Ž . Biochimica et Biophysica Acta 1383 1998 9–36 Review Legume lectin structure Remy Loris ) , Thomas Hamelryck, Julie Bouckaert, Lode Wyns Laboratorium Õoor Ultrastruktuur, Vlaams InteruniÕersitair Instituut Õoor Biotechnologie, Vrije UniÕersiteit Brussel, Paardenstraat 65, B-1640 Sint-Genesius-Rode, Belgium Received 16 July 1997; revised 13 October 1997; accepted 16 October 1997 Abstract The legume lectins are a large family of homologous carbohydrate binding proteins that are found mainly in the seeds of most legume plants. Despite their strong similarity on the level of their amino acid sequences and tertiary structures, their carbohydrate specificities and quaternary structures vary widely. In this review we will focus on the structural features of legume lectins and their complexes with carbohydrates. These will be discussed in the light of recent mutagenesis results when appropriate. Monosaccharide specificity seems to be achieved by the use of a conserved core of residues that hydrogen bond to the sugar, and a variable loop that determines the exact shape of the monosaccharide binding site. The higher affinity for particular oligosaccharides and monosaccharides containing a hydrophobic aglycon results mainly from a few distinct subsites next to the monosaccharide binding site. These subsites consist of a small number of variable residues and are found in both the mannose and galactose specificity groups. The quaternary structures of these proteins form the basis of a higher level of specificity, where the spacing between individual epitopes of multivalent carbohydrates becomes important. This results in homogeneous cross-linked lattices even in mixed precipitation systems, and is of relevance for their effects on the biological activities of cells such as mitogenic responses. Quaternary structure is also thought to play an important role in the high affinity interaction between some legume lectins and adenine and a series of adenine-derived plant hormones. The molecular basis of the variation in quaternary structure in this group of proteins is poorly understood. q 1997 Elsevier Science B.V. Keywords: Lectin; Protein–carbohydrate interactions; Quaternary structure; Carbohydrate recognition Contents 1. Introduction ................................................... 10 2. The legume lectin monomer ......................................... 10 Abbreviations: PHA-L, phytohemagglutinin-L from Phaseolus Õulgaris; PHA-E, phytohemagglutinin-E from Phaseolus Õulgaris; SBA, soybean agglutinin; Con A, concanavalin A; GS-IV, Griffonia simplicifolia lectin IV; GS-II, Griffonia simplicifolia lectin II; LOL, Lathyris ochrus lectin; LOL I, Lathyris ochrus isolectin I; LOL II, Lathyris ochrus isolectin II; PNA, peanut agglutinin; DBL, Dolichos biflorus seed lectin; UEA-I, Ulex europaeus lectin I; UEA-II, Ulex europaeus lectin II; MDP, N-acetylmuramyl dipeptide; NMR, nuclear magnetic resonance; NMRD, nuclear magnetic resonance dispertion ) Corresponding author. Fax: 0032-2-3590289; E-mail: [email protected][email protected]0167-4838r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. Ž . PII S0167-4838 97 00182-9
Transcript
Ž .Biochimica et Biophysica Acta 1383 1998 9–36
Review
Legume lectin structure
Remy Loris ), Thomas Hamelryck, Julie Bouckaert, Lode WynsLaboratorium Õoor Ultrastruktuur, Vlaams InteruniÕersitair Instituut Õoor Biotechnologie, Vrije UniÕersiteit Brussel, Paardenstraat 65,
B-1640 Sint-Genesius-Rode, Belgium
Received 16 July 1997; revised 13 October 1997; accepted 16 October 1997
Abstract
The legume lectins are a large family of homologous carbohydrate binding proteins that are found mainly in the seeds ofmost legume plants. Despite their strong similarity on the level of their amino acid sequences and tertiary structures, theircarbohydrate specificities and quaternary structures vary widely. In this review we will focus on the structural features oflegume lectins and their complexes with carbohydrates. These will be discussed in the light of recent mutagenesis resultswhen appropriate. Monosaccharide specificity seems to be achieved by the use of a conserved core of residues thathydrogen bond to the sugar, and a variable loop that determines the exact shape of the monosaccharide binding site. Thehigher affinity for particular oligosaccharides and monosaccharides containing a hydrophobic aglycon results mainly from afew distinct subsites next to the monosaccharide binding site. These subsites consist of a small number of variable residuesand are found in both the mannose and galactose specificity groups. The quaternary structures of these proteins form thebasis of a higher level of specificity, where the spacing between individual epitopes of multivalent carbohydrates becomesimportant. This results in homogeneous cross-linked lattices even in mixed precipitation systems, and is of relevance fortheir effects on the biological activities of cells such as mitogenic responses. Quaternary structure is also thought to play animportant role in the high affinity interaction between some legume lectins and adenine and a series of adenine-derivedplant hormones. The molecular basis of the variation in quaternary structure in this group of proteins is poorly understood.q 1997 Elsevier Science B.V.
Recognition between proteins and carbohydrates isof prime importance in many biological processes,such as viral, bacterial, mycoplasmal and parasiticinfections, targeting of cells and soluble components,fertilisation, cancer metastasis and growth and differ-entiation. Legume lectins are the model system ofchoice to study the molecular basis of these recogni-tion events because they are not only easy to purifyin large quantities, but also exhibit a wide variety ofcarbohydrate specificities despite strong sequence
w xconservation 1 .Legume lectins have, for decades, been a paradigm
in the area of protein–carbohydrate recognition.Lectin activity in plant seeds has been demonstrated
w xas early as 1888 2 . Despite the lack of knowledgeon their in vivo activity, they have proven to beuseful tools in immunology and glycobiology be-cause of their wide range of specificities for complexcarbohydrates. Their carbohydrate specificity has alsoallowed them to be used in such applications aspurification and characterisation of complex carbo-hydrates and glycoconjugates and bone marrow trans-plantation. Thus, the in vitro function of these pro-
Ž .teins carbohydrate recognition is well documentedand the molecular basis of this interaction has beenstudied with a variety of biophysical techniques, in-cluding X-ray crystallography, NMR and mi-crocalorimetry.
Legume lectins are not only of interest because oftheir carbohydrate binding properties. Related to theirpotential to agglutinate cells and to precipitate multi-valent carbohydrates is the fact that these proteins areoligomeric, forming either dimers or tetramers. Re-cent structural data have demonstrated that an essen-tially strongly conserved monomeric unit canoligomerise in a variety of ways, making these pro-teins also of interest to analyse the details ofprotein–protein interaction. Furthermore, quaternarystructure relates to activity, as has been shown inrecent years for a variety of lectins: highly orderedhomogeneous cross-linked lattices are formed whenlectins are mixed with multivalent carbohydrates, re-sulting in a higher form of specificity than can beachieved on the level of the monomer. In this review,we will focus on the structure of legume lectins andanalyse the structural basis of their carbohydratespecificity.
2. The legume lectin monomer
The legume lectins form a large family of homolo-gous proteins. Of about 50 legume lectin sequencesthat have been determined all show pairwise se-quence identities not lower than 35%. Recently, it hasbeen suggested that also in the animal kingdom
w xlegume lectin homologues may be present 3–5 . For11 members of the legume lectin family, crystallo-
( )R. Loris et al.rBiochimica et Biophysica Acta 1383 1998 9–36 11
graphic co-ordinates have been deposited at the pro-Ž .tein data bank see also Tables 1 and 2 . These
w x w xinclude concanavalin A 6,7 , PHA-L 8 , the lectinsw x w x w xfrom pea 9 , lentil 10,11 , Lathyrus ochrus 12 ,
w x w x w xpeanut 13,14 , soybean 15 , coral tree 16 , Griffo-w xnia simplicifolia lectin IV 17 . The structures of the
lectin-related a-amylase inhibitor from Phaseolusw x w xÕulgaris 18 and arcelin 19 , which have no known
carbohydrate binding activity, are also known. Thebest refined structure to date is that of concanavalin
˚ w xA, which has been refined at 1.2 A 20 and presently˚ w xbeing further refined against 0.94 A data 21 . Not
surprisingly, the legume lectin monomer is struc-turally well conserved. It consists of two large b-pleated sheets that form a scaffold on which the
Ž Ž ..carbohydrate binding region is grafted Fig. 1 a .The same architecture and topology is found in awide variety of carbohydrate recognising proteins
w xsuch as the galactins 22,23 , serum amyloid proteinw x24 , the lectin-like domains Wing-1 and Wing-2attached to the catalytic domain of Vibrio cholerae
w xneuraminidase 25 and Charcot–Leyden crystal pro-w xtein 26 . The topology of the legume lectin fold,
Ž .which is shown in Fig. 1 b , is complex and isstructurally related to the jelly-roll topology com-
w xmonly found in viral coat proteins 27,28 . The archi-tecture of the legume lectin monomer is usuallydescribed as consisting of two b-sheets. As was
pointed out by Banerjee and co-workers, the correctdescription involves three b-sheets: the 6-strandedback sheet, the 7-stranded front sheet and a smaller
Ž .5-stranded sheet which we will call the S-sheet , thatplays a major role in holding the two large sheets
w xtogether 14 . The main hydrophobic core is locatedbetween the back and the front sheet. No a-helix ispresent and about 50% of the residues are in loop
Žregions. One of these loops an V-loop containingthe conserved glycine of the monosaccharide binding
.site – see below curls over the front sheet, resultingin the formation of a second hydrophobic core be-tween the front sheet and this loop.
Carbohydrate binding activity of legume lectinsdepends on the simultaneous presence of both acalcium and a transition metal ion. These metal bind-ing sites were first described in detail for concana-
w xvalin A 29 and have been found to be extremelywell conserved in all other legume lectin structures.A schematic drawing of the metal binding sites in
Ž .concanavalin A is given in Fig. 2 b . The calcium˚and the transition metal ion are approximately 4.5 A
apart and bridged by two aspartate residues. Bothmetals have four protein ligands and two water lig-ands. One of the water ligands from the calcium ionforms a bridge with the carbonyl group of an aspar-
Ž .tate residue Asp208 in concanavalin A that is pre-ceded by a cis-peptide bond.
Table 1Crystal structures of uncomplexed legume lectins
Species Comments Resolution R-factor R -factor PDB codefree
aCanaÕalia ensiformis Containing both a calcium 2.0–1.2 0.142–0.178 nr 1JBC 1CON 1ENRand a transition metal ion 1SCS 1SCR 2CTV
CanaÕalia ensiformis Triclinic form 2.4 0.205 0.265 1VLNCanaÕalia ensiformis Containing only a 2.5–2.8 0.198–0.207 0.249–0.284 1CES 1ENS 1ENQ
transition metal ionbCanaÕalia ensiformis Demetallised 2.5 0.180 0.247 1APN
cCanaÕalia brasiliensis 3.0 0.163 0.246 NDaLathyrus ochrus I. 1.9 0.185 nr 1LOEaPisum satiÕum 1.7 0.177 nr 2LTNaLens culinaris 2 crystal forms 1.75r1.8 0.175r0.184 nr 1LEN 2LAL
a bCanaÕalia ensiformis Arabinose 2.7 unrefined nr NDw xCanaÕalia ensiformis Mana1–3 Mana1–6 Mana1-Me 2.3r2.4 0.205ro.201 0.255r0.255 1CVNr1ONA
a bVicia faba Glucose 2.8 0.37 nr NDaw xPisum satiÕum Mana1–3 Mana1–6 Mana1-Me 2.6 0.183 nr 1RINaLathyrus ochrus I Methyl a-D-Mannopyranoside 1.96 0.182 nr 1LOBaLathyrus ochrus I Methyl a-D-Glucopyranoside 2.2 0.179 nr 1LOAaLathyrus ochrus I Muramic acid 2.05 0.197 nr 1LODaŽ .Lathyrus ochrus I Mur-D-Ala-D-iGln MDP 2.05 0.189 nr 1LOC
Lathyrus ochrus I Mana1–3Manb1–4GlcNAc 2.1 0.175 nr a 1LOGaLathyrus ochrus I Biantennary octasaccharide 2.3 0.190 nr 1LOFaLathyrus ochrus II Biantennary glycopeptide from human 2.8 0.185 nr 1LGC
lactotransferrinaLathyrus ochrus II N2 fragment of human lactotransferrin 3.3 0.210 nr 1LGBaLens culinaris Glucose 3.0 0.206 nr 1LEMaLens culinaris Sucrose 1.9 0.188 nr 1LESaŽ .Erythrina Galb1–4Glc Lactose 2.0 0.190 nr 1LTE
corallodendronaw xGriffonia Fuca1–2Galb1–3 Fuca1–4 GlcNAc 2.0 0.181 nr 1LED
Ž .simplicifolia IV Lewis bw xGriffonia Fuca1–2Galb1–4 Fuca1–3 GlcNAc 2.0 0.185 0.242 1LED
Ž .simplicifolia IV Lewis YaŽ .Arachis hypogaea Galb1–4Glc Lactose 2.2 0.164 nr 2PEL
Ž .Arachis hypogaea Galb1–3GalNAc T-antigen disaccharide 2.5 0.175 0.251 1TEPaŽ w xGlycine max Galb1–4GlcNAcb Galb1–4GlcNAcb - 2.6 0.201 nr 1SBA
Ž .Galb-O CH COOCH2 5 3
a nr: not reported.b ND: not deposited at the time of writing.
Recently, the structure of demetallised concana-˚valin A has been refined at 2.5 A resolution and the
structures of several intermediate steps in the remet-w xallisation process have been presented 30,31 . As
w xpredicted by earlier NMRD measurements 32 , thekey event in the metal-induced activation of concana-valin A is the trans to cis isomerisation of anAla–Asp peptide bond. The metal binding region in
concanavalin A is partly unfolded upon demetallisa-tion, and the conformations of several loops in thedemetallised structure seem to depend on packingcontacts. No calcium binding site is present in demet-allised concanavalin A, but a proto-transition metalbinding site is formed by the side chains of residues
Ž Ž ..Glu8, Asp10 and His24 Fig. 2 b . This metal-freeconformation is called the ‘unlocked’ form of ConA,
Ž .Fig. 1. The legume lectin monomer. a Ribbon diagram of the concanavalin A monomer. The cis-aspartate Asp208 is labeled and shownin ball-and-stick, while the manganese and calcium ions are indicated by grey and black spheres. The 6-stranded back sheet is coloured
Ž .black, the 7-stranded front sheet light grey and the small S-sheet in dark grey. b Topology diagram of the legume lectin fold.Ž .Colour-coding of the strands is identical as in a . The N- and C-termini are those found in concanavalin A. The position of these termini
in the circularly permuted structures of most other legume lectins are indicated by an arrow. The cleavage site between the b-chain andŽ Ž . Ž . .the a-chain of te Vicieae lectins is indicated by an asterix. All figures except Fig. 1 b and Fig. 2 a , Fig. 6Fig. 10Fig. 11 were drawn
w xwith MOLSCRIPT 156 .
( )R. Loris et al.rBiochimica et Biophysica Acta 1383 1998 9–36 13
while the active, metal-bound conformation is calledthe ‘locked’ form. Binding of zinc or cobalt in thisproto-site initially leads to the movement of Asp19and to an ordering of the loop Pro13–Pro23, which is
partly disordered in the structure of apo-concanavalinA, but overall the protein stays in its unlocked apoconformation. Solution NMRD studies showed thatabout 10% of the molecules in demetallised concana-
( )R. Loris et al.rBiochimica et Biophysica Acta 1383 1998 9–3614
Žvalin A are in the locked conformation capable of.carbohydrate recognition and that this percentage
can increase significantly in the presence of highw xconcentrations of mannose 33,34 . Thus, locked and
unlocked conformations are in equilibrium and cal-Žcium but also to a lesser extent manganese and
. w xcadmium stabilises the locked conformation 35,36 .Indeed, presence of a metal bound in the calciumbinding site is essential for observing a native confor-mation for the loops Pro13–Pro23 and Thr97–Glu102of the metal and carbohydrate binding region and forobserving a cis-peptide bond between Ala207 andAsp208 in crystal structures of concanavalin A. Theall-or-nothing conformational transition observed inthe remetallisation process of concanavalin A thusresembles to a certain extent protein folding, in thesense that one goes from an essentially unfolded andflexible metal binding region in the apo structure to aunique well ordered structure in the metal-boundform of the protein. The time constants in the con-canavalin A system are, however, much larger thanthose observed for the folding of small globularproteins, due to the large energy barrier provided bythe requirement of a cis–trans isomerisation step ofan Ala–Asp peptide bond.
In this context, it is noteworthy that binding ofmonosaccharides to concanavalin A stabilises its na-tive conformation and protects against chaotropic
w xagents such as urea 146 . Since high concentrationsof mannose are also capable of shifting the locked–unlocked equilibrium towards the locked conforma-
w xtion in the absence of metal ions 34 , this stabilisa-tion can be explained. The unlocked structure can beconsidered as partly unfolded, and saccharide bindingtherefore stabilises a conformation that has a higherdegree of folding.
The legume lectin family also contains two pro-teins with no known carbohydrate recognition activ-ity that do not possess the otherwise strictly con-served metal binding sites: the a-amylase inhibitorŽ . w xa-AI from Phaseolus Õulgaris 37,145 and arcelin,a seed defence protein found in certain wild acces-
Ž .Fig. 2. a Schematic representation of the double metal bindingsite of concanavalin A. Carbon atoms are shown in white,nitrogen in light grey and oxygen in dark grey. The two metalions are shown as larger light grey circles and are labeled.Functional groups that are esential for monosaccharide bindingare drawn on a light-grey shaded background. Oxygen and
Ž .nitrogen atoms from the conserved Asp-Asn–Arg Gly triad thathydrogen bond to a bound monosaccharide are labeled. Only sidechain atoms are shown, except for Tyr12, Asp208 and Arg228. Inthe case of Arg228, side chain atoms were omitted for the sake of
Ž .clarity. b Superposition of the metal binding region in holoŽ . Ž .grey and apo concanavalin A black .
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Table 3Ž .Residues involved in metal binding in two typical legume lectins concanavalin A and PHA-L and in a-AI and arcelin
Con A PHA-L Arcelin a-AI
Pure transition metal ligands Glu8 Glu122 Val123 Glu101His24 His137 Arg130 Arg108
Ligands bridging both metals Asp10 Asp124 Asn125 Asp103Asp19 Asp132 y y
Pure calcium ligands Tyr12 Leu126 y yCalcium ligands involved in carbohydrate recognition Asn14 Asn128 y yCarbohydrate ligands bridged to calcium via a water Asp208 Asp86 Tyr85 Val80
Arg228 Gly104 Arg103 y
w xsions of the same plant 38 . Both proteins showabout 50–60% sequence identity to the two lectinsfrom Phaseolus Õulgaris PHA-L and PHA-E. Thecrystal structures of arcelin variant 5a and a-AIvariant 1, as well as that of PHA-L have recently
w xbeen solved 8,18,19 . Both a-AI and arcelin lack theloop corresponding to residues Pro13 to Pro23 inconcanavalin A. Also, most of the other residues thatin other legume lectins play a role in metal and
Žcarbohydrate binding are not conserved Table 3 and
Ž . Ž .Fig. 3. Superposition of the metal binding region of PHA-L grey on the corresponding structure or arcelin white from the same plantŽ .Phaseolus Õulgaris . PHA-L residue are labeled normal, arcelin residues in italic.
( )R. Loris et al.rBiochimica et Biophysica Acta 1383 1998 9–3616
.Fig. 3 . No bound metals are observed in the crystalstructure of arcelin or a-AI. Nevertheless, in arcelinTyr85, the residue corresponding to the cis-aspartatein the other legume lectins, is also preceded by a cis
w xpeptide bond 19 . This contrasts with the resultsobtained on demetallised concanavalin A and sug-gests that calcium binding is one way, but not theonly way in which this particular cis peptide bondcan be stabilised. Stabilising interactions includestacking between the aromatic rings of Tyr85 andPhe127 and two main chain – main chain hydrogen
Žbonds Ala84 NH to Thr205 O and Gly207 NH to.Ala84 O . The presence of this unusual and unpre-
dicted cis-peptide was interpreted in terms of thepossible involvement of Tyr85 in an active role in thedefence function of this protein.
The lectin-related a-amylase inhibitor fromŽ .Phaseolus Õulgaris a-AI shows an even larger trun-
cation of the loops involved in metal- and monosac-w xcharide binding by ordinary legume lectins 37,145 .
In the recent structure of a-AI in complex withporcine pancreatic a-amylase, no cis-peptide is ob-served, although the binding site for porcine a-amylase is located in the same region of the proteinas the carbohydrate binding sites in the lectins and
w xthe putative active site in arcelin 18 .
3. Quaternary structure
3.1. The canonical legume lectin dimer
Lectins were originally defined as carbohydratebinding proteins from non-immune origin that areable to agglutinate cells or to precipitate complexcarbohydrates, without having any enzymatic activity
w xtowards their carbohydrate ligands 39,40 . As a con-sequence, legume lectins have multiple binding sitesand indeed, they are found to form dimers and te-tramers. The only known exception to this rule in thelegume lectin family is arcelin 5a, the lectin-relateddefence protein from Phaseolus Õulgaris, which hasno known carbohydrate binding potential. Althoughoriginally identified as a dimer at pH 7.0, its recentlydetermined crystal structure showed a monomer.
Most known legume lectins contain a structuretermed the ‘canonical legume lectin dimer’. Thisdimer, which was first described for concanavalin Aand pea lectin, is characterised by a large 12-stranded
b-sheet resulting from the association of the twoŽ . w x6-stranded back sheets Fig. 4 6,7,9 . Further con-
tacts occur through side chain–side chain and sidechain–main chain interactions of residues 14b to17b, the hydrated b-bulge from residues 46b to 49b
Žin the front sheet and residues 90b and 18a pea.lectin numbering .
In pea lectin, lentil lectin as well as Lathyruslectin, the dimer interface is quite hydrophyllic and
w xcontains a number of buried water molecules 41 .Within a single species, the positions of these watermolecules are well conserved, as is evident from ananalysis of different crystal forms of lentil lectin andLathyrus lectin. However, these waters are onlypoorly conserved among lectins from different speciesand are modulated by point mutations at the interface,and also by disturbances from perfect 2-fold symme-try by strong packing interactions, as demonstratedby the crystal structure of pea lectin.
3.2. Other dimers
Two dimeric lectins of known structure do notform the canonical legume lectin dimer. These are the
Ž .lectins from coral tree Erythrina corallodendronw x w x16 and lectin IV from Griffonia simplicifolia 17 .These structures are compared with the canonicaldimer in Fig. 4. In the case of the E. corallodendronlectin, it was suggested that the canonical dimercannot be formed due to sterical hindrance from a
w xcarbohydrate covalently bound to Asn17 16 . Analternative dimer is formed with a buried surface of
˚2only 700 A per monomer, compared to the about˚21100 A buried in the interface of the canonical dimer.
GS-IV forms a heterodimer, with both monomersdiffering in their covalently bound carbohydrates:Asn5 is glycosylated in only one of the two while
w xAsn18 is glycosylated in both 17 . Inter-subunit in-teractions consist of side chain–side chain and sidechain–main chain interactions as well as water bridgesbetween residues Ser71, Thr159–Arg160, Thr177–Thr186, Ile198–Ser200 and Asp239, with a buried
˚2surface area of 900 A per subunit. Formation of thecanonical dimer is thought not to be impaired by theglycosylation state. Instead, formation of the standarddimer may be prevented because this would bury a
Ž .charged residue Glu58 in the subunit interface. Thecorresponding residue in the lentil lectin dimer isThr48b and is located close to the local dyad axis
( )R. Loris et al.rBiochimica et Biophysica Acta 1383 1998 9–36 17
relating the two subunits in the canonical dimer. Inthe lentil lectin dimer, the Thr48 side chains fromboth subunits hydrogen bond to each other, while inthe homologous pea and Lathyrus lectin interfacesthe corresponding Ser48b are differently oriented,resulting in a different solvent network in the inter-faces of these two proteins. Formation of a canonicalGS-IV dimer would thus bring two negatively charged
˚groups within 4 A from each other. Furthermore, in a
canonical dimer environment, the side chain of Glu58would clash with the side chain of Tyr56 of the othermonomer.
3.3. The concanaÕalin A tetramer
The quaternary structure of concanavalin A is pHdependent, the protein being tetrameric above pH 7.0
w xand dimeric below pH 5.0 42 . The dimer formed at
Ž . Ž . Ž .Fig. 4. The legume lectin dimers and the PNA tetramer. a the canonical dimer. b the GS-IV dimer. c The dimer formed by ErythrinaŽ .corallodendron. d the peanut agglutinin tetramer. For the ease of comparison, one of the monomers is always shown in an identical
orientation.
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low pH is the canonical dimer, and at neutral orhigher pH two such dimers associate with their back
w x Ž .sheets 30,43 . This structure is shown in Fig. 5 a .Because of the concave course of the back b-sheets,the centre of the tetramer is formed by a large
˚3water-filled cavity of about 25=25=8 A . Severalcharged and ionisable side chains are buried upontetramer formation, some of which are involved insalt-bridges. Interestingly, the tetramer interface also
Žcontains two pairs of arginines Arg60 from each of.the four subunits in close contact. In the structure of
demetallised concanavalin A at pH 5.0 they actuallyare involved a stacking interaction with each otherw x30 .
All known crystal structures of concanavalin Aw xhave been obtained around pH 7 20,44–47 , except
for demetallised concanavalin A and its monomet-allised Co2q- and Zn2q-derivatives, that were crys-
w xtallised at pH 5 30,31,48 . Although dimeric insolution, demetallised concanavalin A, through crys-tal packing interactions, forms in the crystal a te-tramer very similar to that observed at pH 7.0. The
number of van der Waals and hydrogen bondingcontacts between the two dimers is, however, drasti-
w xcally reduced 48 . This reduction in inter-dimer in-teractions can largely be explained by the differentprotonation state of aspartates and glutamates at theinterface.
The different crystal forms of concanavalin AŽnative, a mannose complex and two trimannoside
.complexes and the almost identical lectin fromŽCanaÕalia brasiliensis two amino acid substitutions
of which one on the interface between the two canon-.ical dimers have shown that the structure of the
concanavalin A type tetramer is variable to somew xextent 45,49–51 . The two dimers may rotate several
degrees relative to each other and may even show asmall relative translation, leading to a different hy-drogen bonding scheme on the dimer–dimer inter-face. In the concanavalin A–trimannoside complexw x49 , a small relative rotation of the two monomers inthe canonical dimers is observed as well. This sug-gests the concanavalin A tetramer to be a dynamicalstructure in solution.
Ž . Ž .Fig. 5. Comparison of the tetramers formed by a concanavalin A and b PHA-L and soybean agglutinin. The presumed adenine bindingsite is indicated by arrows. Both tetramers consist of two canonical dimers.
( )R. Loris et al.rBiochimica et Biophysica Acta 1383 1998 9–36 19
3.4. The peanut agglutinin tetramer
Ž .Peanut lectin Arachis hypogaea – PNA was thesecond tetrameric lectin of which the structure wasdetermined. Although its crystallisation was reported
˚as early as 1982, a structure refined at 2.2 A was onlyw xdescribed recently 13,14 . Despite its homology with
other known legume lectins, this structure could onlybe solved using isomorphous replacement. The peanutlectin tetramer can be considered to consist of two
Ž .GS-IV type dimers Fig. 4 . Two monomers, onefrom each of these GS-IV type dimers, then associatein a fashion similar but not identical to the canonicaldimer, resulting in the only known tetrameric proteinthat does neither contain 4-fold or 222 symmetry.However, no continuous 12-stranded b-sheet isformed along the dimer interface. Instead, the twoN-terminal strands of the two monomers that associ-ate in the canonical dimer-like fashion are interca-lated by a series of 6 water bridges.
The PNA tetramer is thus unique among all ho-motetrameric proteins in the sense that its internalsymmetry does not lead to a closed point group. Itshould also be noted here that peanut agglutinin isnot a glycoprotein. Therefore, its unique quaternarystructure cannot be due to steric effects of covalentlybound carbohydrate and must be due to intrinsicproperties of the protein itself as coded into its aminoacid sequence. Modelling studies on the concanavalin
w xA and peanut agglutinin tetramers 14 suggested thatthe deciding factor in the choice of the type ofsubunit association might be the amount of non-polarsurface area buried upon oligomerisation.
3.5. The PHArSBA tetramer: an adenine bindingmotif?
Ž . w xThe tetramers of soybean agglutinin SBA 15and the leuko-agglutinin from Phaseolus ÕulgarisŽ . w xPHA-L 8 have in common with concanavalin Athat they also consist of two canonical dimers. Theresulting tetramers are, however, distinctly differentŽ Ž ..Fig. 5 b . Tetramer formation is not pH dependentfor PHA-L and SBA. In the PHA-L and SBA te-tramers, the two curved 12-stranded back sheets in-teract with their two outmost strands, creating a largechannel in the middle of the tetramer. This channelprobably contains the disordered C-terminus of theselectins and protects it from proteolyic degradation.
The interface formed by these two outmost strandsconsists mainly of a number of relatively short sidechains that intercalate in a zipper-like fashion, anarchitecture which is unusual for b-sheet packing.
Many legume lectins contain, in addition to theircarbohydrate binding site, one or more binding sites
w xfor hydrophobic ligands 52–54 . Of special interesthere is the binding of adenine and adenine-related
w xplant hormones 55–59 . Not all legume lectins con-tain an adenine binding site, but those that do recog-nise adenine and certain adenine-related cytokininswith an affinity of 10y5–10y6 M, i.e. several ordersof magnitude stronger than their affinities for
w xmonosaccharides and even oligosaccharides 57–59 .Structural data on these lectin hormone interactions islimited, but it has been determined that the stoi-chiometry is either one or two binding sites per
w xtetramer 54,58 . Affinity labelling experiments iden-tified amino acid residues neighbouring the bindingsite and thus allowed for a crude localisation of theadenine binding site on the PHA-L tetramer as a2-fold symmetric binding site on the interface be-tween different monomers in the tetramer, inside the
w xcentral hole of the tetramer 8 . The broad location ofŽ .this binding site is shown in Fig. 5 b .
The PHArSBA tetramer has recently been ob-served in the crystal structures of Vicia Õillosa lectinw x147 as well as for the seed lectin of Dolichos
Žbiflorus and lectin II of Ulex europaeus our unpub-.lished results . Of these, the Dolichos biflorus lectin
is known to bind adenine, while in the case of ViciaÕillosa and Ulex europaeus adenine binding has neverbeen investigated. The PHArSBA tetramer seemsthus to be the default or canonical tetramer, while theconcanavalin A and peanut agglutinin tetramers arethe exceptions.
3.6. Post-translational processing
Legume lectins are generally synthesised as a pre-Žcursor, which subsequently after an initial removal
.of an N-terminal signal peptide is post-translation-ally processed into a mature protein. This post-trans-lational processing may consist of proteolytic cleav-
w xage of the precursor chain 60 , C-terminal trimmingw x w x62–64 , removal of covalent carbohydrate 65,66and even ligation of the original C- and N-terminiw x67,68 . Depending on each specific lectin, the differ-
( )R. Loris et al.rBiochimica et Biophysica Acta 1383 1998 9–3620
ent precursors may or may not possess carbohydratebinding activity.
Undoubtedly, the most complex and intriguingtype of processing is the chain of events leading to acircularly permuted structure in concanavalin Aw x67,68 . After removal of a signal peptide followed
w xby deglycosylation 69 , a loop segment is removedin the middle of the sequence, presumably by an
w xasparagine-specific protease 70,71 . In about twothirds of the molecules, the original N- and C-terminiare then ligated, resulting in a circularly permuted
w xprotein 67,68 . This relegation was recently shown tobe catalysed by jack bean asparaginyl endopeptidasew x72 .
Another well known form of post-translationalprocessing is the one giving rise to the so-calledtwo-chain lectins of the Vicieae tribe. Here, an aminoacid segment of 6 residues is cut out of the pro-lectin,resulting in a protein containing a short C-terminala-chain and a long N-terminal b-chain. Examples arethe lectins from pea, lentil, broad bean and Lathyrus
w xspecies 60,61 .Less well known is that many so-called single
chain lectins that do not undergo the above process-ing contain ragged C-terminal ends, as determined bymass spectrometry. Examples are soybean agglutinin,
wPHA-E and the lectins from Dolichos biflorus 62–x64 . This phenomenon has also been observed for
w xrecombinant lectins expressed in E. Coli 64 . Someof these lectins were shown to consist of an apparentequimolar mixture of long and short variants. In thecase of the Dolichos biflorus seed lectin and theclosely related stem and leaf lectin, it has beensuggested that C-terminal truncation of half of thesubunits of the respective tetramer and dimer is es-sential for carbohydrate binding activity and that only
w xthe untruncated subunits recognise carbohydrates 73 .The molecular basis of this phenomenon is not known,but may be related to the quaternary structure ofthese proteins. A more detailed explanation probablywill have to await the determination of the crystalstructure of these two lectins.
3.7. Relationship between oligosaccharide specificityand quaternary structure
Many plant lectins, covering a wide range ofcarbohydrate specificities, have been shown to pre-
cipitate glycoproteins and branched multivalentw xoligosaccharides 74 . This precipitation process often
leads to the formation of homogeneous cross-linkedlattices of a crystalline nature and in some cases leads
w xto macroscopic crystals 75–77 . The earlier men-tioned structure of soybean agglutinin in complexwith the biantennary analogue of the blood group I
wcarbohydrate antigen Galb1–4-GlcNAcb Galb1–4-x Ž .GlcNAcb -Galb-O CH COOCH is such an ex-2 5 3
w xample 15 . This type of lectin–carbohydrate interac-tion is extremely specific as in mixed precipitationsystems, distinct and homogeneous lattices can beobserved, even if the individual monovalent con-stituents of the carbohydrate or glycoprotein are bound
w xonly with weak affinity 78 . This type of interactionthus represents a new source of binding specificity
w xwith a high biological relevancy 79,80 . Binding ofmultivalent lectins to the surface of cells often leadsto cross-linking and aggregation of specific glyco-protein and glycolipid receptors, which in turn isassociated with a variety of biological responses suchas mitogenic activities and a variety of biologicalsignal transduction processes.
The formation of homogeneous lattices can in partbe explained by the multimeric nature of the lectins.As discussed previously, different lectins possess dif-ferent types of quaternary structures, independent oftheir specificity for monosaccharides. The differentspacings between the carbohydrate binding sites ofthe different lectins combined with specific distancesbetween the different epitopes on multivalent ligandsare probably the reason for this type of specificity.This is in agreement with observations on otherinteracting systems such as the interaction of choleratoxin B with the GM1 ganglioside in a lipid layer andactin interacting with actin-cross-linking proteinsw x79,80 .
4. Molecular basis of carbohydrate specificity
In the following paragraphs, we will give a de-tailed description of the structural basis of mono- andoligosaccharide binding to legume lectins. Importantsimilarities and differences between the differentlectins will be highlighted. Because of the circularhomology between concanavalin A and the otherlectins and because the carbohydrate binding residues
( )R. Loris et al.rBiochimica et Biophysica Acta 1383 1998 9–36 21
are distributed over several loops, the equivalentresidues for the lectins that are discussed below aregiven in Table 4. These equivalences are based on
Žsuperpositons of the different coordinate files ob-tained from X-ray crystallography or molecular mod-
.elling , except for Lima bean lectin, GS-II and Ulexeuropaeus lectin II, where no three-dimensional co-ordinates were available. For these three lectins, theequivalent residues were determined by aligning thepublished amino acid sequences.
4.1. GlcrMan specific lectins
4.1.1. ConcanaÕalin A: an extended binding siteConcanavalin A, the lectin from Jack bean
Ž .CanaÕalia ensiformis was the first legume lectin tow x w xbe isolated 81 , sequenced 82,83 and its three di-
mensional structure determined by X-ray crystallog-w xraphy 6,7 . The structure of a concanavalin A –
˚methyl a-D-mannopyranoside complex at 2.9 A reso-lution in 1989 provided the first detailed view onhow a legume lectin specifically interacts with a
w xmonosaccharide 84 . This complex has recently been˚ w xrefined at 2.0 A 46 and the interactions in the bind-
ing site are typical for all legume lectin carbohydratecomplexes elucidated by X-ray crystallography todate. The essential interactions involve key hydrogenbonds with three extremely well conserved residues:
Ž .an aspartate Asp208 that is preceded by a cis-peptidebond and is held in place via a water bridge with a
Ž .structural calcium ion, an asparagine Asn14 thatdirectly interacts with this calcium and the backboneNH of Arg228, which in most other legume lectinsequences is a glycine and which also interacts withthe calcium ion via a water bridge with its backbone
Ž .oxygen Figs. 6 and 7 . Also essential are van derŽWaals interactions between aromatic residues Tyr12
in concanavalin A, but often also Phe and sometimes.Leu or Cys and the sugar ring. Further interactions
Ž .involve a loop segment Thr97–Glu102 , of whichthe conformation and length is variable among theknown legume lectin structures and that is not incontact with the calcium ion.
Conformational changes in the binding site ofconcanavalin A upon saccharide binding are small, incontrast to the large differences between holo- andapo-concanavalin A. Two differences between thecomplexed and the uncomplexed structures are never-
theless significant. The first one concerns a move-ment of the side chain of Arg228. In all structures ofuncomplexed concanavalin A, this side chain par-tially blocks the monosaccharide binding site. Thishas been related to the apparent co-operativity of the
w xbinding of Mana1–3 Mana1–6 Man-a-Me by con-canavalin A, which may have a dynamic origin, as is
w xsuggested by recent calorimetric studies 85–87 . Itcan however not be excluded that the observed block-ing of the binding site by Arg228 results from crystalpacking forces, since the crystal form of uncom-plexed concanavalin A is densely packed. Most otherlegume lectins have a glycine at the position ofArg228. The second conformational change concerns
˚Ž .a small movement about 0.5 A of the loop Thr97–Glu102. Strikingly, in all other legume lectins whereboth a complexed and uncomplexed structure are
˚Ž .available, small movements -1.0 A of the corre-sponding loop are observed upon carbohydrate bind-ing.
The effect of the presence of a bulky hydrophobicsubstituent on O1 of mannose and glucose has been
w xinvestigated 88,89 . In the complexes of concana-valin A with 4X-nitrophenyl a-D-mannopyranoside and4X-nitrophenyl a-D-glucopyranoside the nitrophenylgroup hydrogen bonds to the side chain hydroxyl ofTyr100, and makes favourable hydrophobic interac-tions with side chain atoms of Tyr12, Leu99 and
Ž .Tyr100 Fig. 7 .Concanavalin A, as well as the closely related
lectin from Dioclea grandiflora, has its highest affin-w xity for the trimannosyl core Mana1–3 Mana1–6
w xMan found in N-glycans 90,91 . Recently, twogroups independently determined the structure of acomplex between concanavalin A and the trimanno-
w xside 49,92 . The terminal mannose on the a1–6 armis bound in the monosaccharide binding site, in a waysimilar as observed in the concanavalin A – methyla-D-mannopyranoside complex. The reducing coremannose and the a1–3 terminal mannose are bound
Ž Ž ..in an extension of this primary site Fig. 8 a . Thecore mannose interacts with the side chains of Tyr100and Asp16 via its O4 and O2 oxygens respectively.These results are consistent with recent thermody-namic data on the concanavalin A – trimannosyl
w xsystem 93,94 .Both published structures agree with each other on
the interactions between the core mannose and the
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Table 4Residues involved in carbohydrate recognition
Concanavalin A Vicieae lectins Erythrina corallo- Soybean Dolichos biflorus Lima bean Peanut GS-IV Functiondendron lectin agglutinin seed lectin lectin agglutinin
Tyr12 Phe123b Phe131 Phe128 Leu127 Cys127 Tyr125 Trp133 a,bPro13 Tyr124b Ser132 Arg129 Ser128 His128 Ser126 Ile134 bAsn14 Asn125b Asn133 Asn130 Asn129 Asn129 Asn127 Asn135 aThr15 Ala126b y y Ser130 Leu130 Ser128 Lys136 bAsp16 Ala127b y y Gly131 Asp131 Glu129 Asp137 bThr15–Ile17 Ala126b –Trp128b Pro134–Trp135 Ser131– Ser130– Leu130– Ser128– Lys136– c
a Ž .Residues essential for monosaccharide binding: the conserved Asp–Asn–Gly Arg triad and an hydrophobic residue in van der Waals contact with the sugar ring.b Ž .Residues systematically implicated in the higher affnity for oligosaccharides such as the trimmanose core in the case of concanavalin A .c Ž . Ž .Residues identified by NMR, modelling and crystallography to be implicated in interactions with an aglycon Vicieae lectins , an N-acetyl group Dolichos biflorus or a
Ž .fucose residue Erythrina corallodendron . In the case of E. corallodendron, Pro134–Trp135 is also involved in the recognition of a dansyl group.d Hydrophobic pocket that in the case of ConA interacts with aglycons on O1.
( )R. Loris et al.rBiochimica et Biophysica Acta 1383 1998 9–36 23
Ž .Fig. 6. Monosaccharide recognition. left Schematic diagram ofŽ .the binding of mannose to concanavalin A. right Similar dia-
gram for the recognition of galactose by the lectin from Eryth-rina corallodendron.
protein, but differences are seen for the mannose onŽ .the a 1–3 arm. In the structure reported by Nai-
smith and Field, all trimannose molecules are boundin the same conformation, while Loris and co-workers
Ž .observed two conformations for the a 1–3 connec-tion. Interestingly, these two conformations resultedfrom the same hydroxyls interacting with the protein.The existence of two conformations in a ligand that isconsidered a specific binder is unusual and was there-fore extensively verified using the experimental data.Despite the convincing electron density for this situa-tion, no satisfying explanation could be found as towhy this is the case. In fact, the trimannose moleculewith the alternative conformation is not involved incrystal packing interactions and seems not to beprevented in any way to adopt the same conformation
as the other trimannose molecules in the asymmetricunit.
Concanavalin A also has a high affinity for struc-tures containing Mana1–2-Man linkages. No crystal-lographic or NMR data is available, but molecularmodelling has been attempted with the disaccharide
w xMana1–2-Man 95 . From this study, it was con-cluded that the non-reducing mannose most likelyoccupies the monosaccharide binding site, while thereducing mannose interacts with the side chains ofAsn14 and Arg228.
4.1.2. Mimicri between carbohydrate and peptiderecognition.
A couple of years ago, it was reported by twogroups that peptides binding to the carbohydratebinding site of concanavalin A could be isolated from
w xa random peptide library 148,149 . These peptidesbind to concanavalin A with an affinity similar to thatof methyl a-D-glucopyrannoside and inhibit dextranprecipitation of concanavalin A. Conversely,monosaccharides such as a-D-mannopyrannoside in-hibit peptide binding. These peptides, that contain theconsensus sequence Tyr–Pro–Tyr, do not bind toother closely related GlcrMan specific legume lectinsindicating interactions both with the monosaccharidebinding site and some surrounding residues that arenot conserved within the GlcrMan specificity group.On the other hand, anti-DVFYPYPYASGS antibod-ies recognise carbohydrate ligands and anti-a-D-man-
Fig. 7. Stereoview of the hydrophobic binding site of concanavalin A adjacent to the monosaccharide binding site. The saccharide moietyhydrogen bonds to the side chains of Asn14 and Asp208, as well as to the main chain NH groups of Arg228, Leu99 and Tyr100. Furtherimportant van der Waals interactions are observed between the sugar ring and the side chain of Tyr12. The aglycon of 4X-nitrophenyl
Ž .a-D-mannopyranoside labeled PNM is bound in a hydrophobic cavity formed by Tyr12, Leu99 and Tyr100.
( )R. Loris et al.rBiochimica et Biophysica Acta 1383 1998 9–3624
nopyrannoside antibodies recognise the peptide DV-FYPYPYASGS that also binds to the carbohydrate
w xbinding site of concanavalin A 150 . This situation isanalogous to the mimicri found between the cytoker-atin peptide SFGSGFGGY and N-acetyl b-D-gluco-
w xsamine 151 . Also here, antibodies against GlcNacand the peptide are interchangeable and the peptide isrecognised by some GlcNac-specific lectins such aswheat germ agglutinin.
4.1.3. Vicieae lectins: Õariations on a themeCrystal structures of the lectins from fava bean
Ž . Ž . ŽVicia faba , pea Pisum satiÕum , lentil Lens culi-. Žnaris and two isolectins from Lathyrus Lathyrus
.ochrus – LOL I and LOL II with monosaccharidesŽhave shown that these very homologous lectins over
.85% pairwise sequence identity bind mannose andglucose in a way essentially identical to concanavalin
w xA 96–99 .
Ž .Fig. 8. Oligosaccharide specificity in GlcrMan specific lectins. a Stereo figure of the trimannose core bound in the binding site ofconcanavalin A. The 1–6 terminal mannose that occupies the monosaccharide binding site is shown in white, the other two mannose
Žresidues in grey. The amino acid side chains that hydrogen bond to either of the three mannose residues Tyr12, Asn14, Asp16, Tyr100,. Ž .Asp208 and Arg228 are shown in black. b Similar view of the trimannose part of an octasaccharide bound to Lathyrus ochrus lectin.
Ž .Colour-coding is identical to that used in a . The 1–6 teminal mannose and the core mannose stick into the solvent, while the 1–3terminal mannose occupies the monosaccharide binding site. The black residues are the equivalents of those of concanavalin A: Phe123b,
. Ž .Asn125b, Ala127b, Glu31a , Asp81b and Gly99b . c Trimannose part of a glycopeptide bound to Lathyrus ochrus lectin. The situationŽ .is identical to b , except for the different conformation of the trisaccharide. In addition, the specificity-determining fucose is also shown
Ž .in grey and labeled ‘‘F’’. Its position roughly coincides with that of the 1–3 terminal mannose in a and it interacts with the equivalentamino acid side chains.
( )R. Loris et al.rBiochimica et Biophysica Acta 1383 1998 9–36 25
In the case of LOL I, the effect of an aglycon onenhanced affinity was studied in the complexes withN-acetylmuramic acid and a N-acetylmuramyl dipep-
Ž .tide Mur-D-Ala-D-iGln – MDP . It was found thatthe aglycon of both compounds interacts with theside chains of Tyr100b and Trp128b and with
w xresidues Gly97b–Gly98b of LOL 100 . These arethe same residues that according to a recent molecu-lar modelling study are responsible for the enhancedaffinity of lentil lectin for 2-O- and 3-O-m-nitro-ben-zyl derivatives of mannose and glucose as illustrated
Ž . w xin Fig. 9 a 101 . This hydrophobic subsite is differ-ent from the one described above for concanavalin A,which cannot bind mannoses or glucoses containing
w xsubstituents on O3 102 .The binding of sugar residues extending from the
mannose or glucose in the monosaccharide bindingsite is however fundamentally different from what isseen in concanavalin A. Sugar residues immediatelyattached to the mannose or glucose in the monosac-charide binding site are either not visible as in thecomplex of pea lectin with the trimannoside corew x98 , or only interact with the protein via an extendedwater network, as found in a sucrose complex of
w xlentil lectin 99 and the complex of Lathyrus lectinw xwith a trisaccharide 103 . This is in agreement with
the affinities of lentil, pea and Lathyrus lectin forthese oligosaccharides being similar to their affinitiesfor a-D-mannopyranoside. In all cases, the carbo-
Ž .hydrates are in a low but not necessary the lowestw xenergy conformation 105 and are involved in or
located close to crystal lattice interactions. Crystalpacking forces nevertheless seem to have little influ-ence on the interaction between the lectin and theoligosaccharide, as shown in an extensive study ofthe lentil lectin–sucrose interaction that comparedresults from crystallography, solution NMR and
w xmolecular modelling 99 . The conformation of thesucrose molecule, which is known to be highly flexi-ble in solution, as well as the mode of interactionwere found to be essentially identical when studiedwith the three techniques.
In contrast to concanavalin A, the Vicieae lectinshave not significantly enhanced affinity for the tri-
w xmannose core over a-D-mannopyranoside 91,106 .This can be explained by comparing the trimannosidecomplex of concanavalin A with the different oligo-
w xsaccharide complexes of Lathyrus lectin 103,104 .
Ž .LOL and also pea and lentil lectin lacks the func-tional groups that in the concanavalin A trimannosidestructures interact with the core mannose: Tyr12 andTyr100 in concanavalin A are substituted by Phe123b
and Glu31a in LOL. As a consequence, the triman-noside cannot bind in the same orientation in LOL as
w x Ž .in concanavalin A 92 . This is illustrated in Fig. 8 bŽ .and c . Instead, the a1–3 linked mannose binds in
the monosaccharide binding site. The conformationsof the a1–3 and a1–6 linkages in the LOL com-plexes are variable and depend on the exact nature of
w xthe longer oligosaccharide 100 .Lectins from the Vicieae tribe show a high affinity
for N-acetyllactosamine-type glycans that have a fu-cose residue a1–6 linked to the N-linked N-
w xacetylglucosamine 91,106 . The structural basis ofthis specificity is explained by two recent crystalstructures of LOL complexed with fragment N2 ofhuman lactotransferrin and with an isolated bianten-
w xnary glycopeptide 107 . The fucose residue was foundto be bound in a subsite, interacting with Phe123b
and Glu31a , i.e. the same residues that in concana-valin A are responsible for its specificity for the
Ž .trimannose core. Indeed, as shown in Fig. 8 c , theposition of this fucose roughly coincides with that ofthe terminal 1–3 mannose residue in the concana-valin A–trimannose complexes. In absence of thefucose, the affinity of the lectins from the Vicieae
Ž .tribe LOL, favin, lentil, lectin and pea lectin for thelactotransferrin-derived oligosaccharide is somewhatlower, but still about 100-fold higher than for the
w xmonosaccharides 91 . This is explained by the crys-w xtal structure of a LOL–octasaccharide complex 104 ,
in which the non-fucosylated lactotransferrin-derivedoctasaccharide adopts a conformation upon bindingto LOL that is different from the one adopted incomplex of LOL with the fucosylated biantennary
w xglycopeptide 107 . Recently, it was shown that boththe fucosylated and the non-fucosylated biantennaryglycans are highly flexible molecules and that uponcomplex formation, the Vicieae lectins select out a
w xconformation that is already present in solution 155 .The differences in conformation of the fucosylatedand non-fucosylated biantennary oligosaccharidesbound to LOL as seen in the different crystal struc-tures are therefore not due to inherently differentsolution conformations of these oligosaccharides, butrather due to a different lectin-mediated selection of a
( )R. Loris et al.rBiochimica et Biophysica Acta 1383 1998 9–3626
Ž .Fig. 9. A multi-purpose subsite. The same residues a tryptophane, a tyrosine and a glycine are responsible for high affinity binding ofŽ . Ž .substituted monosacchrides. a The nitrophenyl group of 3-O-nitrophenylmannose in complex with lentil lectin. b The fucose moiety of
Ž . Ž .Fuca1–2-Galb1–4-GlcNAc complexed to the Erythrina corallodendron lectin The GlcNAc residue is not shown for clarity . c TheN-acetyl group of GalNAc complexed with the Dolichos biflorus seed lectin. A similar subsite was found four these three substituentsusing modelling calculations, and in the case of Dolichos biflorus also by NMR. The tyrosine and tryptophane are conserved in many, butnot all legume lectins. Notable exceptions are concanavalin A and peanut agglutinin.
particular conformation depending on the presence orabsence of fucose.
4.1.4. Other ManrGlc specific lectinsThe above two fine specificity groups within the
ManrGlc specific legume lectins are the only ones
for which crystallographic information is available.There are, however, other ManrGlc specific lectinsthat exhibit fine specificities different from thosealready described, notably lectins from sainfoinŽ .Onobrychis Õicifolia , the Japanese pagoda treeŽ .Sophora japonica and from the West African
( )R. Loris et al.rBiochimica et Biophysica Acta 1383 1998 9–36 27
w xlegume Bowringia milbraedii 108,109 . Of these,only for the B. milbraedii lectin has a detailed study
w xon sugar specificity been published 110 . This lectindiffers from the other ManrGlc specific lectins inthat it is only poorly inhibited by glucose, and not atall by methyl b-D-glucopyranoside and has a highaffinity for structures containing Mana1–2-Manlinkages. B. milbraedii on the other hand does notseem to have a specific binding site for the trimanno-
w x w xside core Mana1–3 Mana1–6 Man 110 .
4.2. GalrGalnac specific lectins
4.2.1. Structural features of galactose complexesThe amount of structural data available for the
GalrGalNAc specific legume lectins is also growingsteadily. At present, the structures of four legumelectins with a galactose residue in the monosaccha-ride binding site have been refined: E. coralloden-
w xdron lectin in complex with lactose 16 , peanutw xagglutinin in complex with lactose 14 and the T-an-
w xtigen disaccharide 152 , Griffonia simplicifolia lectinŽ .IV GS-IV complexed with the Lewis b tetrasaccha-
w x w xride Fuca1–2-Galb1–3 Fuca1–4 GlcNAcb-Me 17and a cross-linked lattice between soybean agglutininŽ .Glycine max and the biantennary pentasaccharideanalogue of the blood group I carbohydrate antigen
w xGalb1 – 4-GlcNAcb Galb1 – 4-GlcNAcb -Galb-Ž . w xO CH COOCH 15 . Of these complexes, the GS-2 5 3
IV complex is special in the sense that GS-IV doesw xnot bind free galactose or GalNAc 111 . GS-IV thus
belongs sensu stricto to the complex specificity groupand will be discussed in detail later. From the struc-tures of E. corallodendron lectin, peanut agglutininand soybean agglutinin, it can be seen that the hydro-gen bonding network in the binding site in the pres-ence of galactose is similar to the one found for themannose-binding lectins. Since mannose and galac-tose differ in the orientation of their C4 hydroxylŽ .axial or equatorial , the orientation of galactose inthe binding site needs to be different from the one of
w xmannose, as first pointed out by Shaanan et al. 16w xand further discussed by Sharon 153 . As can be
seen in Fig. 6, this results in the conserved Asp–Asn–Gly triad interacting with O4 and O6 in theGlcrMan specific lectins and with O3 and O4 in theGal specific lectins. A similar situation is found forglucose bound to the glucosergalactose binding pro-
w xtein from E. coli 112 , as compared to the galactosew xorientation in the L-arabinose binding protein 113 .
The mode of binding of galactose is essentiallyidentical in the structures of E. corallodendron lectinand soybean agglutinin. In neither of these structures,direct hydrogen bonds are observed with anothercarbohydrate residue than the galactose in themonosaccharide bonding site. A major difference be-tween E. corallodendron lectin and soybean agglu-tinin on the one hand and peanut agglutinin on the
Žother involves the loop Thr216–Glu224 Erythrina.numbering , the conformation of which is strikingly
w xdifferent in the peanut structures 14 . Although simi-lar hydrogen bonds are formed with the conservedAsp–Asn–Gly triad in peanut agglutinin, the exactposition of the galactose residue in the monosaccha-ride binding site of peanut agglutinin is shifted some-what on the peanut agglutinin structure compared tosoybean agglutinin and E. corallodendron lectin.
All available crystal structures contain a galactosein their monosaccharide binding site and therefore donot explain the higher affinity of most of these lectinsfor GalNAc. In order to understand this, Imberty and
Ž .co-workers modelled the strictly GalNAc specificŽ .seed lectin from Dolichos biflorus DBL and used
this structure to dock various saccharides, includingw xGalNAc 114 . It was proposed that the carbonyl
oxygen of the N-acetyl group of GalNAc hydrogenbonds to the main chain NH group of Gly103, whilethe methyl group forms favourable van der Waalsinteractions with the aromatic side chains of Tyr104
Ž Ž ..and Trp132 Fig. 9 b . These results have beencorroborated by a recent NMR study on the interac-tion between DBL and the Forssman pentasaccharideGalNAca1–3-GalNAcb1–3-Gala1–4-Galb1–4-Glcw x115 . In this study, intermolecular NOE’s betweenthe protein and the carbohydrate ligand could beobserved for the terminal GalNAc residue that indeedsuggest that the N-acetylamine group interacts withTyr104 and Trp132.
The blood group A trisaccharide GalNAca1–w x3 Fuca1–2 -Gal occurs in solution in two main con-
formational populations. In the same modelling studyw xinvolving the Dolichos biflorus seed lectin 114 , it
could not be determined with certainty which of thetwo conformations binds to the lectin. This ambiguitywas resolved using an NMR study on the DBL-blood
w xgroup A trisaccharide complex 116 . Combination of
( )R. Loris et al.rBiochimica et Biophysica Acta 1383 1998 9–3628
the modelling and NMR studies indicate that whenbound to DBL, the O4 hydroxyl of the fucose residuehydrogen bonds to the main chain carbonyl of Ser128.This residue corresponds to Tyr124b in the Vicieaelectins and Pro13 in concanavalin A. It is therefore ofrelevance that in the concanavalin A–trimannosidecomplexes, the carbonyl of Pro13 also hydrogenbonds with the 1–3 terminal mannose residue. Fur-ther van der Waals contacts are observed between thefucose and Leu127, the equivalent of Phe123b in theVicieae lectins and Tyr12 in concanavalin A. Theseresults strongly suggest similar interactions not onlyin the monosaccharide binding sites of lectins withdifferent specificities, but also in a conserved subsiteresponsible for high affinity binding of oligosaccha-rides.
The structural basis of oligosaccharide specificitywas also investigated for the E. corallodendron lectinw x117 . This lectin has a high affinity for Fuca1–2-Galb1–4-GlcNAcb-terminated glycosphingolipids.Modelling studies with this trisaccharide suggest thatthe a1–2-linked fucose binds in a cavity extendingfrom the monosaccharide binding site. In this cavity,the fucose residue forms hydrophobic contacts withPro134 and Trp135, as well as hydrogen bonds with
Žthe NH2 group of Asn133 to the 2-OH of fucose as. Žwell as to the glycosidic linkage , NH of Gly107 to
.the 2-OH of fucose and the hydroxyl group ofŽ .Tyr108 to the 3-OH of fucose . These residues
correspond to those that form the so-called ‘‘hydro-phobic binding site’’ adjacent to the monosaccharide
Ž .binding site in both the ManrGlc see above andŽ . Ž Ž ..Gal see below specific lectins Fig. 9 c .
Very recently, the complex of peanut agglutininŽ .with the T-antigen disaccharide Galb1–3GalNac
provided the first crystallographic data of a galactosespecific legume lectin recognising an oligosaccharidethat binds with an affinity that is significantly better
w xthan the lectin’s affinity for Gal or GalNAc 152 .This structure is remarkable because it suggests thatthe 20-fold increase in affinity compared to lactose isentirely due to water-mediated protein–carbohydrateinteractions, the amount of direct protein–carbo-hydrate hydrogen bonds and non-polar contacts being
Ž .identical Fig. 10 . While specific water-mediatedrecognition has been observed previously in protein–DNA interactions, the peanut agglutinin-T-antigendisaccharide complex for the first time shows that
water-mediated specificity is also possible for carbo-hydrate recognition.
4.2.2. Site directed mutagenesisThe galactose specific lectins are also the only
ones for which their specificity has been successfullymodulated by site directed mutagenesis. The lectinfrom E. corallodendron has a 500-fold higher affin-ity for galactoses containing a bulky substituent on
Ž .C2, such as dansylamido NDns compared to thew xunsubstituted monosaccharide 118 , and also a 2-fold
w xhigher affinity for GalNAc than for galactose 119 .In contrast, the affinity of peanut agglutinin forGalNAc is 5 times lower than for galactose, and itsaffinity for MebGalNDns is only 20-fold higher than
Ž .Fig. 10. Schematic diagrams of the binding of lactose a and theŽ .T-antigen disaccharide b to peanut agglutinin. The main differ-
ence between both complexes are the additional water-mediatedinteractions between the protein and the N-acetyl group of theGalNAc residue in the T-antigen disaccharide. Hydrogen bondsare shown as dashed lines and water molecules are indicated by‘‘W’’.
( )R. Loris et al.rBiochimica et Biophysica Acta 1383 1998 9–36 29
w xfor galactose 120 . The structure of the E. corallo-dendron lectin revealed an hydrophobic cavity nearthe C2 carbon of the bound galactose which wasthought to be responsible for its affinity for galac-toses containing substituents on C2. This cavity isformed by Tyr108, Pro134 and Trp135. Arango andco-workers substituted the dipeptide Pro–Trp by thecorresponding tetrapeptide Ser–Glu–Tyr–Asn found
w xin the related sequence of peanut agglutinin 121 .This resulted in a recombinant lectin with an un-changed affinity for galactose, a slightly diminishedaffinity for GalNAc and a markedly lower affinity forMebGalNDns. This change in specificity could berationalised as resulting from steric hindrance by thetwo additional amino acids present in the cavityregion of the mutant.
Similar results were obtained by Jordan and Gold-stein, who modified the binding site of lima beanŽ .Phaseolus lunatus lectin, another GalrGalNAcspecific lectin for which at the moment no crystal
w xstructure is available 122 . This lectin has its highestaffinity for the blood group A immunodominant
w xtrisaccharide GalNAca1–3 Fuca1–2 Galb1-R. Twolima bean lectin mutants showed altered specificityfor C2 substituents on the galactose residue of thistrisaccharide: the mutant His128Pro recognisedGalNAca1–3-GalNAc while the Cys127Tyr mutantshowed specificity for both GalNAca1–3-Gal and
ŽGalNAca1–4-Gal with a necessity for a free hy-.droxyl group at the C2 position of Gal . It is of
interest to note here that Cys127 corresponds toTyr12 in concanavalin A and Phe123b in Lathyrusochrus lectin, suggesting again that the same residuesor loops that are responsible for determining theoligosaccharide specificity in the ManrGlc specificlectins have a similar role in the GalrGalNAc speci-ficity group. Interestingly, the same is true for thethree residues that make up the above mentionedhydrophobic cavity. Both Tyr108 and Trp135 areconserved in the lentil, pea and Lathyrus sequencesŽ .as Tyr100b and Trp128b and were identified as theamino acids responsible for the higher affinity ofmannoses and glucoses with hydrophobic substituents
w xon C2 or C3 101 . In the Dolichos biflorus seedlectin, these residues correspond to Tyr104 andTrp132, which were proposed to be responsible forinteracting favourably with the N-acetyl group of
w xGalNAc 114 .
4.2.3. Determinants of galactose and mannose speci-ficity
As has been discussed, both the GlcrMan and theGalrGalNAc specificity group lectins seem to makesimilar hydrogen bonds between the monosaccharideand the protein upon binding. Indeed, the key hydro-gen bonding partners belong to the conserved core of
w xresidues identified by Young and Oomen 123 .Therefore, despite the well-known directionality ofhydrogen bonds, hydrogen bonding does not seem tobe an important determinant for the selectivity of therecognition process.
When the structures of the GlcrMan specific com-plexes are optimally superimposed onto the complexof the E. corallodendron lectin with lactose, it be-comes clear that also the shapes of the monosaccha-ride binding sites are well conserved. The only excep-tion seems to be the loop from Thr97 to Glu102 in
Žconcanavalin A corresponding to Thr216–Glu224 inErythrina and Thr28a–Ala33a in the lectins from
.Lathyrus, lentil and pea . Clear steric clashes withthese loops are observed if a monosaccharide that isnormally not recognised is placed into the binding
Ž .site Fig. 11 . Since these loops mainly hydrogenbond to the bound carbohydrate with their backboneNH and CO groups, one may assume that the maindeterminant for choosing between mannose or galac-tose specificity is the length and conformation of thisloop, but not directly by the individual amino acid
Žside chains in the case of E. corralodentron lectin,hydrogen bonds between side chains of this loop andlactose are observed, but proven to be dispensable by
.site directed mutagenesis – see below . We willtherefore call this loop the ‘‘monosaccharide speci-ficity loop’’, as we will show in the following sec-tions that it also plays a similar role in determiningfucose, chitobiose and complex specificity. Essen-tially identical conclusions were also drawn in a
w xrecent comparative modelling study 124 . These au-thors found that the length of the monosaccharide
Ž w x.specificity loop termed ‘loop D’ in Ref. 124 isinvariant in the GlcrMan specificity group and thatthere is a correlation between the length of this loopand monosaccharide specificity.
This idea is further corroborated by recent mutage-w xnesis studies on the E. corallodendron lectin 125 ,
the GlcNAc specific lectin II from Griffonia simplici-Ž . w xfolia GS-II 126 , a bark lectin from Robinia pseu-
( )R. Loris et al.rBiochimica et Biophysica Acta 1383 1998 9–3630
Ž .Fig. 11. Comparison of the monosaccharide binding sites of the lectins from Lathyrus ochrus and Erythrina corallodendron. a Thecombining site of LOL is show together with a docked galactose. The galactose was positioned by calculating the best least-squares
Ž .superposition of the invariant monosacchride binding residues Asp89, Gly107, Phe131 and Asn133 residues involved in metal bindingfrom the Erythrina corallodendron lectin on the corresponding residues of LOL. This transformation was then applied to the galactoseresidue present in the co-ordinate set from Erythrina corallodendron. The resulting clashes between the saccharide and the lectin were
w xvisualised as a semi-transparent surface using the program GRASP 157 and are entirely confineded to the loop from Thr28a to Glu31a .Ž .b similar view of mannose docked into the binding site of the Erythrina corallodendron lectin. van der Waals clashes are observedbetween the docked mannose and the loop from Thr216 to Glu224.
w x w xdoacacia 127 and pea lectin 154 . In these studies,muting the conserved Asp and Asn invariantly resultsin a complete loss of carbohydrate binding activity.Also, in the E. corallodendron lectin, the activity of
Žmutants involving Phe131 which stacks upon the.galactose ring in the monosaccharide binding site
depended upon the nature of the replacement residue:replacement with the isosteric and hydrophobic Tyr isallowed, but the smaller Val and Ala lead to an
w xinactive protein 125 . Similar results were also foundw xfor substitutions of Tyr124 in GS-II 126 . Point
mutations in the monosaccharide specificity loop onthe other hand do not lead to inactivation in the case
w xof E. corallodendron lectin 125–127,154 , but dele-tion mutants in this loop resulted in inactivation ofGS-II.
The similarities between the ManrGlc and Galspecificity groups extend also towards oligosaccha-ride recognition. As described in the previous para-graphs, two sets of residues appear to form recurrentthemes. The first set are those residues that form theso-called hydrophobic binding site, which is presentin members of both specificity groups. This subsite is
not only responsible for the enhanced affinity formonosaccharides substituted with hydrophobic agly-cons, but may also play a role in binding the N-acetylamine group of GalNAc and even in oligo-saccharide specificity in the case of E. coralloden-dron, where they have been suggested to form afucose subsite. The second set involves Tyr100 andAsp16 in concanavalin A and their equivalents in theother lectins. These residues may be the key determi-nants for oligosaccharide specificity in most ManrGlcand Gal specific lectins. In contrast to the monosac-charide binding site, however, the specific side chainsof the residues involved are at least as important asloop lengths, although the latter certainly also plays arole.
4.3. Other specificities
4.3.1. Complex specificitySome legume lectins are not inhibited by any
simple sugar. Therefore, this specificity group hasbeen termed ‘complex’. The crystal structures of twomembers of the complex type of legume lectins are
( )R. Loris et al.rBiochimica et Biophysica Acta 1383 1998 9–36 31
known: that of lectin IV from Griffonia simplicifoliaŽ .GS-IV and that of PHA-L from Phaseolus Õulgaris.Only in the case of GS-IV, a structure of a carbo-
w xhydrate complex has been reported 17 . This struc-Žture reveals that the Lewis-b tetrasaccharide Fuca1–
w x .2-Galb1–3 Fuca1–4 GlcNAcb1-Me binds to thelectin with its galactose in the monosaccharide bind-ing site in a way essentially identical to what hasbeen observed for the lectins from coral tree and
w xsoybean 15,16 . The main difference between GS-IVand the E. corallodendron lectin is again the
Žmonosaccharide specificity loop Val221–Glu225 in.GS-IV and Thr216–Glu224 in E. corallodendron .
This loop hydrogen bonds to the galactose in thecombining site of E. corallodendron lectin but inGS-IV hydrogen bonds with the a1–4 linked fucoseŽ .Fig. 12 . In the latter lectin, the corresponding loopŽ .residues Val221 to Glu225 has a rather unusualconformation, including a non-proline cis-peptidebond between Val221 and Gly222, which was identi-fied as essential for the specificity of GS-IV.
Legume lectins that can be inhibited by simplemonosaccharides show only a low affinity for these
Ž y2 y3 . Žsaccharides K s10 –10 M for some re-dis
cently determined values of the thermodynamic pa-rameters of the association between legume lectins
w x.and monosaccharides, see 128–130 . It is therefore
not a surprise that the removal of one or two hydro-gen bonds abolishes monosaccharide binding. This isexactly what seems to have happened with GS-IV:the hydrogen bonds between the galactose in themonosaccharide binding site with the monosaccha-ride specificity loop have been removed and arecompensated by protein–carbohydrate hydrogenbonds with other residues from the tetrasaccharide.This structure therefore suggests that molecularrecognition by the legume lectins with a so-calledcomplex specificity is similar to that occurring inlectins with a defined monosaccharide specificity, butwith some of the interactions that normally occurwith the monosaccharide removed, so that thismonosaccharide cannot bind anymore on its own. Itis therefore likely that all lectins of complex speci-ficity will be classifiable into one of the monosaccha-ride specificity groups when structural informationbecomes known.
4.3.2. Fucose specific lectinsSeveral fucose specific legume lectins have been
isolated, but only two have been analysed in detail aswhat concerns the mechanism of carbohydrate speci-
Ž .ficity: lectin I from gorse Ulex europaeus – UEA-Iw x131 and the lectin from Lotus tetragonolobusw x132,133 . The former lectin has also been crys-
Fig. 12. Superposition of the monosaccharide binding site of Erythrina corallodendron lectin and GS-IV. The open ball-and-stick modelŽ .represents the Erythrina corallodendron lectin including a bound galactose residue , while the grey ball-and-stick model corresponds to
the GS-IV structure. The monosaccharide specificity loops are drawn in grey for GS-IV and black for the Erythrina corallodendronlectin. Labels correspond to the Erythrina structure.
( )R. Loris et al.rBiochimica et Biophysica Acta 1383 1998 9–3632
w xtallised 134 , but no structure has been published sofar. Some evidence comes from binding studies withdeoxy-derivatives of L-Fuc a1–2-D-Galb1–4-D-GlcNAc, the highest affinity ligand known to UEA-Iw x131 . From these studies, all three hydroxyls of thefucose were identified as involved in key polar inter-actions, although the effect of deleting the O2 hy-
w xdroxyl is less dramatic 135 , suggesting that O3 andO4 might interact with the conserved Asp–Asn–Glytriad, as is the case for the Gal specific lectins.
Recently, Gohier and co-workers modelled thew xbinding of fucose by UEA-I 136 . This study re-
sulted in several possible orientations for the fucosering in the binding site, and it was not possible todecide which mode of binding was the most probableone. Therefore, a definite answer on how fucosespecificity is achieved will have to await the determi-nation of a crystal structure.
4.3.3. Chitobiose specific lectinsLess information is available on the molecular
Ž .basis of the specificity of the chitobiose or GlcNAcspecific legume lectins. Such lectins are found in the
Ž .seeds of Ulex europaeus UEA-II , Laburnumw xalpinum and Cytisus species 137–140 and in the
Ž .seeds and leaves of Griffonia simplicifolia GS-IIw x141 . UEA-II is the only one for which the saccha-ride specificity has been investigated in more detailw x142 . It was found that UEA-II has its highest affin-ity for L-Fuca1–2-D-Galb1–4-D-GlcNAcb1–6-R,the same trisaccharide that is the best known inhibitorof UEA-I, which is fucose specific and shares lessthan 50% sequence identity with UEA-II. This simi-larity in oligosaccharide specificity is remarkable inview of their differences in monosaccharide speci-ficity. UEA-I is not inhibited by chitobiose whileL-fucose does not at all bind to UEA-II. UEA-II has
Ž .now been crystallised our unpublished results andcomparison of this structure with the one of UEA-Iwill undoubtedly reveal how these two lectins recog-nise the same oligosaccharide using different epi-topes.
Chitobiose specific lectins also weakly interactwith some glucosides such as cellobiose, indicating
w xthat the N-acetyl group is not essential 141,142 .One might therefore speculate that in the case ofUEA-II, the monosaccharide binding site is occupiedby a GlcNAc or glucose residue in a way similar to
the ManrGlc specific lectins, but that the monosac-Žcharide-specificity loop residues Val223 to Phe229
.in UEA-II does not interact with this GlcNAc orglucose, but rather with one or more residues furtheron the carbohydrate chain, such as for the complexspecificity type of legume lectins. It remains howeverquestionable whether this is compatible with its highaffinity for L-Fuca1–2-D-Galb1–4-D-GlcNAc andL-Fuca1–2-D-Galb1–4-D-Glc. The GlcrMan spe-cific lectins involve O4 in two key hydrogen bonds,which seems incompatible with any bulky substituenton O4. It should also be noted here that in theiroriginal paper describing the fine specificity of UEA-II, Pereira and co-workers showed that these twosaccharides have a large area in common with chito-biose that is structurally identical and that is mostlikely the part of the carbohydrate with which the
w xlectin interacts 142 .Some further evidence comes from a mutagenesis
w xstudy on lectin II from Griffonia simplicifolia 126 ,which also weakly binds GlcNAc. As expected from
wsimilar studies on pea lectin and PHA-L 125,140–x144 , substitution of the conserved Asp88 and Asn136
in the monosaccharide binding site abolishes carbo-hydrate binding. On the other hand, the effect ofmutating Tyr134 depends upon the nature of thereplacement residue: while the mutation Y134F seemsto have no effect, Y134D and Y134G eliminatesbinding of GlcNAc. This can be rationalised whenone considers that Tyr134 corresponds to Trp132 inthe Dolichos biflorus seed lectin, one of the keyresidues suggested to interact with the N-acetyl moi-ety of GalNAc. Perhaps in the chitobiose or GlcNAcspecific lectins, the same amino acids are responsiblefor interacting with the N-acetyl moiety of the Glc-NAc that is bound in the monosaccharide bindingsite. Interestingly, this study also gives evidence forour hypothesis that monosaccharide specificity is de-termined largely by the length and conformation ofthe so-called monosaccharide specificity loopŽThr97–Glu102 in concanavalin A, Thr28a–Ala33a
in the Vicieae lectins and Thr216–Glu224 in the E..corallodendron lectin : Point mutations in the corre-
Ž .sponding loop of GS-II Ser225–Ser230 such asL226K and Q227E had no effect on GlcNAc binding,while the deletion of the dipeptide Leu226–Gln227destroyed GlcNAc binding. In other words, again
Ž .loop length and probably conformation is a more
( )R. Loris et al.rBiochimica et Biophysica Acta 1383 1998 9–36 33
important determinant than the exact amino acid se-quence.
5. Conclusion
Despite the slow start in the seventies and eighties,structural knowledge on legume lectins has boomedin the last five years. Although originally considereda diverse group of proteins, the initial structures ofconcanavalin A and some Vicieae lectins suggestedthat all of them would have a highly conservedtertiary and quaternary structure. The variation in thequaternary associations now known came as a realsurprise. The work by the group of Brewer furthersuggests that this variation in quaternary structuremay be the basis of a form of oligosaccharide speci-ficity previously unrecognised in lectins. The molecu-lar origin of subunit associations in legume lectins,however, is far from clear. Steric hindrance due tobulky covalently attached carbohydrate was initiallyproposed in the case of GS-IV and the lectin from E.corallodendron, but has been revised in the case ofGS-IV. Furthermore, PHA-L forms the canonicaldimer and is glycosylated at the same positions as theE. corallodendron lectin. The lectin from peanut isnot glycosylated but nevertheless forms the mostremarkable of all tetramers. It is likely that the manylegume lectin crystal structures that will be solved inthe next years will add to the variety of quaternarystructures already available and it is hoped that asynthesis of the available structural information willthen lead to a satisfactory explanation of this phe-nomenon.
A great deal has been learned about the molecularbasis of carbohydrate recognition. The principles de-rived from the early structures have been confirmednumerous times and we have seen glimpses on howdifferent mono- and oligosaccharides can be selectedby a conserved scaffold. It appears that a basic set ofessential and conserved residues is surrounded by alimited number of variable residues that direct thespecificity of the lectin, thus opening interesting per-spectives for engineering novel specificities into alegume lectin. Interesting parallels can be drawnbetween the different lectins, not only for the archi-tecture of the monosaccharide binding sites, but alsofor oligosaccharide recognition. A further understand-
ing will undoubtedly require a combination of crys-tallography, site-directed mutagenesis, NMR andmodelling. The continuing interest in these proteinsnevertheless suggests that further advances may bemade in the near future.
Acknowledgements
We wish to thank F. Brewer, A. Imberty, F Casset,˚M. Etzler, J. Angstrom, E. Moreno, C. Reynolds, J.
Naismith and J. Helliwell for kindly providing coor-dinates of modelling and crystallographic studies aswell as for giving us access to results prior to publi-cation. This work was supported by the VlaamsInteruniversitair Instituut voor Biotechnologie. R.Loris and J. Bouckaert are postdoctoral fellows of theF.W.O. T. Hamelryck received financial support fromthe Instituut voor Wetenschap en TechnologieŽ .I.W.T. .
Note added in proof
The predicted location of the adenine binding sitehas recently been confirmed by the structure of acomplex between the seed lectin from Dolichos bi-florus and adenine. A crystal structure has also be-come available of the dimeric stem and leaf lectinŽ .DB58 of Dolichos biflorus. This lectin is unusualas it is dimeric but still shows adenine binding activ-ity. In the crystal structure of DB58, a novel type ofdimer is observed, in which the adenine binding siteof the seed lectin is structurally conserved.
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