d g e a b o u t t h e t r i p l e -h e l i c a l s t ru c -tu re , s e rv in g a s m o d e l s o f c o l l a g e n s e q u e n c e s a c c e s sib l e tox - r a y c r y s t a ll o g r a p h y a n d N M R s p e c tr o s c op y . I n t hi s r e-v iew, we focus on the recen t advances in de f in ing co l lagent r ip l e -h e lix s t ru c tu re a s a b a s i s fo r u n d e r s t a n d in g i ts m o le c -u la r p ro p e r t i e s , s e l f - a s s o c ia t io n a n d s p e c if i c b in d in g o f o th -e r molecu les .

    S t r u c t u r e o f t h e Triple-HelixF i b e r d i f f r a c t i o n s t u d i e s o n c o l l a g e nT h e t r ip l e -h e l ix m o d e l fo r c o l l a g e n w a s b a s e d o n f ib e r

    d i f f r a c t io n s tu d ie s . T h e e x c e l l e n t o r i e n ta t io n o f c o l l a g e nm o le c u le s in s t r e t c h e d t a i l t e n d o n g iv e s h ig h ly o r i e n te d f i -b e r x - ra y d i f f r a c t io n p a t t e rn s o f c o l l a g e n . T h e in d e x in g o fl a y e r l i n e s f ro m th i s p a t t e rn , t o g e th e r w i th c o l l a g e n ' s d i s -t i n c t iv e a m in o a c id f e a tu re s , l e d R a m a c h a n d ra n a n d K a r -th a (1 9 5 4 ) to p ro p o s e a th re e - s t r a n d e d m o d e l fo r c o l l a g e n ,w h ic h w a s th e n m o d i f i e d to g iv e th e g e n e ra l ly a c c e p te ds u p e rc o i l e d t r ip l e -h e l i c a l s t ru c tu re (R ic h a n d C r i c k , 1 9 6 1 ;R a m a c h a n d r a n , 1 9 6 7; s e e F r a s er a n d M a c R a e , 1 9 7 3 , f o rre v ie w o f e a r ly stu d ies ) . T h i s m o d e l w a s fu r th e r r e f in e d u s -in g a l i n k e d a to m l e a s t s q u a re s r e f in e m e n t o n im p ro v e d x -ra y d a ta b y F ra s e r e t a l . (1 9 7 9 ) . T h e f ib e r d i f f r a c t io n p a t -t e rn r e p re s e n t s a n a v e ra g e o v e r th e w h o le c o l l a g e n m o le -c u le , a n d th e m o d e l o b ta in e d p ro v id e s c o o rd in a te s fo r a na v e r a g e G l y - P r o - H y p t r i p e p t i d e u n it . T h e s e c o o r d i n a t e ss e rv e d a s a b a s i s fo r e n e rg y m in im iz a t io n s tu d ie s to r e f in eth e s t ru c tu re fu r th e r (N S m e th y . , 1 9 8 8 ) . F ro m f ib e r d i f f r a c -t ion s tud ies , i t i s no t po ss ib le to de te rm ine i f t r ip le -he l ica lp a r a m e t e r s v a r y w i t h d i f f er e n t G I y - X - Y t r i pl e ts . T o o b t a i ns u c h in fo rm a t io n , i t i s n e c e s s a ry to d e t e rm in e th e m o le c u la rs t ru c tu re f ro m s in g le c ry s t a l s t ru c tu re s .

    A p e p t id e m o d e l o f c o l l a g e n (P ro -P ro -G ly ) l 0 w a s c ry s t a l -l ized , bu t the da ta ind ica ted tha t the co l lagen-l ike he l icesw e re a l ig n e d e n d to e n d to fo rm a c o lu m n a r , p o ly m e r i c ty p eo f a s s o c ia t io n r a th e r t h a n p re c i s e c ry s t al l i n e p a c k in g(O k u y a m a e t a l . , 1 9 8 1 ) . A n a v e ra g e m o d e l w a s o b ta in e dth ro u g h a l i n k e d a to m l e a s t s q u a re r e f in e m e n t th a t s h o w e da b a c k b o n e c o n fo rm a t io n v e ry s im i l a r t o th a t o f d ry ,s t r e t c h e d c o l l a g e n , b u t w i th a d i f f e re n t s y m m e t ry (7 /2 )

    f ro m th a t s e e n fo r t h e t a i l t e n d o n (1 0 /3 ) (F ra s e r a t a l . ,1 9 7 9 ) . T h re e w a te r m o le c u le s p e r t r i p e p t id e w e re a l s o id e n -t i f i e d f ro m th e d a t a .

    C r y s t a l s t r u c t u r e d e t e r m i n a t i o n -t h e t r ip l e - he l i ca l c o n f o r m a t i o nA p re re q u i s i t e fo r t h e d e t e rm in a t io n o f t h e h ig h r es o lu -

    t io n s t ru c tu re o f t h e c o l l a g en t r ip l e h e l ix i s o b ta in in g t ru es in g le c ry s t a l s s u i t a b le fo r x - ra y c ry s t a l lo g ra p h y , a n d B e l l ae t a l . (1994) on ly recen t ly so lved the f i rs t c rys ta l s t ruc tu refo r a t r i p l e -h e l i c a l m o le c u le . T h e s t ru c tu re w a s s o lv e d fo r a3 0 -m e t p e p t id e , d e n o te d th e G ly ~ A la p e p t id e , w h ic h c o n -s i s t s o f t e n r e p e a t in g P ro -H y p -G ly u n i t s w i th a s in g le s u b -s t i t u t io n o f a G ly b y a n A la r e s id u e in th e m id d le . T h eGly ~Ala pep t ide d if f r ac ted to a h igh reso lu t ion (1 .9 ,~),a n d h a d th e u n iq u e m o le c u la r p a c k in g e x p e c te d fo r a s in g lec rys ta l . I t i s l ike ly tha t the p resence o f a s ing le Ala re s iduein e a c h p e p t id e c h a in e s t a b l i s h e d a r e c o g n iz a b le f e a tu reth a t l o c k e d in th e a x ia l a r r a n g e m e n t , r a th e r t h a n fo rm in gth e f ib e r- l i k e a s s o c ia t io n s fo u n d fo r t h e r e p e a t in g (P ro -P ro -G ly)x 0 (O k u y a m a e t a l ., 1 9 8 1 ) .

    T h e G ly ~ A l a p e p t id e w a s fo u n d to b e a tr i p l e -h el i c a l ro d -l ike molecu le 87 A long and 10 ~ . in d iamete r (Be l la e t a l . ,1 9 9 4 ) . T h e s t ru c tu re o f t h e P ro -H y p -G ly r e g io n s o f t h eG ly ~ A la p e p t id e c o n f i rm e d th e g e n e ra l p a ra m e te r s o f th e f i-b e r d i f f r a c t io n m o d e l (F ra s e r e t a l . , 1 9 7 9 ) , b u t n o w v a r i a -t i o n s in in d iv id u a l t r i p e p t id e u n i t s a lo n g th e c h a in c o u ld b ed e te rm in e d . A d ja c e n t t r i p l e t s i n a n in d iv id u a l c h a in h a v e atw i s t c lo s e to 6 0 a n d a h e ig h t n e a r 8 . 4 A , w i th r a th e r s m a l lv a r i a t io n s a m o n g th e d i f f e re n t G ly -P ro -H y p t r ip l e t s , e x c e p tin th e t e rm in a l r e g io n s a n d n e a r t h e A la s u b s t i t u t io n . A G ly -P ro -H y p t r ip l e t i n o n e c h a in i s r e l a t e d to th e G ly -P ro -H y pt r ip l e t i n a n a d ja c e n t c h a in in th e m o le c u le b y a s c re w s y m -m e t ry , w i th a tw i s t c lo s e to 1 0 0 a n d a 2 . 8 6 A u n i t h e ig h t . As o m e w h a t g re a t e r v a r i a t io n i s s e e n in th e s e s u p e rh e l i c a l p a -ra m e te r s a s o n e g o e s a lo n g th e h e lix . T h e ~ , a n g le s arec lo s e to th o s e o b ta in e d fo r m o d e l s o f (P ro -P ro -G ly )1 0 a n dco l lagen .

    T h e c ry s t a l s t ru c tu re a l lo w e d th e f i r s t d i r e c t v i s u a l i za t io no f t h e e x p e c t ed N H C - - O h y d r o g e n b o n d s i n v o lv i n g t h ep e p t i d e b a c k b o n e , s h o w i n g t h e N H o f gl y ci n e h y d r o g e nb o n d e d to th e C = O o f p ro l in e , t h e r e s idu e in th e X p o s i t i o n .T h i s p a t t e rn i s i n t e r ru p te d in th e G ly ~ A la s u b s t i t u t io n r e -g io n . T h e re i s n o in fo rm a t io n c o n c e rn in g p o s s ib l e h y d ro -g e n b o n d in g o f a n a m id e in th e X p o s i t i o n , s in c e th i s p o s i -t i o n i s a lw a y s o c c u p ie d b y P ro in th e G ly ~ A la p e p t id e .

    T h e h e l i c a l s y m m e t ry s e e n in th e G ly ~ A la t r ip l e h e l ix i s7 /2 , g iv in g 3 . 5 r e s id u e s / tu rn , t h e s a m e s y m m e t ry fo u n d fo rth e (P ro -P ro -G ly ) l 0 p e p t id e ( se e F ra se r a n d Mc R a e , 1 9 7 3 ,fo r e x p la n a t io n o f n o ta t io n ) . C o h e n a n d B e a r (1 9 5 3 ) h a ds u g g e s t e d th a t c o l l a g e n h a d a 7 /2 s y m m e t ry f ro m th e f ib e rd i f f r ac t i o n p a t te r n , b u t t h e R a m a c h a n d r a n ( 1 9 67 ) a n dR ic h a n d C r i c k (1 9 6 1 ) m o d e l s w e re b a s e d o n 1 0 /3 s y m m e -t ry (3 .3 3 r e s id u e s / tu rn ). C a re fu l e x a m in a t io n o f th e f ib e r

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    T h e C o l l a g e n T r ip l e -H e l ix 5 4 7( a ) D i r e c t H - b o n d i n g

    ( G l y ) N H " " C O ( P r o )

    interchain

    C o ) W a t e r m e d i a t e d H - b o n d i n g l i n k i n gc a r b o n y l g r o u p s

    COfflyp) ... W ... CO(Gly)intrachain intcrchain

    ( c ) W a t e r m e d i a t e d H - b o n d i n g l i n k i n gH y p - O H g r o u p s a n d c a r b o n y l g r o u p s

    O H ( H ~ ) ... w --. CO(GJy)0 1 l O ' l y p ) . " W . . . C O ( H ) ' p )intrachain and intetchain

    c:.O o.L

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    ,K )~ . . . . w . . .

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    :2 ...... . .....;=O ." ( :,,O3 - - " : : ' " ~ cO : >O

    ~. o ~ v . . . . . ( N -

    Fig. 1. A schematic drawin g illustrating the types of hydrog en bondin g pattern s found in the triple-helix: (a) direct peptide group h ydro-gen bonding; (b) water mediated hydrogen bond ing linking carbonyl groups; and (c) water mediated hydrogen bonding linking hydro-xyproline O H groups and carbonyl groups. Variations in the num ber of w ater molecules linking the groups shown in {b) and (c) are seenin the crystal structure, and the water-mediated hydrogen bonds shown are not fully occupied (Bella et al., 1995).

    d i f f r a c t io n p a t t e r n o f s t r e t c h e d ta i l t e n d o n p a t t e rn in d ic a t -e d th a t i t i s v e ry c lo s e to 1 0 /3 s y m m e t ry (F ra s e r e t a l . ,1 9 7 9 ) . I t i s p o s s ib l e th a t t h e re a re lo c a l v a r i a t io n s a lo n g th ec o l l a g e n h e l ix a n d th a t t h e s t r e t c h in g o f t h e t e n d o n m a y re -duce these va r ia t ions , favoring the 10 /3 he l ix . Be l la e t a l .(1 9 9 4 ) h y p o th e s i z e d th a t t h e h ig h im in o a c id c o n te n t o f t h eG ly -+A la p e p t id e a n d (P ro -P ro -G ly ) l 0 m a y re s u l t i n t h e 7 /2s y m m e t ry , a n d i t i s p o s s ib l e th a t s y m m e t ry v a r i e s a lo n g th ec o l l a g e n c h a in , w i th 7 /2 s y m m e t ry in im in o a c id r i c h r e -g io n s a n d 1 0 /3 s y m m e t ry in o th e r r e g io n s . T h e s e tw o s y m -m e t r i e s a re o n ly s l ig h t ly d i f f e re n t i n t e rm s o f t h e lo c a l a p -p e a ra n c e o f t h e t r i p le -h e l ix a n d th e s p a t i a l r e l a t io n s h ip sb e t w e e n n e a r b y a t o m s . H o w e v e r , t h e y a r e d i s t i n c t f r o m ac ry s t a l lo g ra p h ic p o in t o f v i e w a n d w o u ld r e s u l t i n d i f f e re n ta p p e a ra n c e s o f t h e t r i p l e -h e l ix o v e r l o n g d i s t a n c e s .

    Tr ip le -h e l ix h yd r a t io n n e tw o r kT h e a n a ly s i s o f P r iv a lo v (1 9 8 2 ) e m p h a s iz e d th e d o m i -

    n a n c e o f e n th a lp y a n d h y d ro g e n b o n d in g in s t a b i l i z a t io n o fth e t r i p l e h e l ix . T h e r e c e n t c ry s t a l s t ru c tu re o f t h e t r ip l e h e -l ix h a s c o n f i rm e d a n d g iv e n d e ta i l s t o th i s a n a ly si s , s h o w in gth e s t ru c tu re to b e s t a b i l iz e d b y s e ts o f r e g u la r h y d ro g e nb o n d in g p a t t e rn s . A m a jo r s t a b i l iz in g f e a tu re o f a -h e l i c a la n d [~ -s he et p ro te in s t ru c tu re s i s t h e p a r t i c ip a t io n o f e v e ryb a c k b o n e c a r b o n yl a n d a m i d e g r o u p i n N H C = O h y d r o-

    g e n b o n d s . E x a m in a t io n o f t h e t r i p le -h e l ix s h o w s a s er io u sd e f i c i e n c y in th i s r e g a rd . In e a c h G ly -X-Y t r ip e p t id e u n i t ,t h e a m id e g ro u p o f t h e G ly fo rm s a h y d ro g e n b o n d w i th th ec a rb o n y l g r o u p o f t h e r e s id u e in th e X p o s i t i o n in th e a d ja -c e n t c h a i n o f t h e t r ip l e -h e l ix , ( G I y ) N H C = O ( X ) ( Fi g. l a ).T h i s l e a v e s th e c a rb o n y l g ro u p o f t h e g ly c in e r e s id ue s a n dth e c a rb o n y l o f t he r e s id u e in th e Y p o s i t i o n w i th n o a m id eh y d ro g e n b o n d in g p a r tn e r s . I f t h e X o r Y p o s i t i o n s a re o c -c u p ie d b y a m in o a c id s , r a th e r t h a n P ro o r H y p ' , t h e n th e i ra m id e g ro u p s a re a l s o a v a i l a b le fo r h y d ro g e n b o n d in g , b u tn o c a rb o n y l i n th e t r i p l e h e lix i s w i th in b o n d in g d i s t a n ce . Ina d d i t io n , t h e h y d r o x y l g r o u p o f h y d r o x y p r o l i n e p o i n t s o u tf ro m th e t r ip l e h e l ix a n d c a n n o t d i r e c t ly h y d ro g e n b o n d toa n y o th e r g ro u p w i th in th e m o le c u le . T h e m a n n e r in w h ic hth e s e g ro u p s a re s a t i s f i e d i s s e e n in th e G ly ~ A la p e p t id es t ru c tu re , w h e re a n e x te n s iv e a n d o rd e re d w a te r n e tw o rkf o r m s h y d r o g e n b o n d s w i t h a l l a v a i l ab l e c a r b o n y l a n d H y ph y d ro x y l g r o u p s (F ig . 1 ). T h e s t ru c tu re a l s o s h o w sC c ~ H C - - O b o n d s , w h i ch c o n st i t u te a n e t w o r k o f w e a k b u ts y s t e m a t i c h y d ro g e n b o n d s (B e l l a a n d B e rm a n , 1 9 9 7 ) .

    A d e ta i l e d a n a ly s i s w a s c a r r i e d o u t o n th e o rd e re d w a te r sin th e G ly ~ A la t r ip l e -h e l ix s t ru c tu re s (B e l l a e t a l . 1 9 9 5 ) .A l l a v a i l a b le g ro u p s o f t h e p e p t id e b a c k b o n e a n d H y p a res e e n to b e in v o lv e d in b in d in g w a te r m o le c u le s th ro u g h av a r i e t y o f a r r a n g e m e n t s . O n t h e a v e r ag e , t h e C = O g r o u p so f G ly r e s id u e s a re b o u n d to o n e w a te r , w h i l e t h e C =O

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    5 4 8 B . B r o d s k y a n d J . A . M . R a m s h a w

    . : . , . : _ , . . ' 7

    /'

    -. - . 1 . 4

    Fig. 2. Illustration of the packi ng of adjacent triple helices in thecrystal structure of the Gly 'A la peptide. The wate r moleculesbetween the chains are indicated by dots, and the lack of directcontact between neighboring molecules can be seen. (Bella, Brod-sky and Berman, 1994)

    g r o u p s o f H y p s h o w t w o s it es f o r h y d r o g e n b o n d i n g t o w a -te r. T h e O H o f H y p c a n b i n d t w o w a t e r m o l e c u l e s a t t w od i s t in c t s i t e s , b u t n o t a l l p o s i t i o n s a re fu l ly o c c u p ie d . Wa -t e r s b r id g e tw o c a t e g o r i e s o f g ro u p s in th i s p e p t id e : w a te rm o l e c u l e s m a y l i n k t w o c a r b o n y l g r o u p s o r t h e y m a y l i n ko n e c a r b o n y l g r o u p w i t h a h y d r o x y l g r o u p o f H y p . T h en u m b e r o f w a t er s i n v o l v e d i n b ri d g i n g t w o g r o u p s a p p e a r sto v a ry a lo n g th e m o le c u le , s u c h th a t tw o , t h re e , fo u r , o re v e n f iv e w a te r m o le c u le s m a y fo rm a c h a in l i n k in g th e tw og r o u p s . T h e w a t e r m o l e c u l e s m a y b e l i n ki n g t w o g r o u p s o nt h e s a m e c h a i n , e .g . , ( G ly ) C = O W " O = C ( H y p ) ( Fi g. l b )o r ( G I y ) C = O W O H ( H y p ) ( Fi g. lc ) . L i n k a g e s a r e a l sos e e n b e tw e e n g ro u p s o n tw o d i f f e re n t c h a in s w i th in a m o le -c ul e, e .g ., C h a in A H y p ( O H ) W O = C ( H y p ) C h a i n B ( Fig .l c ) .

    In th e c ry s t a l s t ru c tu re , w a te r b r id g e s a re a l s o o b s e rv e dto b e th e c r i t i c a l e l e m e n t i n c o n n e c t in g a d ja c e n t t r i p l e -h e l i -c a l m o l e c u l e s a n d m a i n t a i n i n g t h e i n t e r m o l e c u l a r s p a c i n g(B e l l a e t a l . , 1 9 9 4 , 1 9 9 5 ) . T h e d i s t a n c e b e tw e e n a d ja c e n tmolecu les in the c rys ta l i s 14 A, a va lue ve ry s imila r to tha ts e e n fo r t h e l a t e ra l p a c k in g o f c o l l a g e n m o le c u le s in t e n d o n ,s k in a n d a l l o th e r t i s s u e s e x a m in e d b y f ib e r d i f f r a c t io n . I tw a s s u rp r i s in g to f in d l i t t l e o r n o d i r e c t c o n ta c t b e tw e e nn e ig h b o r in g m o le c u le s (F ig . 2 ) . R a th e r , t h e d i s t a n c e

    b e tw e e n m o le c u le s i s m a in ta in e d b y th e w a te r m o le c u le sw h ic h c o n n e c t a d j a c e n t h e l i c e s . T h i s s u g g e s t s t h a t t h e l a t e r -a l m o le c u la r p a c k in g o f c o l l a g e n m o le c u le s m a y b e d et e r -m in e d l a rg e ly b y th e i r h y d ra t io n s h e l l , w h ic h i s l i n k e d tob a c k b o n e c a r b o n y l s a n d H y p .

    T h e r o l e o f h y d r o x y p r o l i n e i n t h e t r ip l e h e li xC o l l a g e n i s u n iq u e a m o n g a n im a l p ro te in s in i t s h ig h c o n -

    t e n t o f h y d r o x y p r o l i n e , w h i c h i s f o r m e d a s a p o st - t r a n s la -t i o n a l m o d i f i c a t i o n o f p r o li n e s w h i c h a r e i n c o r p o r a t e d i nth e Y p o s i t i o n o f G Iy -X -Y tr ip l et s . B o th p ro l in e a n d h y -d ro x y p ro l in e s t a b i l iz e th e p o ly p ro l in e I I e x te n d e d c o n fo r -m a t io n o f t h e in d iv id u a l c h a in s in th e t ri p l e h e l ix b e c a u s e o fth e s t e re o c h e m ic a l r e s t r i c t io n s o f t h e im in o a c id r in g s. B u th y d r o x y p r o l i n e w a s s h o w n t o c o n f e r a g r e a t e r s t a b i l i t yth a n p ro l in e in th e Y p o s i t i o n , a n d th i s s t a b i l i z a t io n w a ss h o w n to b e s t e re o s p e c i f i c a n d d e p e n d e n t o n b e in g in th e Yp o s i t i o n ( B e rg a n d P r o c k o p , 1 9 7 3 ; B u r j a n a d z e, 1 9 8 2 ) . T h er e a l iz a t i on t h a t t h e h y d r o x y l g r o u p o f H y p c o u l d n o t d i -r e c t l y h y d r o g e n b o n d t o t h e c a r b o n y l g r o u p s w i t h i n t h es a m e m o le c u le l e d to th e p ro p o s a l t h a t i t s e f f e c t w a s m e d i -a t e d th ro u g h b r id g in g w a te r m o le c u le s (F ra s e r e t a l . , 1 9 7 9 ;P r iv a lo v , 1 9 8 2 ) . T h i s h y p o th e s i s h a s n o w b e e n c o n f i rm e dw i th th e c ry s t a l s t ru c tu re , w h ic h n o w o f fe r s d e t a i l s a b o u tth e s p e c ial ro l e o f t h i s u n u s u a l r e s id u e . T h e h y d ro x y l g ro u pc a n a c t as a h y d r o g e n b o n d a c c e p t o r a n d d o n o r , a n d w a t e rmolecu les can b ind a t two d iffe ren t s i te s (Be l la e t a l . , 1995) .I t p l a y s a p iv o ta l ro l e in c re a t in g th e o rd e re d w a te r s h e l la ro u n d th e t r i p l e -h e l ix . A s p o in te d o u t b y B e l l a e t a l .( 1 9 9 5 ) , H y p h y d r o x y l g r o u p s l i n k ed b y w a t e r m o l e c u le s t oa G l y C = O w i t h i n t h e s a m e c h a i n , a n d t o t h e H y p C = O o fth e a d ja c e n t c h a in , a re s u f f i c i e n t t o s a t i s fy a l l h y d ro g e nb o n d in g p o te n t i a l i n t h e c h a in s (F ig . l c ) . A h ig h c o n te n t o fH y p in th e Y p o s i t i o n o f c e r t a in c o l l a g e n ty p e s ( ty p e Il l ,t y p e IV ) m a y th u s e n h a n c e s t a b i l i t y v ia t h i s e x te n s iv e w a te rn e t w o r k .

    A m i n o A ci d S e q u e n c e D e p e n d e n c eo f t h e T r i p l e - H e l i x S t r u c t u r eT h e f ib e r d i f f r a c t io n -b a s e d s t ru c tu re s (F ra s e r e t a l . ,

    1979) , and the Gly *Ala pep t ide s t ruc tu re (Be lta e t a l . ,1 9 9 4 ) fo c u s o n G ly -P ro -H y p t r ip e p t id e s e q u e n c e s in c o l l a -gen . In the (Gly-X-Y)338 sequ ence o f type I co l lag en , on lya b o u t 1 0 % o f th e t r ip l et s a r e G l y - P r o - H y p . T h e r e m a i n i n gt r ip l et s c o n s i s t o f a b o u t 2 0 f r e q u e n t t r i p l e t s fo u n d m o ret h a n 1 % i n t h e se q u e n ce ( D o l z a n d H e i d e m a n n , 1 9 8 6 ) ,a n d a p p r o x i m a t e l y 7 0 o t h e r t r i p l e t s f o u n d o n e t o t h r e et im e s in th e s e q u e n c e I t s e e m s l ik e ly th a t t h e c o m m o n G ly -P r o - H y p , G l y - P r o - A l a a n d G l y - A l a - H y p t r ip l et s a r e r e-q u i re d fo r s t a b i l it y o f t h e t r i p l e -h el ix , w h i l e t r i p l et s c o n -t a in in g c h a rg e d a n d h y d ro p h o b ic r e s id u e s fu n c t io n a s s i t e so f r e c o g n i t io n in v o lv e d in b io lo g ic a l s p e c i f i c i ty . C la r i f i c a -

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    The C o l l agen Tr ip le -He l ix 549t ion o f the mole cu la r ba s is o f r e c ogn i t ion and b ind ing o f aco l l agen s e quence to ano the r mo lec u le requ i re s an unde r -s t and ing o f how va r i ab le (GIy -X-Y)n amino ac id s equencesaffec t bas ic propert ies of the t r ip le-he l ix , such as the he l ica lpa rame te r s , hydra t ion , dyna mics and po ten t i a l i n t e rac -t ions .

    Table II. The therm al stability of host-guest peptides with non-po-lar GIy-X -Y guest triplets, together with the num ber of occur-rences of each triplet in the cd(I) chain (based on Shah et al., 19 96).Triple t T m n hos t-gues t pe pt id e Occ urre ncesn~l(l) (% )Gly-Pro-Hyp 44 C 42 (12 % )

    St ab i l i t y s c a l eA s ca le o f t r ip l e -he l ix p ropens i ty fo r d i f fe ren t GIy -X-Y

    t r ip l e t s wou ld be a f i r s t s t ep toward unde rs t and ing va r i a -t ions in t r ip l e -he l ix fe a tu re s a long the l eng th o f the t r ip l ehe l ix . B a c h inge r a nd Dav i s (1991) p ropos ed a s t ab i l i t ys ca le fo r d i f fe re n t t r ip l e t s , ba s ed on the c lus t e r ana lys i s o fH e i d e m a n n a n d a v a r i e ty o f m o d e l p o l y p e p t i d e d a t a . T h i ss cheme was us ed to p re d ic t r eg ions o f r e l a t ive g rea te r andlowe r s t ab i li t y in c o l l a ge n , and to exp la in the re l a t ive s eve r-i ty o f d i f fe re n t c a s e s o f o s t eogenes i s impe r fec ta (B ach inge re t a l . , 1993). This c lass i f ica t ion scheme i l lus t ra tes the ut i l -i t y o f hav ing s uch a s ca le fo r unde rs t and ing the e f fe c t s o fmuta t ion s , b u t l a cks a f i rm e xpe r im en ta l ba si s.

    The us e o f a " hos t -gues t " pep t ide s e t fo r examin ing thes t ab i l i t y o f the t r ip l e -he l ix p re s en t s an oppor tun i ty fo r e s -t ab l i sh ing an expe r im en ta l ly ba s e d s ca le o f t r ip l e -he l ix p ro -pens i ty as wel l as c lar i fying the in terac t io ns s tabi l iz ing thet r ip l e -he l ix . Fo l lowing the concep t s u s ed to de te rmine thes tabi l iz ing inf luence of individual amino ac ids for the c~-he-l ix (O ' Ne i l a n d D eGr ado , 1990) and ]3- sheet (Smi th et a l. ,1994) , a " hos t -gu es t " pep t ide de s ign ha s re cen t ly been ap -pl ied to the t r ip le he l ix , us ing GIy-X-Y t r ip le ts as the bas icuni t (Shah e t a l. , 1996). T he des ign used for a hos t-gues t se to f t r ip l e -he l i c a l pe p t ide s was ace ty l (G ly -Pro -Hyp)3Gly -X-Y -(GIy-Pro -Hyp)4GIy-Gly -NH 2, whe re GIy -X-Y i s thegues t t r ip l e t s ubs t i tu t ed in to a s t ab i l i z ing cons ta n t (G ly -Pro -Hyp)8 env i ronmen t w i th bo th ends b locked . In i t i a lhos t -gue s t s tud ie s (Sha h e t a l . , 1996) focus ed on the mos tf reque n t non-po la r r e s idues found in co l l age ns , pu t t ingPro , A la , Le u a nd Phe in the X po s i t ions and H yp , A la , Leuand Phe in the Y pos i t ions . A l though Leu and Phe a refound a lmos t e xc lus ive ly in the X pos i t ion in co l l agens ,pep t ide s we re a l s o ma d e wi th them in the Y pos i t ion , t o s eethe c ons e quences . The 12 pep t ide s s tud ied cove r 35 % o fthe seq uence o f the 0~1(I) t r ip le he l ix . Al l pept ides fo rm eds ta b le tr ip l e -he l i c a l s t ruc tu re s and s howe d a w ide range o ftherm al s tabi l i t ies (Tm = 21 C - 44 C), depend ing on theiden t i ty o f the g ues t t r ip l e t (Tab le II ). Th e re s u l t s con f i rm edthe s tabi l iz ing inf luence of imino ac ids , the grea ter s tabi l iz -ing e ffec t o f Hyp c om pared w i th Pro , a nd the l a ck o f hy -d rop hob ic s t a b i l i z a t ion o f the t r ip l e he l ix . Thes e s tud ies a l -s o de mons t ra t ed tha t t r ip l e t s w i th Leu and Phe in Y pos i -t ions c an fo rm s t ab le t r ip l e he l i c e s , even though the y a rel e s s s t a b le tha n whe n the s e re s idues a re in the X pos i t ion(Ta b le I I) . Th e re s u l ts s ugges t t ha t t he p red om inance o f Leuand Phe in the X pos i t ion o f co ll a ge ns i s l i kely to be a con -s eque nc e o f the i r po ten t i a l fo r in t e rmolecu la r in t e ra c t ions

    Gly-Ala-Hyp 40 C 20 (6 % )Gly-Pro-Ala 38 C 31 (9 % )Gly-Leu -Hyp 39 C 11 (3 % )Gly-Pro-Leu 33 C 0 (0 % )Gly-Phe-Hyp 34 C 7 (2 % )Gly-Pro-Phe 28 C 0 (0 % )

    in tha t pos i t ion , r a the r than a re s u l t o f mo lecu la r s t ab i l i t yfac to r s (B ans a l and R amachandran , 1978) .

    The s tudy o f ind iv idua l t r ip l e t s i s a f i r s t s t ep towa rdunde rs t and ing the e f fe c t o f amino ac id s equence , bu t i t isl ike ly tha t i n t e ra c t ions occu r be twee n ad ja ce n t t r ip l e t s fo rs ome s equences . Thu s , i nco rp ora t ion o f two a d jace n t gues tt r ip l et s in to hos t -gues t pep t ide s e ts ma y be requ i re d be fo rethe re c an be an accu ra te s t ab i li t y s ca le fo r c o l l a gen aminoac id s e quences. The l a rge num ber o f pos s ib le GIy -X -Y t r ip -l e t s p re s en t s an obs tac le to e s t ab l i s h ing a c omprehe ns ivesca le of a l l t r ip le ts , but the re la t ive ly smal l number of t r ip-l e ts found wi th a h igh f requency , and the s imp le add i t iv i tyre l a t ions h ips s e en fo r s ome pep t ide s , make s i t pos s ib le toaddre s s m any a s pec ts o f t r ip l e -he l ix s t ab il i ty u s ing a l imi t -ed num ber o f tr ip l e ts .

    E x p e r i m e n t a l s t u d i e s o f d if f e r e n t t r i p e p t id e s e q u e n c e s :N M R s p e c t r o s c o p yThe e f fe c t o f d i f fe ren t t r ipep t ide s eque nces on the t r ip l e

    he l ix i s now acce s s ib le to d i rec t expe r imen ta l app roaches ,th rough the us e o f s yn the t i c pep t ide s o f va ry ing s e que nce" cap ped " by s t ab il i z ing G ly -Pro -Hy p un i t s (L i e t a l ., 1993 ;F ie lds , 1995) . Thes e pep t ide s a re a me nab le to NM R a ndc i rcu la r d i ch ro i s m s pec t ros copy s tud ie s and a re good ca n -d ida te s fo r c rys t a l log raphy . Suc h s tud ie s p rov ide expe r i -m e n t a l d a t a t h a t m a y b e c o r r e l a t e d w i t h r e c e n t c o m p u t e rca lcu la t ions on va r i ed t r ipep t ide s eque nce s (V i t a g l i a no e ta l . , 1993 ; Pa te r l in i e t a l . , 1995) . He re , r e c e n t NM R ap-p roaches to s equence dependen t f e a tu re s a re re v ie wed .

    NM R has the po ten t i a l t o s tudy p rope r t i e s o f the t r ip l e -he l ix in s o lu t ion , a l lowing inve s t iga t ion o f the dyna m ic s o fmolecu le s , and to focus on the p rope r t i e s o f ind iv idua l l a -be l l ed re s idues . NM R s tud ie s in the 1970s and 1980s byTorch ia (1982) cha l l enged the concep t tha t co l l age n f ib r i l sa re rod - l ike mo le cu le s packed in to r ig id f ib r il s by s howingr o t a t i o n a l a n g u l a r m o b i l it y f o r t h e p o l y p e p t i d e b a c k b o n ein col lagen f ibr i ls , together with cons iderable s ide chain

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    5 5 0 B . B r o d s k y a n d J . A . M . R a m s h a wm o t i o n . M o r e r e c e n t l y , m u l t i - d i m e n s i o n a l N M R s p e c t r o s -co p y h as b een ap p l i ed t o co l l ag en - l i k e t r i p l e -h e l i ca l p ep -t ides (Fan et a l . , 1993; Li e t a l . , 1993; Melacin i e t a l . ,1996) . In theory , i t i s possib le to use d is tance in format iono b t a i n e d f r o m N O E a s s i g n m e n t s t o d i r e c t l y d e t e r m i n e t h et r i p le -h e l i ca l co n fo rm at io n i n so lu t io n , b u t i n p rac t i ce , t h isi s h in d ered b y p eak b ro a d en in g f ro m th e ro d - l i k e t r i p le h e -l i x an d b y o v er l ap p in g r eso n an ces f ro m th e h ig h ly r ep e t i -t i ve Gly -X -Y seq u en ces .

    Al th o u g h a co mp le t e s t ru c tu ra l so lu t i o n h as n o t b eenpossib le , pep t ides may be syn thesized wi th select ively ~SNen r i ch ed amin o ac id s , a l l o win g h e t e ro n u c l ea r NM R s tu d i eso n t h e s t ru c tu re an d d y n amics o f sp ec i f i ca l l y l ab e l l ed r es i -d u es . Reso n an ce sy s t em s co u ld b e as s ig n ed fo r t h e r ep ea t -i n g t r i p ep t i d e u n i t P ro -Hy p -G ly p ep t i d e o f (P ro -Hy p -Gly )1 0an d co n n ec t i v i t y sh o wn a lo n g t h e t r i p ep t i d e u n i t (L i e t a l .,1 9 9 3) . A s m a l l n u m b e r o f N O E p e a k s o b s e r v e d b e tw e e n a t -o ms i s co n s i s t en t w i th t h o se ex p ec t ed fo r t h e t r i p l e -h e l i ca lmo d e l an d i n d i ca t e t h a t t h e t h r ee ch a in s a r e c l o se ly p ack edin so lu t i o n as we l l a s i n t h e so l i d s t a t e . NM R s tu d i es werea l so ca r r i ed o u t o n t h e p ep t i d e (P ro -Hy p -Gly )~ - I l e -Th r -Gly -A l a - A r g - l S N G l y J S N L e u - l S N A l a - G l y ( P r o -H y p - G l y )4 , de s -i g n a t e d T 3 - 7 8 5 , w h i c h c o n t a i n s a n i m i n o a c i d p o o r n i n e -r es id u e seq u en ce f ro m ty p e I I I co l l ag en t h e C- t e rm in a l t oth e co l l ag en ase c l eav ag e s i te . lSN r es id u es were i n c o rp o ra t -ed fo r the Gly , Leu and Ala s i tes ind icated , so that these res-i d u es co u ld b e sp ec i f i ca ll y i n v es t ig a t ed u s in g h e t e ro n u c l ea rN M R t ech n iq ues . Th e Gly, I , eu an d Ala r es id u es co u ld b eassigned fo r each of the th ree ch ains in the t r ip le hel ix , andth e NOE p eak s b e tween t h ese r es id u es co n f i rmed t h e o n e-r es id u e s t ag g er an d c lo se p ack in g o f t h ese th r ee ch a in s . H y -d ro g en ex ch an g e s t u d i es i n d i ca t ed t h a t t h e NH g ro u p s o fGly in t he cen t r a l r eg io n o f T 3 - 7 8 5 h av e a f a s t e r ex ch an g era t e t h an t h e N H o f Gly i n (P ro -Hy p -Gly )1 0 (Tabl e I II ) (F ane t a l ., 1 9 9 3 ) . Th i s sh o w s t h e e f f ec t o f n e ig h b o r in g seq u en c-es o n t h e m o b i l i t y o f t h e cen t r a l G ly r es id u e o f t h e h e l i x , i n-d i c a ti n g t h a t t h e G l y - P r o - H y p e n v i ro n m e n t c r e a te s a v e r yr ig i d h el ix , a s e x p ec t e d . N M R a p p r o a c h e s h o l d g r e a t p r o m -i se fo r c l a r i fy in g t h e e f f ec t o f su r ro u n d in g am in o ac id se -q u e n c e s o n s t r u c t u r e a n d d y n a m i c s . H e t e r o n u c l e a r N M Rhas also been used to moni to r the fo ld ing of speci f ic LSN-la-bel led res idues at d i f feren t posi t ions in a pep t ide, d i f feren-t i a t i n g r es id u es i n v o lv ed i n n u c l ea t i o n f ro m th o se i n v o lv edin p ro p ag a t i o n (L iu e t al . , 1 9 9 6 ) .

    In fo rmat io n ab o u t t h e s t ru c tu ra l p ro p en s i t i e s an d d y -n amics o f s i n g l e ch a in mo lecu l es co n t a in in g co l l ag en Gly -X - Y s e q u e n c e s h a s a l s o b e e n o b t a i n e d f r o m N M R s t u d i e s(M ay o e t a l . , 1 9 9 1 ) . Th e r esu l t s sh o w th e i r p o t en t i a l t oa d o p t b e t a - b e n d s t r u c tu r e s w h i c h m a y b e i m p o r t a n t f o r b i -o lo g i ca l si t u a t io n s su ch as h y d ro x y l a t i o n o f t h e u n fo ld edch a in s o r r eco g n i t i o n . Th e co n fo rmat io n a l p ro p en s i t i e s o fco l l ag en t e l o p ep t i d es h av e a l so b een i n v es t i g a t ed b y NM R,ind icat ing the p resence of ex tended s t ructures and [3 - tu rns(Liu et a l ., 1993) .

    S e l f - A s s o c i a t i o n o f T r i p l e - H e l i c e s i n t o F i b r il sCo l l ag en m o lecu l es se l f - asso c i a t e to fo rm th e i r f i n a l fu n c-

    t i o n a l s t a t e i n t i s su es . Th e mo s t we l l ch a rac t e r i zed an dmo s t co l l ag en co mmo n fo rm i s t h e D-p er io d i c f i b r i l , o b -se rv ed as t h e ma jo r s t ru c tu ra l co mp o n en t i n t en d o n , sk inan d mo s t o th e r co n n ec t i v e t i s su es . Ho wev er , n o n - f i b r i l l a rco l l ag en s fo rm a wid e a r r ay o f o th e r su p ramo lecu l a r s t ru c -t u res , i n c lu d in g n e two rk s , an t i p a ra l l e l a r r ay s an d su p er -co i led s t ructures (Kiel ty et a l. , 1993; Bro dsky and S hah ,1 9 9 5 ) . Th e r ecen t c ry s t a l s t ru c tu re o f a t r i p l e -h e l i ca l p ep -t i d e su g g es t s t h a t l a t e r a l a s so c i a t i o n b e tween mo lecu l esmay b e med ia t ed i n l a rg e p a r t b y wa t e r , r a t h e r t h an b y d i -r ec t v an d e r Waa l s co n t ac t s b e tween ad j acen t t r i p l e -h e l i ces(F ig . 2 ) . P rev io u s mo d e l s wh ere P ro an d Hy p u n i t s o f o n emo lecu l e i n t e r ac t d i r ec t l y wi th imin o ac id s o f t h e ad j acen tmo lecu l e mu s t b e r ep l aced wi th imag es o f n e ig h b o r in gmo lecu l es co n n ec t ed t h ro u g h wa t e r b r i d g es , w i th d i r ec ti n t e r ac t i o n s l imi t ed t o t h e l o n g es t s i d e ch a in s . I t ap p ear sth a t t h e h y d ra t i o n sh e l l f o rms a ro u n d t r i p l e -h e l i ces b e fo rethei r sel f -associat ion and p lays a cr i t ical ro le in th is in terac-t i o n . In co n t r as t t o t h e l a t e r a l p ack in g , t h e ax i a l mo lecu l a rs t ag g er o f ad j acen t mo lecu l es i s li k e ly t o b e d e t e rmin ed b yin teract ions involv ing long s ide chains which can br idgean d i n t e r ac t b e twee n ad j ace n t m o lecu l es . I t i s p oss ib le t h a tt h e asy mmet r i c d i s t r i b u t i o n o f Leu an d P h e an d Glu r es i -d u es (X p o s i t i o n p re fe r en ce) an d Arg r es id u es (Y p o s i t i o np re fe r en ce) p o s i ti o n s t h em to i n t e r ac t d i r ec t ly wi th ad j acen tt r ip le-hel ices .

    T h e i m p o r t a n c e o f w a t e r - m e d i a t e d i n t e r a c ti o n s b e t w e e nt r ip l e -h e li ces h as a l so b een d o c u me n ted b y t h e e l eg an t s t u d -ies o f Leik in et a l . (1994 , 1995) . By measur ing the ef fect o f

    Table III. The hydrogen exchan ge rates, protection factors and therm al stability for ~SN-GIy n two different environments in triple-heli-cal peptides (based on F an et al., 1993).Peptide H- D exchange Protection Thermal StabilitySequence rate (*SN-Gly) Factor (tSN-Gly) of peptide(Pro-Hyp-Gly)~lle-Thr-Gly-Ala-Arg- SNGly-Leu-Ala-Gly-(Pro-Hyp-Gly)4 0.5 1077 25 "C

    (Pro-Hyp-Gly)4 Pro-Hyp-SlNG ly-(Pro-Hyp-Gly)s < 0.04 > 104 60 C

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    os mot ic s t re s s on co l l a gen in t e rmolecu la r d i s t ance s , t h i sg r o u p h a s r e p o r t e d s h o r t r a n g e e x p o n e n t i a l r e p u l si o n s t o -g e t h e r w i t h a l o n g e r r a n g e t e m p e r a t u r e d e p e n d e n t a t t ra c -t ion . The s e re s u l t s a re in t e rp re t ed in t e rms o f a nece s s a ryr e a r r a n g e m e n t o f a h y d r o g e n b o n d i n g n e t w o r k o f w a t e r a sco l l agen mole cu le s pack toge the r , i nd ica ting a c r i t ic a l ro l efo r wa te r s t ruc tu re in p ro te in fo ld ing and a s s e mbly .

    Tr ip le -Hel ica l Binding DomainsB i n d i n g d o m a i n s a n d i n t e r a c t i o n sThe c o l l age n t rip l e he l ix ha s been c ons ide re d a s a p ro to -

    type o f a ro d - l ike p ro te in who s e ro le i s s e l f -a s s oc ia tion tofo rm f ib r i l s , bu t the re i s an inc rea s ing apprec ia t ion o f i t srole in spec if ic b inding of o ther molecules (Table I) . Col la-gen in t e ra c t s w i th m any d i f fe ren t k inds o f mo lecu le s (Kad-l e r , 1994) , a nd d i s t inc t p ro te in b ind ing doma ins have beenloc a l i z ed a long the co l l a gen t r ip l e -he l ix , i nc lud ing theun ique b ind ing and c l eavage s it e fo r co l l agenas e, c e l l b ind -ing s i tes for in tegrins , and an ant ibody binding s i te (Fie lds ,1995; Hori e t a l . , 1992) . The binding s i tes a re a l l l inear se-quences a long the cha in , and s ome in te rac t ions have beens how n to be de p enden t on the na t ive t rip l e -he li c a l s t ruc tu reo f co l l a gen , wh i l e o the rs appea r to be independen t o f con -format ion (Fie lds , 1995).

    Examina t ion o f the ba s i c fe a tu re s o f the t r ip l e -he l ixs t ruc tu re s hows tha t i t i s we l l s u i t e d fo r in t e rac t ing w i thother molecules as wel l as for se l f-associa t ion. Al l res iduesin X a n d Y pos i t ions o f the (G ly-X-Y)n s equence a re h igh lyexpos e d to s o lven t and ava i l ab le fo r in t e rac t ions ( J ones andMil ler , 1991). As a resul t , l inear sequences of X a nd Y res i -dues define binding s i tes . Res idues in the X pos i t ion have as omewha t g rea te r e xpos u re than thos e in the Y ( J one s andM i l l er , 1991) and ap pea r to be mo re fa vorab ly p la ce d fo rin t e ra c t ions in s ome ca s e s . The na tu re o f r e cogn i t ionbe tween a t r ip l e he l ix and i t s l i ga nd i s no t known . Hydro -gen bond ing , e i the r d i rec t o r wa te r med ia ted , i s t he domi -nan t in t e rac t ion s t ab i l i z ing the t r ip l e -he l i c a l con fo rma t ionof col lagen. As discussed above , as a col lagen t r ip le-he l ixapproaches ano the r mo le cu le the re , mus t be s ome reo r i en -t a t ion o f i t s hydra t ion s t ruc tu re . A l l hydrophob ic andcha rge d re s idue s a re ava i l ab le on the s u r face fo r in t e rac -t ions w i th o th e r mo le cu le s , and c ha rges in pa r t i cu la r occu rwi th h igh f re qu ency in iden t i f ied b ind ing s it es (Gu l lbe rg e ta l . , 1992; Fields, 1 995). Th ree exam ples of t r ip le-he l ixin te rac t ions w i th o the r mo lecu le s a re g iven be low to i ll us -t ra t e the cu r re n t s t a t e o f unde rs t and ing .

    C o l l a g e n - c o l la g e n a s e i n t e r a c t io n sC ol la gen i s a n unus ua l ly s t ab le p ro te in , and i ts con t ro l l ed

    degrad a t ion i s e s s en ti a l i n no rma l de ve lopm en t and o the rphys io log ica l p roce s s e s . The t r ip l e -he l ix o f f ib r i l - fo rmingco l l agens is r e s is t an t to m os t p ro tea s e s , and i ts deg rad a t ion

    The C o l l age n Tr ip le -He l ix 551is in i t ia ted b y c leavage of a l l three cha ins a t the s i te be tw eenre s idues 775 a nd 776 by co l l agenas e (ma t r ix me ta l lop ro te i -na s e s M M P 1 , 8 and 13 , L i e t a l . , 1995) ) . The confo rma-t ion of the t r ip le he l ix a t and near the c leavage s i te has dis -t inc t ive fea tu re s tha t may be impor tan t fo r r e c ogn i t ion andbinding (Fie lds , 1991). For example , in type I col lagen, the12 re s idues towa rd the C - te rmina l end o f the c l e a va ge si tecon ta in n o imino ac ids, wh ich cou ld re s u l t i n a loos ene d o rs t ruc tu ra l ly a l t e red confo rma t ion .

    The recen t de te rmina t ion o f the th ree -d imens iona l s t ruc -tu re o f co l l agenas e th rough c rys t a l log raphy ha s ra i s ed theques t ion o f t r ip l e -he l ix re cogn i t ion by the enzyme a t t hemolecu la r l eve l. C o l l agenas e s cons i s t o f th ree doma ins : anN- te rmina l c a t a ly t i c doma in , a C - t e rmina l he mope x in - l ikedom a in , and a p ro l ine - r i ch l inke r connec t ing the s e two do -ma ins (L i e t a l. , 1995) . The ca ta ly t i c dom a in a lone ca nno tb ind to o r c l e ave na t ive co l l agen , and e xpe r ime n t s po in t toa ro le fo r the hemopex in dom a in and pe rhaps the l inke r re -g ion in such b ind ing . The s t ruc tu re de te rm ina t ion o f thefu l l l eng th co l l agenas e fo rmed the ba s i s fo r a t t e mpts tomode l the b ind ing o f a t r ip l e -he l ix be tween the ac t ive s i t ea n d t h e h e m e p e x i n d o m a i n , b u t n o o b v i o u s f a v o r ab l e b i nd -ing s i te was seen (Li e t a l . , 1995). One theory pos tula testha t t he p ro l ine - r i ch l inke r reg ion adop t s a po lyp ro l ine I I -l ike s ing le s t randed confo rma t ion wh ich ca n the n in t e ra c tw i th the c o l l agen t r ip l e -he l ix , s epa ra t ing the th re e s t randsand a l lowing c leavage (De Souza e t a l . , 1996). I t i s notabletha t even though the s t ruc tu re s o f bo th co l l age na s e and thet r ip l e -he l ix a re now known to molecu la r r e s o lu t ion , t hemo de o f in t e rac t ion o f the enzym e wi th the t r ip l e -he l ix tha tde term ines c leavag e specif ic ity is ye t to be e luc ida ted.

    C o l l a g e n c e l l -b i n d i n g s i te sC ell b ind ing to co l l agen p lays a ro l e in num erous p hys io -

    logica l processes . Cel l adhes ion to t r ip le-he l ica l col lagen isme dia ted in m any cases by binding to (xl]31, 0~2151 and 0G131in teg r ins , a nd the re i s a l s o in t eg r in - independe n t b ind ing(Gu llberg e t a l . , 1992; Fie lds, 1995). Various ce l lu lar recog-ni t ion s i tes have been ident i f ied within the t r ip le-he l ica l re -gions o f type I , II , II I and IV col lagens . S tudies on pe pt ides in-co rpo ra t ing the s e s equences s howed tha t t he p re s e nce o f thet r ip l e -he l i c a l con fo rma t ion g rea t ly enhanced ce l l adhes ionac t iv i ty in s ome ca s e s, wh i l e the ac t iv i ty was c onfo rm a t ionindepen den t fo r o the r s i te s (F ie lds , 1995) . The m e c ha n i s m o fp la t e l e t agg rega t ion by co l l agens ha s bee n cha rac te r i z ed inde ta i l t h rou gh s tud ie s on t r ip l e -he li c a l pep t ide s (M or to n e ta l . , 1995) an d has b een show n to inv olve both 0t2[31 in tegrinrecogn i t ion o f s pec if i c type I I I G ly -X-Y s equenc e s a nd an in-t eg r in - independen t p roc e s s wh ich re s ponds to (G ly -Pro -Hyp ) n repea t s in a t r ip l e -he l ix fo rm . Thu s , c e l l b ind ing to thetr ip le-he l ix can req uire sequenc e-specif ic in terac t io ns , as in-vo lved in in t eg r in med ia ted b ind ing , o r con fo rm a t iona l s pe-c i fi c , s equence - inde p enden t in t e rac t ions s uch a s thos e cha r -ac te r i z ed fo r p l a t e l e t agg rega t ion .

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    5 5 2 B . B ro d s k y a n d J . A . M. R a m s h a wM a c r o p h a g e s c a v e n g e r r e c e p t o r : t r ip l e - h e l ix l ig a n db i n d i n gN o n -c o l l a g e n o u s p ro te in s w i th a t r i p l e -h e l ix d o m a in in -

    c l u d e C l q , l u n g s u r f a c t a n t a p o p r o t e i n s A a n d D , m a n n o s eb i n d i n g p r o t e in a n d m a c r o p h a g e s c a v e n g e r r e c ep t o r ( M S R )(H o p p e a n d R e id , 1 9 9 4 ; B ro d s k y a n d S h a h , 1 9 9 5 ) . In th e s ep ro te in s , t h e t r i p l e -h e l ix d o m a in m a y h a v e b o th a s t ru c tu -ra l ro le in de f in ing a rod- l ike domain and a ro le in l igandb in d in g (T a b le I ) . T h e s p e c i fi c i ty a n d n a tu re o f l i g a n d b in d -in g b y th e t r i p l e -h e l ix d o m a in o f th e MS R i s u n d e r a c t iv ein v e s t ig a t io n . MS R s a re in t e g ra l m e m b ra n e g ly c o p ro te in sw h ic h m e d ia t e th e u p ta k e o f o x id i z e d L D L , a p ro c e s s im p l i -c a t e d in th e e t io lo g y o f a th e ro s c l e ro s i s (K r i e g e r a n d H e rz ,1 9 9 4 ) . MS R s b in d to o th e r p h y s io lo g ic a l ly im p o r t a n t l i -g a n d s , i n c lu d in g G ra m -p o s i t i v e b a c te r i a t h ro u g h l ip o tei -cho ic ac id (D unne e t a l . , 1994) , and [3 -amylo id pep t id e (ElK h o u ry e t a l ., 1 9 9 6 ) , a n d a re a l s o c a p a b le o f b in d in g a w id ev a r i e ty o f p o ly a n io n ic l i g a n d s w i th c o n s id e ra b le d i s c r im i -n a t io n . F o r in s t a n c e , MS R s b in d to c h e m ic a l ly m o d i f i e dp r o t e i n s ( o x L D L , A c L D L , m a l e y l a t e d b o v i n e s e r u m a l b u -m in ) b u t n o t t o th e u n m o d i f i e d p ro te in s ; a n d to t e t r a p le xnuc le ic ac ids (po ly ( I ) , po ly (G)) , bu t no t to double - o r s in -g le -s t randed nuc le ic ac ids (e .g . , po ly (A) and po ly (C))(K r ie g e r a n d H e rz , 1 9 9 4 ) .

    MS R c o n ta in s d i s c re t e d o m a in s , i n c lu d in g a m e m b ra n espann ing reg ion , a th ree -s t randed co i led co i l {x -he l ica l re -g ion , and a co l lagen-l ike t r ip le -he l ica l reg ion . De le t iona n d s u b s t i t u t io n s tu d ie s s h o w e d th a t l i g a n d b in d in g a n ds p e c i fi c i ty are d e t e rm in e d b y th e c o l l a g e n q ik e t r ip l e-h e l i -c a l d o m a in (K r i e g e r a n d H e rz , 1 9 9 4 ) . T h e u n u s u a l lyb ro a d l i g a n d s p e c i f i c i ty a n d th e d i s c r im in a t io n b e tw e e nd i f f e re n t p o ly a n io n s a re th e re fo re a r e s u l t o f i n t e ra c t io n sin v o lv in g th e t r i p l e -h e l ix . T h e t r ip l e -h e l ix d o m a in (G ly -X-Y)23 has a h igh ly bas ic cha rac te r , and severa l spec if ic ly -s ine r e s id u e s n e a r t h e C - t e rm in u s o f t h e d o m a in w e re im -p l ica ted in b ind ing . A t r ip le -he l ica l pep t ide con ta in ing theG l y - P r o - L y s - G l y - G l n - L y s - G l y - G l u - L y s s e q u en c e f r o m t h isl ig a n d b in d in g r e g io n w a s fo u n d to fo rm s t a b le t r i p l e -h e l -ices a t neu tra l , bu t no t ac id , pH va lues (Anach i e t a l . ,1 9 9 5 ) . T h i s m o d e l MS R p e p t id e w a s fo u n d to b in d to t e t -r a p le x p o ly ( I ) , b u t n o t t o d o u b le - s t r a n d e d o r s in g le -s t r a n d e d fo rm s o f p o ly ( I ), m im ic k in g th e d i s c r im in a t io ns h o w n b y MS R (Mie le w c z y k e t al . , 1 99 6 ) . In a d d i t io n , are l a t e d c ro s s - l in k e d t r ip l e -h e l i c a l p e p t id e w a s fo u n d tob in d a c e ty l a t e d L D L (T a n a k a e t a l ., 1 9 9 6 ) . T h e s e s tu d ie si l l u s t r a t e t h a t a s h o r t l i n e a r (G ly -X-Y) , s e q u e n c e h a s th ea b i l i t y to m o d e l t h e b in d in g a n d d i s c r im in a t io n o f t h e n a -t iv e m a c ro p h a g e s c a v e n g e r r e c e p to r .

    C o l l a g e n D i s e a s e s : S t r u c t u r a l M u t a t i o n s i nt h e T r i p l e H e l i xS o m e h e re d i t a ry c o n n e c t iv e t i s s u e d i s e a s e s a re c a u s e d b y

    m u t a t i o n s i n f i b r i l f o r m i n g c o l l a g e n s , m o s t c o m m o n l y a

    s u b s t i t u t io n fo r a s ing le gly c ine r e s id u e w h ic h b re a k s th e r e -p e a t in g (G ly -X-Y) , p a t t e rn . T h e m o s t w e l l - s tu d ie d d i s e a s ei s o s t e o g e n e s i s im p e r fe c t a , w h e re o v e r 1 5 0 c a s e s o f G lys u b s t i t u t io n s h a v e b e e n r e p o r t e d , r e s u l t i n g in b r i t t l e b o n ed i s e a s e r a n g in g f ro m m i ld to l e th a l (P ro c k o p a n d K iv i r r ik o ,1995) . I t has been sugges ted tha t the degree o f sever i ty o fth e c l in i c a l o s t e o g e n e s i s im p e r fe c t a p h e n o ty p e m a y re l a t eto th e d i s t an c e o f t h e G ly su b s t i t u t io n f ro m th e C - t e rm in a lnuc lea t i on s i te o f the he l ix (g rad ien t m ode l) o r to the loca le n v i ro n m e n t o f t h e G ly s u b s t i t u t io n s i t e ( r eg io n a l m o d e l ) . I th a s a l s o b e e n h y p o th e s i z e d th a t a m u ta t io n in a n im in o a c -id r i c h e n v i ro n m e n t h a s m o re s e r io u s c o n s e q u e n c e s th a no n e in a n im in o a c id p o o r , m o re f l e x ib l e r e g io n (B a c h in g e rand D avis , 19 93) . I t has been sugges ted tha t the subs t i tu -t ion o f a s ing le Gly res idue may lead to a loop o r k ink , asmal l dec rease in the rm al s tab i l i ty o f the co l lagen , and a de -c re a s e d fo ld in g r a t e (P ro c k o p a n d K iv i r r ik o , 1 9 9 5 ) . S tu d ie so n a sim p le (P ro -H y p -G ly )1 0 p e p t id e w i th a s in g le G ly , A las u b s t i t u t io n s h o w e d a l a rge d ro p in th e rm a l s t a b i l i ty , a l o s so f d i r e c t i n t e rc h a in N H O C b o n d s , w h ic h a re r e p la c e d b yw a te r m e d ia t e d h y d ro g e n b o n d in g , a n d a lo c a l u n tw i s t in ga t the Ala s i te (Be l la e t a l . , 1994 ; Long e t a l . , 1993) . Pep-t ides with more rea l is t ic co l lagen sequences a re needed toin v e s t iga t e th e e f f e c t o f e n v i ro n m e n t o n s u c h a s u b s t i t u t io n .I t is w o r t h n o t in g th a t s o m e c a s e s o f f a m i l i al e a r ly o n s e t o s -t e o a r th r i t i s a n d s p o n d y le p ip h y s e a l d y s p la s i a h a v e b e e nfo u n d to b e a s s o c ia t e d w i th c h a n g e s in th e Y p o s i t i o n(A rg , C y s ) o f G Iy -X -Y re p e a t s in ty p e I I c o l l a g e n (P ro c k o pa n d K iv i r r ik o , 1 9 9 5 ) , a n d i t i s i n t r ig u in g to c o n s id e r w h a te f fe ct s s u c h a s u b s t i t u t io n m ig h t h a v e o n p ro p e r t i e s o f t h etr ip le he l ix .

    F u t u r e D i r e c t i o n sT h e r e c e n t c r y s t a l l o g r a p h y a n d N M R r e s u l t s m a k e u s

    c o n f id e n t t h a t t h e b a s i c m o d e l o f t h e t r i p l e -h e lix i s c o r re c tfo r t h e G ly -P ro -H y p re g io n s b o th in th e s o l id s t a t e a n d ins o lu t io n , a n d e m p h a s iz e th e im p o r t a n c e o f t h e w a te r n e t -w o rk a s a n in t e g ra l p a r t o f i t s s t ru c tu re . T o a p p ro a c h th eb io log ica l p rope r t ie s o f co l lagen , in pa r t ic u la r i t s specif icin t e ra c t io n s in s e l f - a s s o c ia t io n a n d b in d in g o th e r m o le -c u le s, t h e n e x t s t e p i s t o u n d e r s t a n d th e e f f e ct o f v a r i a t io n sin a m in o a c id s e q u e n c e o n t r ip l e -h e l ix c o n fo rm a t io n , d y -n a m ic s a n d fo ld in g . T h e s u c c e s s fu l u s e o f s y n th e t i c c o l l a -g e n - l ik e p e p t id e s fo r c ry s t a l lo g ra p h y a n d m u l t i -d im e n s io n -a l N MR w i l l s e rv e a s a fo o th o ld fo r s tu d y in g t r ip l e -h e l i c e sw i th v a ry in g s e q u e n c e s , i n c lu d in g fu n c t io n a l ly im p o r t a n tb in d in g r e g io n s .

    AcknowledgementsWe thank Dr. R.D.B. Fraser for helpful comments on x-ray dif-fraction and Dr. Jordi Bella for valuable discussions clarifying thehydration network. W e are grateful to Dr. Jordi B ella and Dr.Helen Berman for the diagram on which Figure 2 is based. This

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    T h e C o l l a g e n T r i p l e - H e l i x 5 5 3work was supp or ted by NIH g ran t AR196 26 (B .B .), an NSF U.S .-Aus t ra l i a Coop era t ive Resea rch g ran t and the Aus t ra l i a /U .S . B i la t -e ra l Sc ience and T echno logy Col labora t ion Program.

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    Dr. Barbara Brodsky, Department of Biochemistry, UMD NJ-R ob-ert W ood Johnson Medical School, Piscataway, NJ 08 854Received Novemb er 26, 1996