Review

Legume lectin family, the `natural mutants of the quaternary state',provide insights into the relationship between protein stability

and oligomerization

V.R. Srinivas, G. Bhanuprakash Reddy 1, Nisar Ahmad, Chittoor P. Swaminathan,Nivedita Mitra, Avadhesha Surolia *

Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India

Received 10 January 2001; received in revised form 2 May 2001; accepted 3 May 2001

Abstract

Legume lectins family of proteins, despite having the same `jelly roll' tertiary structural fold at monomeric level, exhibit considerablevariation in their quaternary structure arising out of small changes in their sequence. Nevertheless, their folding behavior and stabilitycorrelates very well with their patterns of assembly into dimers and tetramers. A conservation of their fold during evolution, its widedistribution in many protein families together with the availability of structural information on them make them interesting as proteins toexplore the effect of inter- versus intra-subunit interactions in the stability of multimeric proteins. Additionally, as `natural mutants' ofquaternary association, proteins of legume lectin family provide interesting paradigms for studies addressing the effect of subunitoligomerization on the stability, folding and function as well as the evolution of multimeric structures. ß 2001 Elsevier Science B.V. Allrights reserved.

Keywords: Legume lectins ; Protein stability ; DSC; Quaternary structure; Subunit interaction; Evolution of lectin oligomerization

1. Introduction

Though much e¡ort has gone into the elucidation of thefolding mechanisms of single domain monomeric proteins,as well as the quantitation of their stability, similar studieson oligomeric proteins have been less common and infor-mation regarding their folding mechanisms meager [1].The stability and structural integrity of oligomeric pro-teins, unlike their monomeric counterparts, is determinedby both intra- as well as inter-subunit interactions. Yetdespite this, the unfolding of many, if not most, oligomericproteins appears to be a two-state process, with just thefolded oligomer and the unfolded polypeptides existing inequilibrium, suggesting that intersubunit interactions con-tribute very signi¢cantly to their overall stability andstructural integrity. To cite a typical example, the stabilityof the tryptophan aporepressor under standard conditionsis 22.2 kcal/mol, while the dissociated monomer was esti-

mated to have a stability of just 5.4 kcal/mol monomer [2].Where monomeric intermediates are observed, they aredevoid of a large percentage of their structural elementsand are, in addition, highly solvent exposed. Thus studieson multimeric proteins can provide important insights intothe relative contributions of the various forces that stabi-lize the oligomeric structures of proteins.

Legume lectins can serve as excellent model systems forstudies on the unfolding of dimeric and tetrameric pro-teins, and the e¡ect of oligomerization on their stabilityand structural integrity. Legumes lectins are strikingly sim-ilar in their primary, secondary and tertiary structures[3,4]. Nearly 60% of their secondary structural elementscomprise of L-strands. These strands are connected toeach other by loops. For all the legume lectins known sofar, the tertiary structure is made up of two anti-parallel Lsheets, a six-stranded £at `back' and a seven-strandedcurved `front' L sheet. These sheets are in turn connectedby a ¢ve-stranded L sheet that may be considered to formthe `roof'. This structure has been described as the `jellyroll' motif (Fig. 1a). Yet despite their similarities at theprimary, secondary and tertiary structural levels, theyshow considerable di¡erences, in their quaternary associa-

0304-4165 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved.PII: S 0 3 0 4 - 4 1 6 5 ( 0 1 ) 0 0 1 5 3 - 2

* Corresponding author. Fax: +91-80-360-0683, +91-80-360-0535.E-mail address: surolia@mbu.iisc.ernet.in (A. Surolia).1 Permanent address: National Institute of Nutrition, Biochemistry

Division, Hyderabad 500 007, India.

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tions and modes of monomer organizations in the dimeric/tetrameric assemblage (Figs. 2 and 3). Small di¡erences inamino acid sequences at the monomer interfaces, and pres-ence/absence of glycosylation appears to a¡ect their mono-mer associations, resulting in di¡erent modes of monomerinteractions. In principle, the oligomerization in legumelectins involves the six-stranded back L sheet of the sub-unit in various ways, and consequently it is possible todescribe their quaternary structure in terms of the mutualdisposition of these L sheets in the participating mono-mers. In the case of lectins with the `canonical' quaternaryassociation, the monomers interact quite extensively,through an anti-parallel side-by-side alignment of their£at six-stranded back L sheets, one from each monomer,

so that a relatively large surface area, of about 1000 Aî 2

per subunit is buried at the dimeric interface, e.g., conca-navalin A, pea, lentil lectins, etc. (Figs. 2 and 3). On theother hand, the lectins with the `handshake' kind of qua-ternary structure formed by a 50³ rotation of their £at six-stranded sheets relative to each other, buries only about700 Aî 2 area from each monomer at the interface (e.g.,Erythrina corallodendron) [3]. The monomers of the isolec-tin IV from Gri¡onia simplicifolia (GS IV) associate in aback-to-back fashion, that results in the burial of nearly1920 Aî 2 of the surface area from each subunit at thedimeric interface [5]. Tetrameric assembly in concanavalinA (Con A) involves back to back association of the twoside-by-side dimers inclined in perpendicular manner whilethat in soybean agglutinin (SBA) and phytohemagglutinin-L (PHA-L) such dimers are aligned nearly in a parallelfashion. Yet another kind of quaternary association isdemonstrated by the peanut agglutinin (PNA). It possessesthe back-to-back structural feature of GS IV as well as thecanonical mode of interaction, with the crucial di¡erencebeing the presence of six water bridges through which thecanonical contact takes place [6,7]. An additional impor-tant feature, that appears to be common to all legumelectins, is that their monomers alone carry in their struc-ture all the features necessary for ligand recognition. Thisimplies that oligomerization imparts these lectins the ¢nespeci¢city necessary for their biologic activity, in additionto providing the overall structural stability to the macro-molecule [8]. Thus, in view of the importance of the qua-ternary structure towards stability and structural integrity,as well as biologic activity of lectins, our laboratory hasbeen investigating the relationship between legume lectinstability and their modes of dimer and tetramer formation.Such studies have served to relate the unfolding behaviorof legume lectins with their quaternary structures. Whatwill follow will be a brief review of the results obtainedfrom di¡erential scanning calorimetric (DSC) studies thathave been carried out on some of the legume lectins. Theresults to be discussed are those of Con A, pea, lentillectin, Erythrina corralodendron lectin (ECorL), wingedbean basic agglutinin (WBA I) and winged bean acidicagglutinin (WBA II), and PNA [8^13]. In addition, work

6Fig. 1. (a) Tertiary structure of a legume lectin (e.g. concanavalin A)monomer is best described as a `jelly roll' fold which consists of a £atsix-stranded anti-parallel `back' L sheet (dark gray), a curved seven-stranded `front' L sheet (light gray) and a ¢ve-stranded `top' L sheetlinked by loops of varying lengths. Monomers of di¡erent lectins essen-tially vary in the length and the shape of their loops. (b) Dimerizationin concanavalin A involves antiparallel side by side alignment of their£at six-stranded `back' L sheets giving rise to the formation of a contig-uous 12-stranded sheet. Similar mode of dimerization was seen in manyother legume lectins subsequently. Hence, it is also described as the`canonical' mode of dimerization in legume lectins. (c) The terameriza-tion in concanavalin A consists of back to back association of twodimers, each resulting from the side-by-side association of two subunits.The ¢gures have been generated using MOLSCRIPT [40].

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on two galectins, one conducted by our group and anotherelsewhere will be mentioned, to compare and contrast theresults obtained, and conclusions drawn from the study onlegume lectins [14,15].

2. Thermal denaturation by di¡erential scanningcalorimetry

The di¡erential scanning calorimeter (DSC) consists oftwo cells, one containing the sample and the other an inertreference solution, that are heated at a programmed rateby heaters, controlled to give a zero temperature di¡erencebetween the cells. When a thermally induced change takesplace in the sample, the control system either suppliesmore or less energy to the sample cell, as the case maybe, to maintain a zero temperature di¡erence [16]. Theoutput of the calorimeter is this change in supplied heat

as a function of temperature from which the van't Ho¡enthalpy (vHv), the calorimetric enthalpy (vHc), the tem-perature at the peak maximum, Tp and the temperature athalf the peak area, Tm are obtained. These four parame-ters together de¢ne the stability of a protein (Fig. 4). An-other important parameter describing the unfolding pro-cess is the ratio of calorimetric to van't Ho¡ enthalpy. Formonomeric single domain proteins, the van't Ho¡ enthal-py is expected to be equal to the calorimetric enthalpy. Onthe other hand di¡erential values of calorimetric and van'tHo¡ enthalpies suggests the plausible participation ofmore than one molecular entity in the transition. The ratioof calorimetric to van't Ho¡ enthalpy, vHc/vHv, is hencea measure of cooperativity of the transition, that can pro-vide information on the number and organization of do-mains in a protein that participate in the unfolding reac-tion. In our studies on legume lectins, we observe distinctdi¡erences in this ratio between the three groups of legume

Fig. 2. (Left) (a) Stereo view of side-by-side (canonical) type of dimer in legume lectins. (b) Back-to-back `handshake' kind of dimer observed in ECorL,WBA I and WBA II. The two sheets are inclined at about 47.6^49.4³ with respect to each other. (c) Back-to-back dimer seen in GS IV and in the twophysiologic dimers of PNA, i.e. 1^4 and 2^3 dimers. The two sheets are mutually inclined at 78³. (Right) Stereo diagrams of the tetramers in (a) ConA, (b) SBA and (c) PNA. In Con A the two dimers associate in a back to back fashion perpendicular to the side by side dimers at an angle of roughly82³. In SBA and also in PHA-L the two side-by-side dimers associate in a back-to-back fashion but which are nearly parallel to each other. PNA, how-ever, shows an open quaternary structure as it neither exhibits C4 nor D2 symmetry expected of a tetrameric protein and shows three distinct kinds ofinterfaces. Interface across subunits 1 and 2 is like Con A, etc., but the two `back' L sheets are bridged by six water molecules, the interface betweensubunits 1^4 and 2^3 is akin to GS IV dimer while that spanning subunits 3^4 is an open one and has not been observed, so far, in any other tetramer-ic protein. These ¢gures are partly adapted from [3] with permission from the authors and publishers.

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lectins. These di¡erences correlate well with the generalorganization of the monomers in the oligomeric structure[9^14] and, in fact, in more than one instance led to thecorrect prediction of the mode of monomer association inthis class of proteins [11,12].

3. Denaturation of lectins with `canonical' mode of dimeri-zation and cooperative unfolding of domains

During the dimerization of concanavalin A, pea andlentil lectins, the £at six-stranded back L sheet of eachmonomer interact extensively to from a single large con-tinuous 12-stranded sheet, that, as mentioned above, re-sults in the burial of nearly 1000 Aî 2 area per monomer atthe interface. This appears to impart the dimeric structurewith considerable stability, and this is apparent from thecalorimetric studies performed on these lectins. In the caseof the lectins with this mode of association, di¡erentialscanning calorimetric measurements show that the dimersunfold and dissociate into respective monomers and sub-monomer fragments at the denaturation temperature. Theunfolding pro¢les are distinctly asymmetric. The data ob-tained are presented in Table 1. Importantly the ratio ofthe calorimetric to the van't Ho¡ enthalpies are close toone [9]. When we consider that these are dimeric proteinsthis is quite striking, since it seems to suggests that theseproteins unfold as though they were single molecular en-tities, despite the presence of non-covalently interactingmonomers. The extensive sheet interactions between themonomers appears to be responsible for their high coop-erativity in unfolding. The strongly stabilizing intersubunitinteractions results in the entire dimeric structure behavingas a single molecular species, that dissociates and unfoldsat once (Fig. 5). Similar e¡ects, of extensive monomerinteraction, in the oligomeric assembly, a¡ecting the ther-mal stability and unfolding cooperativity is also seen instudies on two galectins [14,15].

Calorimetric studies on sheep galectin L-14, have shownthe transition to be very highly cooperative in nature. Infact the vHc/vHv is less than 1, implying that the oligo-meric integrity is maintained even at the denaturation tem-perature [14]. Similar results have been obtained independ-ently from a study on the bovine spleen galectin-1 [15].The monomeric structure of galectin is the same `jellyroll' tertiary structure of the legume lectins, consisting oftwo parallel L sheets. But the explanation for their unfold-

Fig. 3. As all the inter-subunit interfaces in legume lectins involve `back'L sheets a schematic representation with them alone illustrates clearlythe di¡erent modes of oligomerization in legume lectins. The back Lsheets participating in oligomerization are shown in gray and black. (a)The `canonical' type dimeric interface in Con A, pea lectins, etc. (b)`Handshake' kind of interface of ECorL, WBA I, etc. (c) Back-to-backdimer of GS IV and PNA (1^4 and 2^3). The interfaces shown in a^care also meant to emphasize their occurrence across the physiologicdimers to distinguish them from dimeric interfaces that are involved intetramerization of the dimers of Con A and SBA (i.e., d and e, respec-tively). The back-to-back interface involved in the tetramer formation inCon A (d) and in SBA and PHA-L (e). Schematic of interfaces between(f) Con A, (g) SBA (7 PHA) and (h) PNA tetramers. Details of themodes of dimerization and tetramerization are discussed in the text andin Fig. 2. These ¢gures are partly adapted from [3] with permissionfrom the authors and publishers.6

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ing parameters becomes apparent when one examines theirdimeric interfaces. In galectins, both the L sheets extendcontinuously across the dimer interface (unlike just one, asin the legume lectins) [14,15]. Thus there is even greatersubunit-subunit interactions in the galectin dimer, provid-ing it the greater stability and unfolding cooperativity. Asa consequence, the oligomer integrity is maintained evenat the denaturation temperature, so that it does not dis-sociate. Similar observations have also been made fromDSC studies on the catabolite activator protein from Es-cherichia coli [17].

4. Folding cooperativity of the lectins with the handshakeinterface

In the ECorL dimer, the two monomers dock to buildan interface of an `handshake' type. The interface is sta-bilized by hydrophobic as well as polar interactions, butjust 700 Aî 2 area per monomer is buried at the interface[18]. The monomers of ECorL thus appear to be moreloosely attached, suggesting lesser dimeric integrity, thanthe canonically associated counterparts. This is re£ected inthe scanning calorimetric studies on ECorL (Table 1). Theratio of calorimetric to van't Ho¡ enthalpy, vHc/vHv, thatis the unfolding cooperativity, is close to 2, implying theparticipation of two unfolding domains in the transition.But the DSC transition is both single and asymmetric,indicating that the domains unfold at the same temper-ature, and are thus identical in nature. These domainsare easily identi¢ed as the protein subunits [10]. As a con-sequence of the less tenacious dimerization, with a smallercontact region (700 Aî 2), the monomers of the protein un-fold and dissociate as independent domains; with eachdomain contributing independently, and signi¢cantly, to

the directly measurable calorimeric enthalpy (Fig. 5).The thermal unfolding parameters of WBA I (wingedbean basic agglutinin) unexpectedly yielded two peaks[11]. At that time it thus appeared that WBA I possessesrelatively lesser dimeric integrity than the `canonically' di-merized lectins [11,19,20]. Subsequent determination of itscrystal structure has borne out this prediction and WBA I,in fact, has the same handshake kind of dimer association,with identical surface area buried at the subunit junction,as that of ECorL [21]. We may mention that the WBA II(winged bean acidic agglutinin) exhibits a DSC transitionvery similar to that of ECorL, viz., a single asymmetrictransition peak with the vHc/vHv close to 2 [12]. Thisagain suggests that, just as in ECorL, there exist two in-dependent unfolding domains. Given that WBA II is ahomodimer like ECorL, we identi¢ed the domains corre-sponding to the two monomers and suggested that themonomers may be as autonomously associated in thedimer as in ECorL again borne out by the solution ofits three dimensional structure recently [22]. It must benoted that, quite apart from the cooperativity ratios, thecanonically associated lectins, in general, have higher tran-sition temperatures (Tp and Tm) and enthalpies than thelectins with the handshake quaternary structure. Thisagain suggests greater stabilization of the former proteinsas a result of more extensive contacts between their sub-units.

5. The unfolding of peanut agglutinin

As mentioned above, the peanut agglutinin has a verydi¡erent and distinct kind of quaternary association, de-scribed as an open quaternary structure. Each PNA tet-ramer may be considered to be a dimer of two dimers,

Table 1Correlation between the quaternary structures of legume lectins and their stabilitiesa

Lectin PDBb code Oligomericstate

Quaternaryassociation

Surface area buried at theintersubunit interface(Aî 2/monomer)

Tm (K) Tp (K) vHc/vHc Ref.c

Con A 2cna 2 and 4 Canonical 1000 360^365 362^365 1.4^1.5 [9]Pea 2ltm 2 Canonical 1000 343^345 345^346 0.78^092 [9,22]Lentil 1lem 2 Canonical 1000 346^348 347^348 0.63^0.73 [9]ECorL 1lte 2 Handshake 700 334^335 334^336 2.12^2.45 [10]WBA I (P1) 1wbl 2 Handshake 700 342^344 332^334 V2 [11]WBA I (P2) 348^350 348^350 V1WBA II 1F9K, r1F9 Ksf,

1FAY, r1FAYsf2 Handshake 700 332^333 332^334 1.92^2.13 [12]

L-14 (P1) 1SLT 2 Galectin 334^335 0.58 [14]L-14 (P-2) 340 0.22^0.25BSG 1 1080 344^345 0.22^0.33 [15]PNA (P1) 2pel 4 Canonical (1^2) 329^334 330^334 3.2^3.4 [8,13]PNA (P2) Back-to-back

(1^4, 2^3)1900 336 336 0.22^0.23

aPredicted from DSC studies. P1, peak 1; P2, peak 2; BSG 1, bovine spleen galectin 1.bPDB protein data bank [37].cReferences pertain to the folding work on these proteins.

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where subunits 1 and 4 and subunits 2 and 3 associate in aGS IV kind of back-to-back fashion. These dimers in turncome together in such a way that monomers 1 and 2associate in a canonical fashion, except that the sheetsdo not interact directly, but by means of six water bridges.Thus while PNA is made up of two GS IV type dimers,both 2-fold symmetric, and related to each other by adyad, these di¡erent dyads fail to intersect [6,7]. Quiteanalogous to its quaternary association, the thermal dena-turation of peanut agglutinin was found to be distinctlydi¡erent from that observed for the other legume lectins.There appears to be two transitions, each with di¡erentdenaturation temperature and cooperativity ratios. The¢rst peak has an unfolding cooperativity (vHc/vHv) closeto 4 (calculated for tetramer concentration), while the sec-ond peak corresponds to the unfolding of the monomers[13]. The ¢rst transition is identi¢ed with the dissociationof the tetramer to monomeric intermediates, while the sec-

ond transition describes the unfolding of these intermedi-ates (Fig. 5). It may be mentioned that solution denatur-ation of PNA using urea and guanidine hydrochloride,where the unfolding changes are monitored spectroscopi-cally, exhibited distinct biphasic and non-overlapping pro-¢les, also suggesting the presence of an intermediate in theunfolding equilibrium [8]. The intermediate was mono-meric and had lost a considerable amount of its tertiarystructure but with signi¢cant secondary structure [8]. Thisis in striking contrast to other legume lectins which exhibittypical two-state monophasic denaturation pro¢les. Thusin the case of PNA, it seems that the back-to-back asso-ciation between the two subunits, 1^4 and 2^3 is intrinsi-cally less stable, than for instance the canonical dimericinterfaces of Con A, pea and lentil lectins. Likewise the 1^2 interface, though resembling the canonical association ofthese legume lectins, would also be intrinsically less stabledue to the presence of six water bridges. Since this 1^2interface is common to both the 1^4 and 2^3 dimer, the

Fig. 4. A typical DSC scan for the unfolding of a protein yields peak(s)that is/are characterized by four parameters : Tp, the temperature of thetransition peak maximum; Tm, the temperature at half peak area; vHv,the van't Ho¡ enthalpy, which is derived from equilibrium constants,which for a DSC pro¢le is obtained from the ratio of the transitionpeak height (Cmax

p ) to the peak area ( = {4RT2mCmax

p /vHc}) ; and vHc,the calorimetric enthalpy, which is the directly measurable heat change,a result of the transition process, and is obtained from the area underthe peak per mole of protein. vCp is the di¡erence in speci¢c heat ca-pacity between the folded and the unfolded protein. The free energy ofunfolding vG³u(T) at temperature T is given by vHm(13T/Tm)+vCpC[T3Tm3Tln(T/Tm)], where vHm is the molar unfolding en-thalpy change at Tm. vHc/vHv gives the cooperativity of the transition.

Fig. 5. In the canonical type of oligomerization (top panel), as seen inCon A, the dimeric interface is extensive. Consequently, it unfolds as asingle entity, i.e., the interface accounts for the stability of the dimer en-tirely, so much so that dissociation of the dimer and unfolding of theconstituent polypeptide chains occur simultaneously. As the handshaketype of interface (middle panel) seen in ECorL, etc., is less extensive.Consequently, its two subunits unfold independently, and dissociate atthe denaturation temperature. In the case of PNA (bottom panel), be-cause of the less tenacious nature of interactions between the subunitsin the tetramer, it dissociates rather easily giving rise to the formationof a stable folded monomeric intermediate, which, interestingly, retainsthe carbohydrate binding property of the native protein. The foldedmonomer subsequently unfolds completely.

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whole tetramer perhaps dissociates quite readily intomonomers.

The solution denaturation studies, in addition, have alsorevealed di¡erences in stabilities between the canonicallyassociated lectins on the one hand, and the lectins withhandshake quaternary structure on the other. The lectinswith the canonical association have greater unfolding freeenergy and heat-capacity then the latter group, implying,not only greater stability, but also lower exposed hydro-phobic surface area (N. Ahmad, V.R. Srinivas, G.B.Reddy, A. Surolia, unpublished observations). Also, pre-liminary investigations on the glycosylated and the non-glycosylated forms of ECorL (the latter is expected tohave a `non-handshake' structure) have shown di¡erencesin unfolding, with the non-glycosylated form being lesscooperative in its folding behavior (N. Mitra, A. Surolia,unpublished observations). These studies thus assume sig-ni¢cance, since the results from two di¡erent biophysicalmethods, calorimetric and spectroscopic, allude to thesame conclusion, that is di¡erences in the stabilities andunfolding properties of the legume lectins, and perhapsmore generally of multimeric proteins, appear to a a¡ectedby the di¡erent modes of dimer and tetramer formation.

6. Some conclusions and general considerations

To summarize, the denaturation studies, conducted sofar in legume lectins with di¡erent modes of quaternaryassociation, strongly suggests that the stability of theselectins is, at least partly, a consequence of their di¡erentmodes of quaternary associations; or more precisely, ofthe strength of the subunit interactions that make up theoligomeric structure [9^13,23]. The denaturation of PNA,in particular, is quite noteworthy in this regard, since itsuggests that the back-to-back dimers (1^4 and 2^3) areless stable. This is rather surprising, when we recall thatthe dimerization in GS IV consists of extensive inter-sub-unit contacts and results in the burial of nearly 1920 Aî 2

surface area, of which 71% is non-polar. But again it maynot be so, when we consider that the 1^2 interface of PNAdespite its super¢cial similarity with that of Con A, etc., itis in fact not quite the same. Unlike in the latter lectins,the `back' sheets from subunits 1 and 2 of PNA do notstretch right across the interface, but are instead connectedby just six water bridges. In an analogous manner, it isquite plausible that the strength of the interactions withinthe 1^4 and 2^3 subunit pairs may not be quite as strongas the `canonical interface'. Hence both these interfacesare intrinsically less stable than their counterparts in theother legume lectins. Denaturation studies on GS IVshould help clarify this. Fortuitously so, folding studieson legume lectins also led to the discovery of a moltenglobular species which retains its activity [8]. The observa-tion highlights that the molten globule, a key intermediate

in protein folding processes, should be considered quiteclose to the native state of the protein [24].

Legume lectins may be considered as `natural mutants'of quaternary structure, where small changes in aminoacid sequence and/or post-translational modi¢cations, re-sult in di¡erent modes of oligomerization. Comparison ofthe primary sequences reveal considerable conservation ofresidues in the inter-subunit contact regions, amongst thelectins with the canonical structure on the one hand, andthe handshake structure on the other [19,20,25]. Detailedanalyses of the amino acids at the inter-subunit interfacesof lectins exhibiting the canonical mode of dimerizationand those with a handshake interaction have shown thatnon-evasion of the charged residue at the interface Lys 55in ECorL, Lys 53 WBA I and a Glu 54 in WBA II isresponsible for the deviation of these proteins from thecanonical mode of dimerization [20,21]. Glu 58 has beenproposed to play a similar role in the departure of GS IVfrom the canonical mode of dimerization [5] (Fig. 6 showsthe interacting residues at the dimeric/tetrameric interfaceof some of the lectins discussed in this paper). The di¡er-ent modes of quaternary associations in turn appear toa¡ect the thermal stabilities of these lectins, where theunfolding mechanisms correlate well with the strengthand degree of monomer interactions in the oligomericstructure. A detailed description of the speci¢c subunitinteractions in the oligomeric structures of legume lectinslies beyond the scope of the present discussion, but onemay consider the e¡ects of altering the quaternary struc-ture on the stabilities of these proteins. Will modifyingECorL, for instance, so that it assumes the canonical di-merization, a¡ect its stability and unfolding? On the otherhand, one may conceive speci¢c changes that a¡ect one ofthe thermodynamic parameters, without altering the other.For instance, one may engineer a single disul¢de link be-tween the handshake monomers, which while not a¡ectingthe quaternary association and the unfolding cooperativ-ity, may stabilize the protein by increasing its unfoldingtransition temperature and the absolute unfolding enthal-py (calorimetric and van't Ho¡). In this regard one mayrefer to the recent work on the homodimeric enzyme, as-corbate peroxidase [26]. In this study, the stabilities of twomutants E112K and E112A were considered. The E112Kmutant, which caused a change from attractive to repul-sive interaction at the dimer interface also resulted in re-duced stability as compared to the neutral E112A mutant.Nonetheless, both the mutants showed none or very smalldrop in enzyme activity. Continuing on the same theme, itmay also be possible to enhance the interactions betweenthe monomers in the `handshake' dimer, for instance byengineering multiple disul¢de links or enhancing the num-ber of attractive non-covalent interactions at the interface.This should conceivably a¡ect both, the unfolding coop-erativity as well as the stability parameters (Tm/Tp andvHc) of the protein. Lastly, one may also ponder on theimportance of quaternary structure vis-a-vis the function

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and stability of a protein; and the possible evolution ofthese di¡erent modes of oligomerization. As mentionedearlier, a feature that appears to be common to all legumelectins, is the architecture of their binding site. Unlike thegarlic bulb agglutinin and the snow drop lectin, wherestrand exchange between neighboring subunits contributesto the formation of one of the binding sites ; legume lectinbinding sites are constructed from the amino acids of thesame subunit [27,28]. Moreover, no post-translationalmodi¢cation of the legume lectins appears to be necessaryto generate, either the active site or its speci¢city. Thelegume lectin binding site is thus preformed and each lec-tin monomer appears to carry all the features necessaryfor carbohydrate recognition. In fact, our recent report onthe isolation of a monomeric molten globule of PNA, thathas retained most of its binding activity, provides directevidence to this [8]. In the case of Con A it is observedthat sugars containing 1, 2 or 5 mannose groups bind todimeric Con A with a¤nities equal to that of tetramericCon A but this similarity is not observed in higher ordersugar groups with 7, 8 or 9 mannose groups where the

a¤nities in the tetrameric protein are much higher as com-pared to the dimeric molecule [38]. Thus oligomerizationappears to impart to these lectins the ¢ner speci¢cityneeded for the recognition of oligosaccharides and com-plex glycoconjugates that is responsible for their speci¢cbiologic activities [29]. One may then consider the possi-bility of altering the biologic activity of a legume lectin,without a¡ecting its monomer speci¢city, by altering itsquaternary association.

It might not be entirely out of context here to mentionan interesting observation made by Hoedemaeker et al.[39]. They observed that a V103A mutation in the sugarbinding site of pea lectin reduced the Tm of the protein by10³C and the enthalpy by 55%. Also, binding to sugar wasnot observed at 37³C but only at lower temperatures.

There are other families of proteins (e.g., K and L che-mokines, galectins and cystine knot growth factors) whichalso exhibit extensive variation in quaternary structure re-sulting from small di¡erences in tertiary structure [30^32].However, the data on their folding behavior unlike thoseon legume lectins are not available.

Fig. 6. Residues at the intersubunit interface(s) in various legume lectins. The interacting residues were determined by their loss of accessible surfaceareas (ASA) upon dimerization. The residue-wise ASA was calculated using NACCESS [41]. All the interacting residues except in peanut lectin aremarked as bold and italicized. The dimerization in the case of WBA1, WBA2 and ECorL is through the handshake mode. Pea lectin and Con A havea canonical interaction. GS4 dimerizes through the back-to-back mode. In peanut lectin, three di¡erent interactions take place, the back-to-back whichgives rise to the dimer (bold and underlined). The dimers come together to give rise to two kinds of interfaces, the canonical (bold and italicized) andthe open interface (lower case and italicized).

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The legume lectin family of proteins can provide con-siderable scope for discussions on the evolution of oligo-meric proteins. Not only is the tertiary structure (the `jellyroll' fold) common to all legume lectins, but several non-legume lectins, as well as non-lectin proteins, seem to pos-sess this fold, underscoring the biological importance ofthe evolutionary conservation of this structure. The do-main-swapping hypothesis provides a fascinating mecha-nism of oligomer assembly and higher order protein evo-lution [33^36]. In three-dimensional domain swapping, onedomain of a monomeric protein is replaced by the samedomain from an identical protein chain. This results in theformation of dimers and higher order oligomers. Thedimers of diphtheria toxin, RNase A, interferon-Q andinterleukin-5 are excellent examples of domain swappedproteins [36]. In the case of legume lectins, one can imag-ine a modi¢ed model, producing the observed array ofoligomer associations (Fig. 7). For instance, to considerthe peanut agglutinin structure that exhibits, features com-mon to the canonical lectins (the 1^2 interface of PNA) aswell as the back-to back GS IV dimers (1^4 and 2^3 inter-face). One can now imagine the development of the canon-ical structure (such as in pea and lentil lectin) from theback-to-back one (or vice versa), through a PNA kind oftetrameric intermediate. Two pairs of back-to-back dimersmay ¢rst associate with one another across one of their`back' L sheets. This intermediate would be PNA like tet-ramer. This could then be followed by a gradual disrup-tion of the back-to-back dimer (to recall, the 1^4 or 2^3dimers of PNA) itself while strengthening the side-by-sideinterface thus resulting, ¢nally in the canonical dimer. The

peanut lectin protein may represent a ¢nal well estab-lished, and stabilized, oligomer in its own right. Also be-ginning with a back-to-back associated dimer of the GSIV type, the canonical dimer could have evolved throughthe `handshake' kind of interface as an intermediate. Al-ternatively, a divergent mode of the oligomeric evolutioncould ensue from the lectins with `handshake' type of as-sociation in opposite directions giving rise to both theback-to-back and the canonically associated dimers re-spectively. Thus as `natural mutants' of quaternary asso-ciation, legume lectin family can serve as a paradigm forstudies addressing the e¡ects of quaternary association onthe stability, folding and function of oligomeric proteins,as well as the evolution of multimeric structures.

Acknowledgements

Grants from the Departments of Science and Technol-ogy, and Biotechnology, Government of India, and theCouncil of Scienti¢c and Industrial Research, Governmentof India to A.S. are acknowledged. The authors thankDrs. M. Vijayan, K. Suguna, and M.M. Prabu for thediscussions, Dr. N. Manoj for help with illustrations,and Ms. N. Valsala for help in preparation of the manu-script.

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