Common cold viruses

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Common cold viruses Michael G. Rossmann, Edward Arnold, James P. Griffith, Greg Kamer, Ming Luo, Thomas J. Smith, GerritVriend, Roland R. Rueckert, Barbara Sherry, Mark A. McKinlay, Guy Diana and Michael Otto The ~tructure o f a human c o m m o n cold (rhmovtrus) serotype, human rhmowrus 14 ( H R V I 4) ts providing extensive mformatton on vtral assembly, stabd~ty, neutrahzatton by annboches and anttwral agents as well as the stte o f receptor attachment

Stephen C H a r m o n m an article for T I B S in 1984~, descnbed the structure and assembly of tomato bushy stunt virus (TBSV) Although a good deal of reformation had been gathered m the previous half century on the structure of the cyhndncal tobacco mosmc virus (TMV)2, TBSV was the first sphencal v~rus to be stud~ed at atomtc resolutmn Many sphencal plant wruses can occur m high concent~:auon m leaves and, after punficauon, can form beauUful smgle crystals Nevertheless, Jt stdl reqmred a great deal of technical and computational development before the structure of Tl3SVwasdetermmedm 19783 Other plant virus structures followed: southern bean mosmc wrus (SBMV) 4, satellite tobacco necros~s virus (STNV) 5 and turmp cnnkle vmrus (TCV)6 It ~s, however, only m the last two years that atomic resolutton results on sphencal ammal v~ruses have become available (although components of influenza viruses had been studied earlier) prananly because ~t ]s far more dtfficult to obtain sufficient quantiues for extenswe X-ray crystallographic stud]es The wealth of avmlable mformaUon, the considerable complex]ty and the dwerse functional demands of ammal vLruses has meant that their structures have provided a depth of understanding not prevmusly achteved There are numerous vu'uses which cause upper respiratory infections A typical cold, w~th a discharge of nasal mucous and perhaps a shghtly elevated body temperature, is frequently caused by the more than 100different serotypes Rtunovlruses are members of the plcornavn'us7 (p=co-RNA-v=rus) family wluch comprises one of the largest fam]hes of M G Rossmann, E Arnold, J P Gnffith, G Kamer. M Luo, T 3 Smith and G Vrlend are at the Departmem of BtologJcaISctences, Purdue Umver511y, West Lafayette, Indiana 47907, USA, R R Rueckert and B Sherryareat the Biophyst¢5 Laboratory, Umverstty of Wtsconsm, Madzson, Wtsconsm 53706, USA, M A McKmla3,. G Dzanamid M Otto are at the Sterhng Winthrop Research hlstaute. Columbm Turnpike, Rensselaer NY 12144. USA

viral pathogens PIcornaviruses cause serious diseases m humans and other animals ranging m seventy from the common cold to paralyuc pohomyehtls Other examples of diseases that can be caused by p,comaviruses are hepatitis, foot-and-mouth disease viruses and encephalomyocard~t]s These viruses are among the smallest RNA animal viruses with a molecular weight of around 8 5 × 106 contmmng approxzmately 30% RNA by weight Their external diameter is roughly 300 ,~ and they form icosahedral shells They d=ffer m vanous physical and chemical properties but also m the number of known serotypes 3 for pohowrus, 7 for foot-and-mouth disease vu'uses and 100 for human rlunov~ruses (HRV) It has been posstble to produce effective vaccmes for pohomyelltlS and, wnh greater difficulty, for foot-and-

mouth d~sease, but the large number of serotypes makes vaccine development difficult for the common cold Pzcomavmons contain 60 protomers, each composed of 4 structural proteins VPI, VP2, VP3 and VP4 The first three have a molecular weight of around 30 000 and the last of about 7 000 The vmons contain a single, positive strand RNA, which ,s translated into a single polyprotein and then processed stepw]se into its component proteins (m part) by xlrally coded proteases The gene order ,s essenhally the same ,n all plcornavlruses Structure and assembly Amongst the greatest surpnses and most e•¢ltlng dlscovenes m the structures of small icosahedral, plant and ammal RNA v=ruses, [s that the folding patterns ol their proteins and the way these proteins organize in the shell are all very similar The plant viruses TBSV s, SBMV 4, TCV 6 and insect black beetle virus (BBV) ° are composed of 180 covalently tdentlcal protein subumts arranged m a ' T = 3' tcosahedral surface latucel ~a In thts arrangement there are three protein subunlts, A. B and C, with the same primary structure and with essenttally the same tertiary structure arranged about aquasi-threefold axts (Fig 1) The shell domain of these protems consists pnmanly of an eight-stranded, antiparallel I] barrel with the wedge end

I~g I Icosahedralcaped the thickly oqlhp'~d VPI VP3 VP2 umt corresponds to thz 6S ( VPI. k'P3. t "PO) protmner and ttze 15-mer cap to the 14S pemamer obsert ed m as~embl) experiments ~)198"/ Ehe,,lerPubhcarton~Cambndgc (1v.76-'~1"~2(11

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314

(b)

VP!

r=r.-.,--,=,--.~-

( ~ =1' I=5

(a)

SBMV

ii

"-',,-.

Fig 2 Dtagrammattc drawings showing the polypeptlde ]'old o[ SBMV and of each o]'the three larger capstd protems of HRVI4 The nomenclatureo]'thesecondary structural elements rs derived from that of SBMV Amino-acid sequence numbers, appropriate for each protein, are a/so shu~n (a) SBMV, (b) VPI, (c) VP2 and VP4 o]`HRVI4, (d) VP3 o[ HRVI4 There are ]'our excumons of the polypepade chains towards the wedge shaped end Each excursion makes a sharp bend or 'corner' The most exterior (top le]'t) corner ts formed between the [l sheets [JB and [tC, the second corner down Is]`ormed between [IH and Ill, the thrrd corner =sbetween [JD and ~JEi and the most internal corner connects ~F and [3G The [JF-[JGcorner ts the stte o]`a 25-residue msertmn m SBMV, including the ot-hehx aC, that is not present m an), o]':he ~rralprotems o]'plcomawruses, norm TBSV or STNV (Adapted from a drawmgofSBMV by Jane Richardson ) (d)

VP3

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(f-2) - ' ' = ' " " ' ||r'g"

T I B S 12 - A u g u s t 1987

315

pomtmg t o an icosahedral fivefold or threefold ax~s In plcornavsrnsesU-13 these subumts consist of three d~stmct proteins VPI, VP2 and VP3, respecuvely These three larger plcornawrus capsid proteins each have a tertiary structure s~mdar to that found m the plant v~ruses and m the insect BBV v~rus P~BC (Fig. 2) although their ammo acid sequences have no obvmus homology. Furthermore, their quaternary organization ts closely smular to that of the T-- 3 plant vu~ses. In the plant viruses TBSV, SBMV, PtAB Pl~B STNV and TCV, the amino-terminal portion (20--80 residues) of each capsld Ftg 3 Stereographtc vlew of the Ca backbone of one HRVl 4 protomer Hypothetlcal hnkages of VPI, VP2, polypeptlde is very bastc and assocmted VP3. and VP4 are mchcated by thinner hnes labeled PI A B, PI BC, and PI CD as they might occur m the with the R N A How tlus RNA Is folded precursorpolyprotempnortopost-lranslat~onalcleavage Dashedpornonscorrespondtotermmalsegments reside the ~nrus cannot be seen crystal- of the native structure that might have rearranged subsequent to processing lograph~cally because, unhke the protein shell, the R N A lacks icosahedral sym- probably needed to permit the protem approxunately four epltopes on the metry The high external synunetry dis- substrate to accommodate itself into the haemagglutmm spike of influenza vlrns courages orientation of the internal enzyme's acUve center to which neutrahzmg antibodies could asymmetric R N A on crystalhzanon The The last step m viral maturation (for- bind Since many humans were hkely to extended amino ends m HRV14 are matron of an mfectmus pamcle) is cleav- have antibodies to one or more of these mtemal to the [~-barreis and mtertwme age of VP0 into VP4 and VP2 This sRes, R required suitable mutations m vath one another Smnlarly the small occurs only when the RNA is bemg each of the four epttopes to produce a VP4 protein, which results from cleav- packaged736 The site of cleavage is virus winch would leave large segments age of VP0 into VP4 and VP2 dunng the deeply buried in the wral capsld and of the population defenseless Indeed tt final stages of assembly, is intertwined therefore not accessible to a protease was possible to show that flu pandemics vdth the ammo ends of VPl and VP3. Nor does the site of cleavage have a were caused by such :hff-ts in the antiAssembly of plcomav~ruses proceeds sequence appropriate to the wral pro- gemc properties of the virus The antlfrom 6S protomers consisting of VPI, tease. The structure of HRVI4, poho- gemc surface of rinnoviros serotype 14 VP3, and VP0, vm 14S pentamers of five wrus and Mungo wrus all show the close has been investigated 192° using tech6S protomers, to mature wrions 7. The proxumty of a serme residue to the car- tuques stnmlar to those that had been mtertwin,.ng of armno and carboxyl ends boxyl-end of VP4, suggesting that the used on influenza viruses. These studies serine might act as a nucleoplnie for an found that there were four different of VPl, VP3, VP0 (VP2 and VP4) (Fig 1) strongly suggests the structural basis autocatalyuc digestion of VP0 l~ssmg, types of epltopes on the viral surface of the 6S assembly unit The 6S proto- however, from the structure is a Insudme Thus a new rhmovlrus serotype may mers are woven into 14S pentamers by base which occurs m other serme pro- occur whenever there has been a slgrufithe VP3 amino ends which form a five- teases. Since the VP0 cleavage occurs cant change m each of the four epitopes fold ~-cylinder surrounded by the amino only during the introduction of RNA, However, the hrmted number of pohothe unpficatmn ss that the RNA bases vu'us serotypes, and the close resemblance ends of VP4. The cleavage s~tes w~thin a protomer ~4 might take the role of the hlst~&ne thus of poho to rhmovuus shows that some producing an enzyme that ~s partly pro- other unknown factors are at work are posiuoned m regmns between the Antibodies can bind to wruses but barrel domains The probable manner in tern and partly nucleic acid 15 The processing of picornavwal pro- they do not necessarily neutralize mfecwhich the carboxyl end of VP3 could be tomers into VP0, VP3 and VPl is a tmty Neutrahzlng antibodies can cause assocmted w~th the amino end of VPl necessary prerequisite to pentamer for- viral cross-hnkmg but more often interbefore proteolysls is shown m Fig 3 After cleavage the new ends would easdy maUonl4.15.17 The amino termnu of VP3 fere w~th wral functions such as cell reposmon themselves into onentatmns released by cleavage, associate with each attachment, membrane penetration or observed m crystallized wnons without other m a parallel five-stranded []-cyhn- uncoating It has been shown that as few d~sruptmg the contacts between [~-bar- der about the i~sahedral fivefold axis as one annbody can neutrahze pohorels The pervasiveness and stablhty of and thus stainhze the formation of 14S v~rus2S.-'2 and that bivalent antibodies the ~-barrel arrangement m R N A vir- pentamers The cleavage of VP0 pro- may be necessary for neutral~atlon 2~ uses suggests that they probably fold and rides a sw~tch from the reqmrements of Neutral,TaUon is frequently accomorgamze themselves w~tlun a protomer assembly (m which the VP0 protein pained by an lsoelectnc change of the even before they are cleaved away from mamtams the integrity of the 6S and 14S vu'us23, indicating that a conformatlonal the nascent polyprotem The vwal pro- assembly umts) to an infectious parUcle change may have occurred Escape mutants to neutrahzmg teases are exceedingly specific For which may require the separate disasmonoclonal antibodies mapped Into four sembly of VP4 and VP2 during meminstance, the vtrally coded protease '3C' chstmct regions on the external vu'us surin pohovirus cleaves pnmardy between brane attachment and penetrahon face of HRVI4, consistent with the nonGIn-Gly paus However, not all the overlapping epltopes found w~th the Neutralization by anfibedies GIn-Gly pairs are cleaved Those wluch In 1981 Don Wiley, lan Wdson and monoclonal antibodies 192° The neutare not he in apparently ng~d pomons of the polypept~de 14 Thus flex~b~hty is John SkeheP 8 showed that there were rahzmg ~mmunogemc s~tes were called

TIBS 12-August 1987

316

FIg 4 The canyon hypothes:s The presence of depressions on the p~cornawrus surface suggests a strategy for the evas~on of ~mmune survedlance The d#menstons of the putauve receptor bmdmg sltes (the canyon m HRVI4, the p,t m Mengop stencally hinders an an:~body's (top right) recogmtlon of residues at the base of the stte, ~hde soil allowing recognmon and bmdmg by a smaller cellular receptor (top left) This would allow for receptor spec~ficay while at the same nme permtmng evoluaon of new serotypes by mutanng restdues about the nm of the canyon or ptt

NIm-IA, NIm-IB (on VP1), NIm-II (pnmanly on VP2) and NIm-III (primarily on VP3) The chstance between the nearest twofold related ]mmunogemc sites are 120, 120, 50 and 60 A respectwely Lower and upper hmlts of the distance of the two ant,body binding s,tes on an ~mmanoglobuhn molecule probably he m the range 50-180 A As an antibody Rself has a twofold axas, the orgamzatlon of ant)gemc rotes on the v~rus surface is consistent w~zh binding across twofold axes It ~s, however, conce)vable that bivalent attachment could also occur across three- or fivefold related axes The resultant torque m~ghtproduce conformatlonal changes suffictenz to interfere w~ththe normal process of refection Experiments are now m progress to study the structure of the Fab components of some of the neutrahzmg monoclonal antibodies to HRVI4 and thmr complexes w~th the appropnate peptades to deterrmne the mode of antibody bradmg that causes wral neutrahzat~on

uon by antibodies Thus, there is a confl~ct between the need for conservation of receptor bmchng and the des]rabahty for change of the ant]gemc surface The surface of HRV14 has a 25 ~ deep canyon c~rculatmg about each fivefold axis I~ wlule Mengo virus 13 has a set of five deep p, ts d~stnbuted about each fivefold ax~s The amino acids that hne the canyon and p~t are far more conserved than res,dues elsewhere on the v~ral surface (the armno acid sequence of HRV14 was independently determmed2S.26), it has therefore been hypothesized that these deep surface depressmns are the s,te of host cell attachment (Ftg 4). Various other pieces of ctrcumstanttal ev,dence support this hypothesm but no completely chrect venfication of the canyon as host cell attachment sRe has yet been possible Neutralization with antiviral agents The Sterhng Winthrop Research Institute has mvesugated a large number

The receptor attachment site

lhcomav]ruses mltmte entry into host cells by attaching to receptors on the host cell membrane Two types of receptors to rl'anov~ruses are known to exist on human (HeLa) cells24 HRV14 belongs to the larger group of rhmovlrus serotypes wlach compete amongst each other for cell attachment, but do not compete w~th serotypes from the minor group The receptor attachment s~te must remmn sufficmently conserved that the virus continues to be able to bind to the same receptor Yet the v~ral surface m wnons that infect animals that have an ~mmune system ~s under pressure to change and thereby to avoid neutrahza-

of compounds27 that prevent uncoatlng after v~ral attachment and membrane penetration Whde most tests of these compounds have so far been made only m tissue culture, it has been shown that the drug WIN 51,711 can reverse symptoms of paralyms m mice due to pohovlrus tf the drug is gwen sufficiently soon after mfecUon2s Two of these compounds have been stuched structurally when complexed to HRV1429. They brad mto a hydrophoblc pocket wttlnn the VPI []-barrel (Rg 5) and are accompained by large (up to 4 A m the mmn chain C a pomtmn) conformattonal changes Various Imes of evidence show that these compounds stab]hze the v~rions by mluhtmg conformat]onal changes that would otherwise occur during the uncoaUng process WIN 52,084 (Fig 5) differs from WIN 51,711 only by an addltaonal methyl group which then creates an asymmetric carbon atom The imtml studies of WIN 52,084 were made with a racewac maxture. However, the X-ray crystallographic results showed that the S isomer was bound to the vinons Subsequent checks of efficacy voth the two purified opt,cal ~somers showed that the S isomer had ten times the antmral acuvtty of the R isomer, conustent vnth the preferred blndmg to HRV14. Predlctmns on the efficacy of various ample modd~catmns of the compounds are now possible based on arguments of steric hindrance within the binding pocket In reverse, s~milar arguments can be apphed to predict the efficacy of antiviral compounds in an homologous HRV serotype where armno acids that hne the binding pocket have been changed However, such pred~cttons are only partially successful, perhaps m part due to the unpredictable effect a compound has on the conformatmn of VP1. An ongoing search for drug resistant and drug dependent mutants may help m understanding the relauon-

C*NVON V,.OOR

i

104

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152

188

Fig 5 D:agromm~ncwpwsento1~onof anavlralcompoundWIN 52,084bindings~te

TIBS 12 August 1987

317

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Fig 6 Poss~ble evolunona 0 scheme for wral evolu. non based on d~fferences m capstd structures Numbers give approx¢mme percentage change o f structurally eqmvalenced amino ands

Prlmordlol Cell Attachment and Pockoglng Protein

conconovolin A

DNA other RNA viruses virus cepsid domains

1 k

T=3 viruses

primitive ptcornoviruses

I0

cowpea mosaic vwus (Stauffacher, C V , Usha, R , Schrmdt, T , Hamngton, M , Arnold, E , Kamer, G , Vnend, G and Johnson, J E , unpubhshed) Another crucial feature m the structure determination of viruses is the rapid collection of good X-ray dlffractmn data pnor to extensive radiation damage The work on HRVI4, Mengo sarus, BBV and CpMV employed synchrotron sources at Cornell and Lure, with earher exploratory data from the Hamburg and Daresbury sources, while the polio studies depended on cooling of crystals to about -20°C in the presence of a cryosolvent to avoid excessive radiation damage

14 primord,al picornovlruses

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various

17

p~c0mavlruses

viruses

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Evolution Three-dimensional protein structures are generally conserved far longer than pnmary protein sequences ~ The major differences between homologous structures usually correspond to delettons and msertmns while the essential polypeptide folding motif is maintained The extent of these changes can be used as a rough measure of evolutionary dwergence (Fig 6) m the same way as thfferences between amino acids in aligned sequences The degree of slmdanty of tertiary structure between capsld protelns of ammal and plant icosahedral RNA wruses is better than, for instance, the s~mdanty between the NAD binding domains in dehydrogenases 3° Thus, it is probable that these wruses have diverged from a pnmordml vtrus Argos etal 3~ suggest that such a precursor may have been related to an ancient receptor binding protem, e g lectm concanavahn A, which also has the same fold and which can compete ~lth pohovlrus for HeLa cell receptors it may therefore be significant that the hexon unit of the DNA adenowrus ~z contains two domains folded like the SBMV coat protein and that the influenza v~rus

a.

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ship between efficacy, binding energy, viral and drug conformauons as well as showmg which pmts of the structure control the stabd~zat~on of the wnons

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haemagglutmm spike also contains this fold as its globular extremity 33 technical comment One of the central procedures which made it possible to determine the structure of lcosahedral wruses utdlzes the symmetry in the wnon that is not incorporated rata the crystal lattice This possibility was recogmzed in a 1962 pubhcatton ~ and was subsequently explored m numerous studies A major advance was made m the apphcatmn of the concepts by the use of real space averaging of electron denqty between non-crystallographically related subumts 35-38 ApphcaUon of tlus 'molecular replacement' method has become progressively bolder In the structure detern'unatmn of HRV 14, the central crystallographic 'phase problem' was solved by extending, in very small steps, the resolutmn from 5 to 3 Rapid exploration of the methodology and application of the techmque to HRVI4 was greatly facdltated by avadability of a Cyber 205 super-computer The computational power of tlus machine allowed the qmck evaluation of several alternative schemes for phase extension of HRVI4 before selectipg an optimal technique Such methodology was subsequently used in the structure determination at pohovlruS 12 and, in an even more daring apphcatmn, tO Mengo VLrUS13 as well as to black beetle virus9 and

A

Acknowledgements We thank all those who have participated in the investigations reported here and S Fateley, S Wilder and K Shuster for help in preparation of this manuscript The work was supported by NIH and NSF grants to MGR, an ACS grant to RRR, and postdoctoral fellowships from Jane Coffins Child Memonal Fund to TJS and from N1H to EA, respectively References I Harmon S C (1984)Trends Blochem Sa 9 .'t4%351 2 Namba K andStubbs G (Iq86)£ctence231 14011406 and Holmes K C (1980) Trends Btochem Sa 5 4-7 3 Harmon S C Olson A J Sehut! C E Wmkler F K andBncogne G (Iq78)Namre276 368-373 4 Abad Zapalero C , AbdeI-Megmd S S Johnson, J E , Leslie A G W Raymenl, I Rossmann M G Suck D andTsukthara T (1980)Nature286 33-39 5 Ldlas L Unge T Jones T A Fndborg K Lovgren S Skoglund U and Strandberg B (1982) J Mol Biol 159,93-108 6 Hogle J M Maeda A aodHamson S C (lq85)J [Viol Brol 191 6~-%638 7 Rueeken R R (1986) m Fundamental Virology (Fields B and Kmpe O K eds) pp 357-390, Raven Press 8 Hamson S C Olsoa A J SchulI,C E Wmkler F K =.d Unc~gne G 0978)Nature 276, 368-373 q Hosur M V,Schmidt "t Tucker R C Johnson J E Gallagher T M Selhng B H and Rueckert R R Protein(m press) I0 Caspar D L D and Klug A (1962) ColdSpnng HarborSymp Quanr Bto! 27 1-24 I I Rossmann M G Arnold E Enckson J W Frankenberger E A Gn[fith J P HeLht H J Johnson J E Kamer G Luo M Mosser A G Rueckerl R R Sherry B and Vnend G (1985) ~amre 317 145--153 12 Htogle J M Chow M andFdman D J (Iq85) Science229.1"4~8-1365 It Luo M Vriend G Kamer G Minor I Anmld E , Rossmann M G Boege U Scfaba. D G Duke G M and Palmenbetg A C (~987)Saence 235 182-191 14 Toyoda H Nicklm M J H Murray M V Anderson C W Dunn J J Studler F W and W,tamer E (1986)Ce1145 761--770 ~'~ Arnold E Luo M Vnend G Rossmann M G Palmcnberg A C Parks G D Ntckhn M J H andWimmer E (1987)Proc Natl..tcad So USA84 21-25 16 JaLobson M F Asso J andBaltlmore D (1970)J Mol Btol 49 6'i7-669 17 Palmenberg A C (1982)1 Viral 44 900-.906 18 Wiley D C Wdson I A andSkehel J J (|a81) Nature 289 373-378 19 Sherry B and RueLkert R R (1985)J Viral 53 1~7-143 20 SherD B Mosser A G Colonno R J and Rueckerl R R (Iq86~J Viral 57 246--257 21 Icenogle J Shtw~n H Duke G Gdberl S

TIBS 12 -August 1987

318 RueckeN. R and Andetegg, J (1983) Vwology 127 412--425 22 Wetz, K Wtlhngman. P, Zelchhardt, H and Harbermehl K V (1986)Arch VIrol 91 207-220 23 En.m.E A Jameson.a A andWtmmer E (1983)

Nature 304 699.-703 24 Abraham G and Colonno, R J (1984)J Virol 51 341k34S 25 Stanway G Hughes P J Mountford, R C Minor. P D and Almond. J W ([984) NuclezcAcrds Bes 12, 7859-7875 26 Callahan P L Mu.,mant, S and Colonno n J (1985)Proc NatlAcad So USA 82 732-736

27 Dmna. G MclGnlay, M Otto M , Akulhan V and Oglesby C (It~841)J Med Chem 28 IqWo 2g McKmlay M A and Steinberg, B A (1986)Anu mlcrob AgentsChemother 29 30 29 Smith T J Kremer, M J Luo M Vnend, G Arnold E Kamer G Rossmann. M G , McKmlay M A Dmna G D and Otto. M J (1986) Science 233, 1233-1356 30 Rossmann M G , Moras O and Olsen. K W (1974) Nature250 194-199 31 Argos. P Tsukthara.T and Ro~smann M G (1980) J Mol E¢ol 15,169-179 32 Robetls M M Wh,te J L , Grutter M G and

Molecules for strenflth and shape:

our fibre-reinforced composite bodies J. E. Scott Soluble polymers (proteoglycans, PGs) associate with the msoluble fibrds (collagen) of connecnve nssues, regularly and specifically Tissue elasncay, calctflcanon, fibril growth and f i e structure, for example, m the transparent corneal stroma, are much mfluenced by these mteracnons The mdependent hvmg cell was a major achievement of early evolution, but as such, a orcumscnbed success An independent cell must do everytlung for itself, and w~tlun so small a volume there can be only a hi]ted amount of metabohc machinery A great leap forward came when cells learnt to collaborate, allowing some to specialize in one funcuon, others in another, m an endless vanety. However, great variety without tight organization equals great confusion, and control systems had to evolve to keep pace. At higher levels of complexity, control systems are ineffective unless the spatial relationships between parts of an organism remam reasonably constant. A central nervous system is inconceivable w~thout a definite shape A tissue system evolved to maintain the shape of the organism, even under the stresses of movement the connective ussue. The story of the evolution of single cells into higher animals is also the story of the evolution of connective tissues The simple plan of connective tissue has remmned unchanged throughout much of evoluhon. There are two mare elements; insoluble hbnls which resist tensile or pulling forces, and the watery ]nterfibnllar material or 'ground substance' which acts hke the stuffing In a cushion, res~stmg compressive forces and inflating the fibrous meshwork. The 2 F. Scott ts at the Department of Chermcal Morphology, Cell and Structural Biology, Chemistry Buddmg, Unwerst0, of Manchester, Oxford Road, Manchester MI3 9PL, UK (~ 1987 ElsevierPubhcattons Camlmdge 0376- 5067187/$020O

cells excrete and absorb metabohtes via the ground substance The connective tissues that take mainly tensile forces, (e g tendon) are mainly fibrous, while those that elastlcafly resist compresswe forces (e g cartilage) are rich in ground substance Advances dunng the last 30 years have elucidated the chemical structures of the protein nbres, collagen and elastlnl, and of the charactensuc soluble polymers of the ground substance, the proteoglycansz (PGs) (Fig 1) Although the fibrils and PGs are intimately associated at the rmcroscoplc level, little was known of whether specific molecular interactions are involved Indications that the primary chemical structures of the participants are important in their interactmns came from m varo experiments using soluble collagens and PGs 3 However, there is a fundamental &fference between such systems and those in functmmng ussues Tissue collagen fibnls are side-by-side and end-to-end aggregates of long thin collagen molecules, often covalently cross-hnked (Fig 2) This close apposition presents completely new, stable arrays of amino acads, extending across many collagen molecules, provichng vastly mcreased opportunlUes for specific PG biding, as compared with single collagen molecules m solution Thus, examination of tassues for interactions yields results that have structural and functional significance Connective tissue ultrastrueture Although upwards of ten different collagen types are known, most of the body

Burner R M {198b)Sc~cnce232 1148-1151 ";3 Wilson. I A . Skehcl J J dnd Wdcy. O C 0981) Nature 289 366--373 34 nossmann M G and Blow D M (1962)Acta Costallogr 15 24--31 35 S~gl,~r P B Blow D M Matthews n w and Henderson. R (1968)./ Mo/ Biol 35. 143-164 "46 Buehner, M , Ford. G C , Moras. D . Olsen. K W and Rossmann.M G (1974)J Mol Bfol 82,563--585 37 Flettenck, R J and Stettz T A (1976)Acta Crystallogr , Sect A 32. 125-132 38 Bncogne. G t1974) A~ta Crystallogr Secl A 30, 395-4O5

collagen ~sof one gene product, the first to be characterized, and hence known as type I Several major connective tissues, for example bone, tendon, skin, sclera and cornea contam little of any other type Thus, the interaction of PGs with type i collagen ~s of particular mterest Tendon t~ssue is ~deal experimental matenal as it ~s morphologically simple and has an uncomphcated function Collagen fibrds are insoluble, permanent structures readily visible, w~th or without staling, by electron microscopy In contrast, the POs in aqueous solution are highly swollen, and very difficult to v~suahze, except after stmmng Tins relatwe mvmbday gave nse to the classical anatonucal descnptlon of the inteffibnllar material as 'amorphous' ground substance. The relevant substrates are PGs containing chondromn sulphate, dermatan sulphate and keratan sulphate (Fig. 1), which can be stamed with Cupromeronlc blue 4, now available commercially Its intense colour, high electron density, and relatively small s~ze allows sensitive and precise Iocahzauon of the substrate. Specificay depends on the application of the 'cnfical electrolyte concentration' (CEC) principle4 Thus, the salt concentration (e g. magnesium chlonde) m the dye bath ]s adjusted so that only sulphated polyanions take up stain The results are confirmed and extended by the use of enzymes which specifically digest PG polysacchandes" keratanase, heparitinase, and chondroltmase ABC or A C The applscataonof Cupromeromc blue to tendon revealsa beautiful regular pattern, m which filamentous PGs are arranged across the outside of the collagen fibrils,separated by a repeat dlstance of about 60 n m ThlS implies that there is a specsfi~btndtng sltefor the P G (shown to be derm~tan sulphate-nch) every 60 n m along the P G Fibril Itwas of great interestto know more of thisbinding rote The collagen flbrl[ t.'...--up heavy metal stmn, e g uranyl, UO2Z+, in a pattern of bands, designated a-e, which arise out of the lateral juxtaposition of