bowman–birk protease inhibitor from the seeds of vigna

1774 (2007) 1264–1273www.elsevier.com/locate/bbapap

Biochimica et Biophysica Acta

Bowman–Birk protease inhibitor from the seeds of Vigna unguiculataforms a highly stable dimeric structure

K.N. Rao1, C.G. Suresh ⁎

Division of Biochemical Sciences, National Chemical Laboratory, Pune-411008, India

Received 1 March 2007; received in revised form 2 July 2007; accepted 16 July 2007Available online 3 August 2007

Abstract

Different protease inhibitors including Bowman–Birk type (BBI) have been reported from the seeds of Vigna unguiculata. Proteaseisoinhibitors of double-headed Bowman–Birk type from the seeds of Vigna unguiculata have been purified and characterized. The BBI fromVigna unguiculata (Vu-BBI) has been found to undergo self-association to form very stable dimers and more complex oligomers, by size-exclusion chromatography and SDS-PAGE in the presence of urea. Many BBIs have been reported to undergo self-association to formhomodimers or more complex oligomers in solution. Only one dimeric crystal structure of a BBI (pea-BBI) is reported to date. We report the three-dimensional structure of a Vu-BBI determined at 2.5 Å resolution. Although, the inhibitor has a monomer fold similar to that found in other knownstructures of Bowman–Birk protease inhibitors, its quaternary structure is different from that commonly observed in this family. The structuralelements responsible for the stability of monomer molecule and dimeric association are discussed. The Vu-BBI may use dimeric or higherquaternary association to maintain the physiological state and to execute its biological function.© 2007 Elsevier B.V. All rights reserved.

Keywords: Bowman–Birk protease inhibitor; Vigna unguiculata; Plant protease inhibitor; Protein–protein interaction

1. Introduction

Inhibitor proteins of proteases are ubiquitous in nature.Animals, plants and microorganisms contain a number of‘protease inhibitors’ that form reversible, stoichiometric pro-tein–protein complexes with their cognate proteolytic enzymes[1,2]. Usually, they are present in multiple forms in differenttissues of organisms. For the last several years protein inhibitors(PIs) have been investigated for various reasons which alsoinclude their utility in the study of protein–protein interactions[3]. Their gross physiological function could be the prevention ofunwanted proteolysis and thereby control the protein turnover and

Abbreviations: BBI-Bowman, Birk protease inhibitor; PIs, Protein Proteaseinhibitors;ES-MS,Electrosprayionizationmassspectrometry;SDS-PAGE,Sodiumdodecyl sulfate-polyacrylamide gel electrophoresis; FPLC, Fast protein liquidchromatography; BT, Bovine Trypsin; NCS, Non-Crystallographic Symmetry⁎ Corresponding author. Tel.: +91 20 25902236; fax: +91 20 25902648.E-mail address: [email protected] (C.G. Suresh).

1 Present address: Biology Department, Brookhaven National Laboratory,Upton, NY 11973, USA.

1570-9639/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.bbapap.2007.07.009

metabolism. Themost abundant sources of PIs are the plants; theirpresence in plants is known since long. A large number of PIshave been isolated and characterized; majority of them are serineprotease inhibitors [4,5]. Most PIs are of comparatively lowmolecular weight (4–20 kDa), soluble in water and theirpolypeptide chains are non-glycosylated. Although, large varia-tion in the overall structure of serine protease inhibitors exist, theirinsertion loop regions display remarkable similarity in confor-mation. To ascertain the general principles of inhibition by proteininhibitors, structural study of a variety of complexes will beprudent. Indeed, an increasing number of structures of complexesanalyzed using X-ray crystallography have been reported.However, the details of the mechanism and fine specificity ofcomplex formation still remain obscure. In this context it isinteresting to note that out of some 70 structures of proteaseinhibitor–enzyme complexes determined to date only few involveBowman–Birk protease inhibitors [3,6].

A Bowman–Birk inhibitor (BBI) was first isolated fromsoybeans byBowman and its biochemical properties were studiedby Birk [7]. Subsequently, many BBIs have been isolated and

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Table 2The percentage of NaCl (B) at which the isoinhibitors eluted from MonoQcolumn, the isoelectric pH values estimated using IEF unit, the molecularweights determined by ES-MS method for the six isoinhibitors and their specificactivities towards trypsin and chymotrypsin, respectively, are listed here

Isoinhibitor Elutionconc. %NaCl(B)

IsoelectricpH values

Mol.wt. inDalton

Specificactivity a fortrypsin(units/mg)

Specificactivitya forchymotrypsin(units/mg)

PI 12.5 4.45 7761 607 40PII 16.0 4.68 8050 413 57PIII 17.5 4.95 7890 345 68PIV 19.0 5.16 8180 432 52PV 20.2 5.38 8005 390 132PVI 21.5 5.71 8418 233 42a Maximum value of estimated standard deviation in activity is 5 units.

1265K.N. Rao, C.G. Suresh / Biochimica et Biophysica Acta 1774 (2007) 1264–1273

characterized from legumes (only Fabaceae family), Gramineaeand many other plants [8]. The special features of dicot membersof BBI include: (1) small molecular weights in the range of 6–9 kDa; (2) occurrence of seven disulfide bridges that stabilize theiractive configurations; (3) presence of two tandem homologousdomains, each bearing an insertion loop and consequently capableof inhibiting two protease molecules simultaneously andindependently, making them “double-headed” inhibitors.

BBIs from dicotyledonous seeds are of 8 kDa size and double-headed. In contrast, the monocots have 8 kDa single-headed and16 kDa double-headed inhibitors [9]. Dicot BBIs could haveevolved from a single-headed ancestral BBI via internal geneduplication, fusion and mutation. It has been suggested thatduring evolution one of the reactive sites of the 8-kDa double-headed BBI became non-functional resulting in an 8-kDa single-headed BBI in monocots [10]. The 16-kDa double-headed BBImight have evolved from this 8 kDa inhibitor, subsequently, bygene duplication and fusion. The intra molecular sequencehomology of BBIs renders credence to this hypothesis.

Presently, sequences of more than 100 Bowman–Birk typeprotease inhibitors from plant sources are available at the PLANT-PIs database accessible at http://bighost.area.ba.cnr.it/ PLANT-Pis[11]. Many BBIs from plants have been isolated and char-acterized. Despite these extensive studies, few three-dimensionalstructures of these proteins are available. The three-dimensionalstructures of BBIs from different plant seeds, both in the nativestate and in complex with trypsin or chymotrypsin, have beenreported [10,12–20]. The structural features and the application ofBBIs are described in a recent review article [6]. Many BBIs,including the Vu-BBI, have been reported to undergo self-association to form homodimers or trimers or more complexoligomers in solution [21,22]. However, adequate structural infor-mation to understand the self-association phenomenon of BBIs is

Table 1Structure refinement details

Parameter ValueaR factor and bR free (%) 19.1 and 25.8cCc and bCcfree (%) 87.6 and 92.8Number of reflections for refinement 3763Number of reflections for Rfree 177Average B-factor for all atoms 46.4Number of protein atoms 798Number of solvent molecules 58

RMS deviation from ideality(a) bond lengths (Å) 0.01(b) bond angles (°) 3.1

Ramachandran plotResidues in: (a) most favored region (%) 84.4

(b) additionally allowed region (%) 12.5(c) generously allowed region (%) 3.1(d) disallowed region (%) 0.0

a R-factor= (∑||Fo|− |Fc||)/(∑|Fo|).b R-free and Ccfree are calculated for about 5% of the data that was excluded

from refinement.c Cc: correlation coefficient={[∑(Fo−bFoN) (Fc−bFcN)] / [∑(Fo−bFoN)

2

(Fc−bFcN)2]½} where Fo and Fc are the observed and calculated structure factor

amplitudes and bFoN and bFcN are their averages over all reflections, respectively.

lacking. Studies on Vu-BBI including detailed characterization,stability, self-assembling tendency, activity and exposed hydro-phobic surface have been reported [21,23–26]. Here we presentthe characterization and study of the dimeric crystal structure ofBBI from the seeds ofVigna unguiculata (‘cowpea’ or ‘black eyedpea’). Since Vu-BBI exists in equilibrium between multimericstates, structural studies have been limited thus far due to difficultyto push Vu-BBI into a single species of molecular association andhelp crystallization. The crystallization and preliminary X-raycrystallographic analysis of BBI from black eyed pea seeds isreported by us [27] and crystallization of an isoinhibitor fromsame seeds in complex with bovine trypsin [28] and its three-dimensional structure has been reported by Barbosa et al. [29].

2. Materials and methods

The BBIs were purified from dry cowpea seeds by extraction, using ion-exchange chromatography (DEAE-Sephadex) and gel filtration (Sephadex-G50)followed by FPLC using MonoQ anion exchanger [27]. The homogeneity of thepreparation was checked using native PAGE and SDS-PAGE, isoelectricfocusing and X-ray film-contact print technique. In the X-ray film-contact printtechnique, after running the native PAGE, the gel was incubated in 0.1 mg/mlprotease solution; then the gel was washed and placed on an undeveloped X-rayfilm. The inhibitor bands appear as unhydrolyzed gelatin against the backgroundof hydrolyzed gelatin. Later the rear side of the film can be cleared with proteaseand then be developed.

The specific activity of each isoinhibitor was determined against both trypsinand chymotrypsin. Kunitz's method [30] was used to assay activity on caseinsubstrate by measuring the absorbance of the supernatant at 280 nm aftertreatment with trichloroacetic acid (TCA). The reaction mixture (2 ml) in 0.1 Mpotassium phosphate buffer pH 7.5 and casein (10 mg or appropriateconcentration) contained the respective protease alone or with inhibitor. Themixture was incubated for 15 min at 37 °C and the reaction was terminated withthe addition of 3 ml of 5% TCA. The mixture was centrifuged for 10 min at10,000 rpm and the absorbance of the clear solution read at 280 nm. Thereadings were corrected for absorbance due to casein, enzyme and inhibitor.

One unit of protease is defined as the amount of protease required for thehydrolysis of 1 μmol/min casein. One unit of inhibitor is defined as the amountrequired for inhibiting hydrolysis of 1 μmol/min casein. Specific activity of theinhibitor is the activity in units per 1 mg of the pure inhibitor. Proteinconcentration was estimated by Lowry method using bovine serum albumin(BSA) as standard [31]. Bovine trypsin, porcine chymotrypsin and BSA arepurchased from Sigma-Aldrich.

We have reported the crystallization and preliminary X-ray studies of the BBIfrom Vigna unguiculata (Vu-BBI) [27]. The structure of tracey bean BBI (pdb,1pi2) was used as model for structure determination using molecular replacement

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1266 K.N. Rao, C.G. Suresh / Biochimica et Biophysica Acta 1774 (2007) 1264–1273

method implemented in AMoRe [32]. The rigid body refinement of solutions frommolecular replacement calculations was carried out using REFMAC program [33]with the data in the resolution range 15–3.0 Å. This was followed by several cyclesof positional refinement using data in the same resolution range. Electron densitymaps were visualized at this stage. Since the molecule was a dimer in theasymmetric unit,molecular averagingwas tried,making use of the presence of non-crystallographic symmetry (NCS). The averaged electron density map lookedbetter and the initial model could be improved by averaging. Side chains wereprogressively fitted as the map quality improved and at positions where the sidechain density was ambiguous alanine residues were retained. Solvent moleculeswere added progressively to the structure usingX-SOLVATEmodule of QUANTA[34]. At the end of the refinement, most of the residues were identified unam-biguously except for those at the chymotrypsin binding loop region and terminalresidues. The electron densitymapwas poor in one of the active-site loop regions ofboth monomers in the dimer. The quality of the structure was checked usingPROCHECKprogram [35]. The refinement statistics is shown in Table 1. Structureis deposited in protein data bank, accession code:2OT6.

3. Results and discussion

3.1. Characterization of BBIs

In the present study, we were able to separate six isoinhibitors,labeled P-I to P-VI (Table 2) to homogeneity and characterize

Fig. 1. Multiple sequence alignment usingCLUSTALW[45] of several representative ‘doand the two binding site loop residues are denoted by Pn to Pn′. Only those sequencescorresponding to that seen in the electron density ofVu-BBI only is listed. The sequencescow pea, S2—Q4VVG2-CPTI from cow pea, S3—Q9S9H8—Isoinhibitor FIV from costructure), S6—Garden pea, TI12-36, S7—Garden bean, PVI-3, S8—Vicia angustifoliabean, PI-IV, S12—Soybean CII (precursor), S13—Macrotyloma axillare seed DE-4, S1PI-II, S17—adzuki bean-IA, S18—horse gram seeds, S19—Lima beans, S20—Mglabra, DGTI-I, S24—apple leaf seed-DE4, S25—Canavalia lineata, CLTI-II, S26—leaves, S29—Peanut A-II, S30—Peanut B-II.

them. These isoproteins differed in charge since multiple bandswere seen on isoelectric focusing gels and thus were separated onFPLC using MonoQ anion exchange column, eluted with variousconcentrations of NaCl. One of the reasons for undertaking theseparation and characterization of the isoinhibitors is to char-acterize them based on their physico-chemical properties. Indeed,the six isoforms could be distinguished by their variation inspecific activity towards trypsin and chymotrypsin, in terms oftheir isoelectric points and molecular weights determined byelectro-spray mass spectrometry (ES-MS) (Table 2). All the sixisoinhibitors showed inhibitory activity towards both trypsin andchymotrypsin and hence are recognized as ‘double-headed’ PIs.However, they differed in terms of their specific activity, againsttrypsin and chymotrypsin (Table 2). The molecular weight ofmajor isoinhibitor P-IV determined by gel filtration techniqueusing standard molecular weight markers kit was 14.8 kDa andthat determined by SDS-PAGE method was 15.2 kDa. However,this estimatewas not in agreementwith that generally observed forBowman–Birk type or Kunitz type PIs. But the isotopicallyaveraged masses measured by ES-MS method showed that theywere close to 8 kDa, the typical molecular weight of classical

uble-headed’ dicot PIs of Bowman–Birk family. The ⁎ indicates conserved residueswith more than 50% sequence homology are included in the list. The chain lengthare, V1—electron density derived sequence ofVu-BBI, S1—gi|124042 -BTCI fromwpea, S4—BBI Pea seeds, PSTI-IVa (precursor), S5—Pea seeds (Chain A, crystal(common vetch), S9—fava bean, S10—Soybean, D-II (Precursor), S11—Tracey

4—Macrotyloma axillare seed DE-3, S15—adzuki beans PI-I, S16—adzuki beans,ung bean beans, S21—Kidney bean-II, S22—Garden bean-II, S23—DiocleaErythrina variegata seeds, EBI, S27—snail medic seeds, MSTI, S28—alfalfa

Fig. 2. The subunit structure of Vu-BBI, disulfide bridges are shown in liquoriceyellow color.


double-headed dicot BBIs [7,9]. The molecular weight of the Vu-BBI isoinhibitor P-IV estimated using gel filtration is double thisvalue. Thus gel filtration experiment suggests the existence of BBIas dimer in solution. Similarly the molecular weight of the banddetermined using SDS-PAGE could be thought as of an undis-sociated dimer. Alternatively, this aberration can also be thought ofas a result of abnormal migration, as previously observed in othersimilar proteins [22,36]. It may be noted that the dimer bandremained same even after boiling the sample in presence of β-mercaptoethanol (β-me) and sodium dodecyl sulfate (SDS) sup-porting the possibility of existing as a very stable dimer. In thepresence of urea multiple bands corresponding to mixed-associations of different number of monomer chains were ob-served. Atomic force microscopic studies and modeling of BBI-chymotrypsin complex have also suggested equilibrium betweenmonomers and several multimers of Vu-BBI in solution [21,26].

By using partial N-terminal sequencing and with the help ofelectron density map combined with sequences of isoinhibitorsreported previously from the same source, we could infer the fullamino acid sequence of the isoinhibitor P-IV that crystallized.There was no electron density in the crystal structure for the first16 residues at the N-terminus. However, the cleavage betweenresidues 74 and 75 which is known to happen in these type ofinhibitors [22] explains the absence of C-terminal residues in themap, which also agrees with the estimated molecular weight(MW) of 8.1 kDa. Thus the inferred full sequence of isoinhibitorP-IV is “SGHHEDSTDEPSESSEPCCDSCVCTKSIP-PQCHCTNIRLNSCHSGCKSCLCTFSIPGSCRCLDIANFCY-KPCKS” matching the estimated MW. Thus, we believe thatP-IV is a different isoinhibitor than those reported by otherauthors [24,29,37,38].

Physiological role and the reason for several isoinhibitors tobe present in the same plant are still not very clear. Propertiesincluding molecular weight established by ES-MS indicate thatthe isoinhibitors are produced either through post-translationalmodification or gene duplication. Also it has been postulatedthat in a co-evolving system, plants and insects evolve with newforms of PIs and proteases to counter each other's defensemechanisms [39]. Therefore it is possible that the plants evolvednumerous isoinhibitors against diverse digestive enzymes ofvarious parasites in nature [5,40].

3.2. Sequence comparison of dicot BBIs

The sequence of Vu-BBI derived from our electron density(e.d.) map was aligned against sequences of reported iso-inhibitors from the same source and various representativesequences of dicot BBIs (Fig. 1). The alignment shows that thesequences are highly homologous, especially the cysteinepositions are conserved throughout. The sequence identityamong these BBIs is more than 50%, which imply that theseBBIs may share a common tertiary fold. Also these proteinsdisplay an intra-molecular sequence identity of 55% betweenthe N and C domains. The conservation of residues in thetrypsin and chymotrypsin binding loop regions is significantlyhigher. These residues are important for the inhibitor to achievethe β-turn ‘VI b’ conformation of polypeptide chain.

3.3. The three-dimensional structure

The Vu-BBI (isoinhibitor P-IV) crystallized in space groupP21 and the asymmetric unit contained two inhibitor molecules.The secondary structure consists mostly of β-sheets and isdevoid of any α-helices. The monomeric structure of BBI can bedivided into two domains, an N and a C-domain. Each domainhas a two-stranded anti-parallel β-sheet and an associated shortstrand (Fig. 2). The monomer fold of this BBI, is the same “bow-tie motif”, found in other BBI structures [10,12–20]. The two β-sheets place the trypsin and chymotrypsin-binding loopstowards two opposite ends of the molecule (Fig. 2). Thetrypsin-binding domain (N domain) is formed from two peptidechain segments (residues Pro17–Thr35 and Asp63–Lys73)separated in the sequence, whereas the chymotrypsin-bindingdomain (C domain) is formed by a continuous segment ofresidues Asn36–Leu62. The interactions of the domains arethrough hydrogen bonds between main chain atoms andhydrophobic contacts between side chains. The individualdomains are further stabilized through seven disulfide bridges.There are four such bridges in the N-domain and three in the C-domain. The connecting disulfide bridges are Cys18–Cys72,Cys19–Cys34, Cys22–Cys68, Cys24–Cys32 in the N domain,and Cys42–Cys49, Cys46–Cys61, Cys51–Cys59 in the Cdomain (Fig. 2). These seven disulfide bridges provide high


rigidity to the structure and in the event of any proteolyticcleavage of the reactive or insertion sites the conformationalchanges can confine to insertion loops alone while the rest of thestructure can remain intact [41].

The structure has no hydrophobic residues in the core. Theside chains of Pro17, Pro29, Ile28, Phe67, Ile64 and Pro71 arefound exposed to solvent, and constitute the hydrophobicpatches on the surface of the protein. Another notable feature isthe buried polar or charged residues Gln31, Asn36, Asp63 andAsn66. Similarly the polar or charged side chains of Asp20,Asn36, Arg60, Asp63 form an electrically charged cluster at theinter-domain region. The interactions of the side chain atoms ofthe invariant aspartates and an arginine in both monomers

Fig. 3. The dimeric structures of (A) Vu-BBI showing the 2-fold NCS symmetry and tWaals spheres, (B) pea-BBI [12] showing the arrangement of 4 binding sites at the cointeracting residues shown in ball and stick in yellow. Hydrogen bonds are show

presumably stabilize the association and orientation of the twodomains.

The exposed hydrophobic patches on the surface of the proteinare an unusual structural feature, but common among BBIs[21,22,42]. The nature of the side chains on the surface of theprotein is thought to be responsible for the commonly observedphenomenon of self-association in BBIs. The structural patternsobserved here are in contrast with that of typical water-solubleproteins. The structural stability of the BBI monomer does notseem to have arisen from the hydrophobicity factor as normallythe case withmost of the globular proteins. The structural stabilityappears to be contributed by a combination of numeroushydrogen-bonded contacts, electrostatic attraction at the inter-

he four binding sites, also shown are the six invariant water molecules as van derrners of a square, (C) the close view of the dimer interface of the Vu-BBI with then in black with dashed lines. Figure is prepared in MOLSCRIPT [46].


domain region and by the rigid framework of disulfide bridgesalong with higher proline content.

3.4. Quaternary association

The asymmetric unit in the crystal structure contains two BBImolecules – subunits A and B – that closely interact to form acompact dimer (Fig. 3A and C). A pseudo 2-fold axis whosedirection is different from the unit cell axes directions relates thetwo subunits. The approximate dimensions of the dimer are 40 X53 X 30 Å. The r.m.s. deviation in Cα positions on superpositionof two subunits of the dimer is 0.52 Å. The overlap of themonomers is poor in the chymotrypsin loop region that isanyway poorly defined in the electron density map. The core ofthe dimer interface involves an extensive network of hydrogenbonds involving residues Gln31, Gly45, Lys47, Asn66, Tyr69and Cys68 (Fig. 3C). The interface region has a dozen hydrogenbond interactions as shown in Table 3 and Fig. 3C.

The strands 64–66 of both monomers come very close toeach other forming an anti-parallel β-sheet (Fig. 3C), withamino and carbonyl of Ile64 and Asn66 forming two hydrogenbonds. The two monomers are symmetrically positioned aboutthis β-sheet. Mainly the trypsin-specific loops of the twomolecules are positioned on one side of the β-sheet plane andthe rest of the molecules on the other side. On the trypsin loopside of the sheet plane, the interactions are between the sidechains of Gln31 and Asn66. On the opposite side of the β-sheet,side chains of Lys47 and Tyr69 interact with the carbonylgroups of Cys68 and Gly45, respectively.

A dimer structure of BBI was first observed in the crystalstructure of pea-BBI [18]. The association of monomers in pea-BBI is different from that of Vu-BBI. Since their monomerssuperposed well, their difference is confined to quaternarystructure. Like the pea-BBI structure, Vu-BBI also forms a tightdimer in the asymmetric unit. In pea-BBI the two monomersorient almost perpendicular to each other (Fig. 3B), whereas inVu-BBI the molecules associate face-to-face (Fig. 3A). In thecase of Vu-BBI the C-terminal tail from the two monomers packtogether closely to form a 2-stranded β-sheet. In pea-BBI the C-

Table 3Possible hydrogen bonded interactions between the subunits A and B of Vu-BBIclassified according to the type of interaction

Nature of thehydrogen bond

Atoms ofA subunit

Atoms ofB subunit

Distance(Å)

(residue/No./atom) (residue/No./atom)

Main chain–Mainchain

Ile aA64 O Asn B66 N 2.93Asn A66 N Ile B64 O 2.96

Main chain–Sidechain

Gly A45 O Tyr B69 OH 2.56Lys A47 NZ Cys B68 O 2.99Cys A68 O Lys B47 NZ 2.58Tyr A69 OH Gly B45 O 2.76

Side chain–Sidechain

Gln A31 OE1 Asn B66 OD1 3.16Gln A31 OE1 Asn B66 ND2 2.81Gln A31 NE2 Asn B66 OD1 2.48Asn A66 OD1 Gln B31 NE2 2.52Asn A66 ND2 Gln B31 NE2 3.30Asn A66 ND2 Gln B31 OE1 3.10

terminal tail from one monomer crosses over to the othermonomer and associates with the N-terminal β-strand to form acontinuous 4-stranded β-sheet. The β-sheet in pea-BBI islengthier compared to that in Vu-BBI and consequently thereare two hydrogen bonds observed between the main chainatoms in Vu-BBI and three in pea-BBI. The two monomers ofVu-BBI are arranged more symmetrically at the dimer interface.All the residues interacting at the dimer interface are related byan approximate 2-fold symmetry and a corresponding symmet-rical hydrogen bonded network is observed (Table 3). The pea-BBI dimer interface lacks the same type of symmetry observedin Vu-BBI. Unlike in the Vu-BBI where the trypsin binding sitescome close to each other, the four binding sites of the pea-BBIcan be imagined to be placed at the four corners of a square withthe molecules oriented along the diagonals. However, it is stillunknown whether these dimers are the functional units of themolecule. Since the association of monomers in Vu-BBI isdifferent from that of pea-BBI, the reported interaction of lysinefrom chymotrypsin binding site with an aspartic acid at the C-terminal, suggested to aid self association to form dimer [22], isnot possible in the case of Vu-BBI. The three-dimensionalstructure of the ternary complex of bi-functional soybean-BBImonomer shows that it can bind two trypsin molecules at thesame time [19]. Similarly, a modeled ternary complex of pea-BBI with trypsin and chymotrypsin indicates that the simulta-neous binding of two proteases is possible only with amonomeric molecule [18]. It is possible that nature has selectedthe present type of dimer to protect one set of proteaseinhibitory sites from any unwanted protease attack.

3.5. Topology of the reactive site loops and inhibitorymechanism

The two protease-binding sites of BBI are located in the turnregion of the two pairs of anti-parallel β-strands that make up thetwo domains of the molecule (Fig. 2). In each subunit the tworeactive site loops are at two symmetrically related ends along thelongest dimension of the subunit, 36 Å apart. This arrangementof the reactive sites facilitates simultaneous inhibition of twoprotease molecules. The two loops project out from the BBI so asto be easily accessible to the active site of proteolytic enzymes.The chymotrypsin insertion loop is poorly defined in the electrondensity map of both the monomers (Fig. 4A and B). We think thepoor density is due to large flexibility and resultant high disorderof these active site loops at their protease insertion regions.Otherwise we expect these loop structures to be more rigid due todisulphide bridges and hydrogen bonds. The trypsin-bindingloops are clearly seen in the map. These latter loops in eachmonomer are stabilized at their tips presumably by the mutuallyclose disposition of them in the dimer.

The reactive site loops are constrained by disulfide bridges,Cys24–Cys32 in the N-domain and Cys51–Cys59 in the C-domain. The disulfide bridges could be playing the role oflimiting the conformational freedom of the loops. In addition,the hydrogen bonds and van der Waals contacts betweenresidues at the N-terminal and C-terminal sides of the loops alsostabilize the rigid structure of the reactive site loops. The

Fig. 4. The electron density (2Fo-Fc) is poorly defined for chymotrypsin-binding site (CBS) loops in the two monomers of the dimer in the crystal structure of Vu-BBI.(A) One of the subunits in which the electron density missing for residues 55 and 56 is shown. (B) The second subunit in which the main chain of the polypeptide couldbe built into the electron density of the same loop, although the density was not excellent. The residue numbers are shown. The residues 51 and 59 are cystines thatform disulphide bridge. The electron densities are drawn at contour level of 1σ.


extremities of the two loops are tethered by two symmetryrelated strong polar interactions, the side chains of Asn36 andAsp63 are hydrogen bonded to the main chain nitrogen atoms ofAsp20 and Ser21 and Lys47 and Ser48. It is interesting to notethat Asn36 and Asp63 are invariant among BBIs [18], theirpositions are related by 2-fold pseudo-symmetry, and may havea role in stabilizing the structure. A conserved cis-proline

residue at the P3′ position in each binding region creates a turnclassified as type ‘VI b’. The residues at P3′ and P4′ positionsand the internal hydrogen bond between Thr at P2 and Ser at P1′positions help the loop to acquire this conformation. This featureis observed throughout the serine protease inhibitor families,despite possessing different topologies and sequence variationwithin the reactive sites themselves [1,3]. The pointed shape of


the turn ‘VI b’ reflects the lock and key motif thought to be themode of binding of serpins to their cognate enzymes [2,6].

One notable difference between the two reactive loops in themonomer is at P3′–P4′ in the sequence. In domain I thesepositions are occupied by two proline residues, whereas indomain II it is Pro–Gly in this position as found in most BBIs(Fig. 1). One reason for the observed differences in specificity ofloops may be attributed to the conformational differences causedby the above difference in residues. The nature of the residue inthe crucial position P1 is selected by the specificity of the loopcorresponding to proteases inhibited. The P1 residue is lysine fortrypsin binding, while the residue could not be identified inchymotrypsin inhibitory loop because of poor electron density.In addition to the P1 residue, the residues at P2 to P2′ are alsoimportant for binding proteases. These positions are occupied byresidues Thr at P2, Ile at P2′ and Ser at P1′. The P2 position innatural BBIs is most conserved and occupied by Thr. Usually alarge aliphatic side chain, frequently Ile, is found at P2′. Theresidues of the trypsin-binding loop seem to be more conservedthan those of chymotrypsin loop (Fig. 1). The loops are solventexposed, hence their surface accessibility and B-factors are high.The small size of the reactive site loop of BBI (9 residues) is incontrast with the large reactive site loop of the Kunitz type PI,which contains as many as 50 residues, suggesting that thedifferences in molecular weights and cysteine content reflectalso on the sizes of the reactive site loops in these PIs.

3.6. Solvent structure

The final refined structure has 58 ordered solvent molecules.Most of the solvent molecules are found on the surface of theprotein. 20 water molecules are found in contact with proteinatoms. Rest interact among themselves only and not directlywith protein. Also no waters are involved in bridging the twosubunits. An analysis of the water molecules common to bothsubunits was carried out. The structurally significant (invariant)

Fig. 5. Overlap of the structure of BTCI–trypsin complex (2g81) on the dimer structu(red) have been used to overlap the two structures. Overlap shows that trypsin (yellohindrance from the second monomer.

water molecules that are common to both were identified. Awater molecule was considered invariant, if it interacted with atleast one common protein atom in the two subunits, and onsuperposition of the subunits along with their hydration shell,the oxygen atoms of the two waters remained closer than 1.8 Å[43]. The positions of the invariant water molecules W2, W4and W13 of subunit A were occupied by W15, W44 and W57,respectively, in subunit B (Fig. 3A). The water moleculesW2 andW4 interact with atoms N and O of Cys34, whereas W15 andW44 interact with atoms O and N of Cys61. The water moleculesW2 andW15 interact with both N of Cys34 and O of Cys61 in thetwo subunits; whereas W4 and W44 interact with O and N of thesame two residues.W4 andW44 also interact with N andO atomsof Asn36. SimilarlyW2 andW15 interact with O of Asp63 whileW13 and W57 interact with O of Gly45. The B-factors of thesewater molecules are lower than the average value implying thatthey are tightly bound to protein atoms and may have a role instabilizing the structure. Similar water mediated interactionsinvolving homologous residues are observed in other BBIstructures [19]. Thus, it is reasonable to conclude that theseinvariant water molecules are an integral part of the structure,essential for preserving the tertiary structure of the BBI.

3.7. Comparison with the reported structure of BTCI–trypsincomplex

The dimer structure of Vu-BBI described here has been com-pared with the recently reported structure of a BBI-isoinhibitor(BTCI) from the seeds of Vigna unguiculata complexed with β-trypsin [29]. The overall monomer structure of the inhibitormolecule in both the structures is similar (r.m.s.d. 1.1 and 1.2 Åfor superposition of 53 Cα atoms of subunit A and 55Cα atoms ofsubunit B, respectively) indicating that only the quaternary struc-ture is affected by trypsin binding. The superposition betweenBTCI–trypsin complex and the Vu-BBI dimer using the co-ordinates of inhibitor molecule as reference are shown in Fig. 5.

re of Vu-BBI (2ot6). The coordinates of one of the dimers in Vu-BBI and BTCIw) binding to one of the monomers in a dimer like Vu-BBI (grey) can have little


The residues 21, 23, 33 and 60 at the interface of BTCI–trypsincomplex are different in Vu-BBI. The superposition based oninhibitor coordinates from both the structures shows that thesecond subunit of Vu-BBI is not involved in any serious stericclashes with trypsin molecule (Fig. 5). It is possible that theinhibition potential of Vu-BBI is unaffected by the dimer forma-tion. Not surprisingly, the samples containing dimers we used forcrystallization and other experiments showed trypsin inhibition.

3.8. Comparison with other PI structures

When the atomic coordinates of the Vu-BBI were subjected tocomparison in DALI database [44], three homologous PI struc-tures were found. They are from the classical dicot BBIs, themonocot BBI from barley and the solution structure of cysteinePI, bromelain inhibitor VI. The Vu-BBI structure was alsocompared with all the known PI structures of Bowman–Birkfamily. The program ALIGN was used for the super-position ofstructures. As expected, theVu-BBI structure has more similaritywithin the dicot family than with monocot BBI or bromelain PI.Also within the dicot family Vu-BBI overlaps best with the pea-BBI monomer and least with the adzuki bean BBI.

4. Conclusion

In the current study, Bowman–Birk type protease inhibitorsfrom the seeds of Vigna unguiculata were purified to homo-geneity. The isoinhibitors were characterized and differentiatedin terms of molecular weight, isoelectric pH and specific pro-tease activity. One of the isoinhibitor could be crystallized and itsthree-dimensional structure could be determined. Althoughmore than 100BBIs have been sequenced, the three-dimensionalstructures determined thus far are for only a few. Self-associationtendency is commonly observed in BBIs. Vu-BBI is the secondstructure reported to form a very tight dimeric structure. Theexposed hydrophobic surface patches in the monomer, stronghydrogen bonded network in the dimer structure explains thereason for BBIs to exist in dimeric as well as other multimericforms. The comparison between the structures of Vu-BBI dimerand recently reported BTCI–trypsin complex shows that trypsincan bind to one monomer of Vu-BBI dimer without hindrancefrom the second monomer. The double-headed BBIs are thoughtto have evolved from a single-headed common ancestral protein.The intra molecular sequence homology of Vu-BBI rendersfurther credence to this hypothesis.

Acknowledgements

Authors thank Department of Biotechnology, New Delhi,India for graphics and computational facilities. KNR thanksCSIR, India for senior research fellowship.

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