Báo cáo khoa học: Structural analysis of the jacalin-related lectin MornigaM from the black mulberry (Morus nigra) in complex with mannose ppt

In contrast to the gJRL, mJRL are not confined to the Moraceae family but are wide-spread among plants as is illustrated by the isolation and characterization of such lectins from jack fr

from the black mulberry (Morus nigra) in complex with

mannose

Anja Rabijns1, Annick Barre2, Els J M Van Damme3, Willy J Peumans3, Camiel J De Ranter1 and Pierre Rouge´2

1 Laboratory of Analytical Chemistry and Medicinal Physicochemistry, Faculty of Pharmaceutical Sciences, K U Leuven, Belgium

2 Surfaces Cellulaires et Signalisation chez les Ve´ge´taux, UMR-CNRS 5546, Poˆle de Biotechnologie ve´ge´tale, Castanet-Tolosan, France

3 Department of Molecular Biotechnology, Ghent University, Belgium

During the last decade numerous lectins that are

struc-turally related to jacalin, the Gal⁄ GalNAc-specific

lec-tin from jackfruit (Artocarpus integrifolia) seeds [1],

have been isolated and characterized from plants

belonging to taxonomically distant families [2–13] At

present, jacalin-related lectins are subdivided on the

basis of their apparent monosaccharide-binding

speci-ficity in Gal-specific (gJRL) and Man-specific

jacalin-related lectins (mJRL) Hitherto, gJRL have been

identified in only a few species of the family Moraceae

such as Osage orange (Maclura pomifera) (MPA), jack

fruit (Artocarpus integrefolia) (jacalin) and a few other

Artocarpus species, and in black mulberry (Morus nigra) (MornigaG) In contrast to the gJRL, mJRL are not confined to the Moraceae family but are wide-spread among plants as is illustrated by the isolation and characterization of such lectins from jack fruit (artocarpin) and mulberry (MornigaM), banana (Musa acuminata; Musaceae), hedge bindweed (Calystegia sepium; Convolvulaceae), bindweed (Convolvulus arven-sis; Convolvulaceae), Jerusalem artichoke (Helianthus tuberosus; Asteraceae), Parkia platycephala (Fabaceae: Mimosoideae subfamily), Japanese chestnut (Castanea crenata; Fagaceae), rice (Oryza sativa; Poaceae) and

Keywords

carbohydrate-binding site; jacalin-related

lectin; mannose; Morus nigra; quaternary

association

Correspondence

P Rouge´, Surfaces Cellulaires et

Signalisation chez les Ve´ge´taux, UMR-CNRS

5546, Poˆle de Biotechnologie ve´ge´tale, 24

Chemin de Borde Rouge, 31326

Castanet-Tolosan, France

Fax: +33 05 62 19 35 02

E-mail: rouge@scsv.ups-tlse.fr

(Received 22 March 2005, revised 25 April

2005, accepted 31 May 2005)

doi:10.1111/j.1742-4658.2005.04801.x

The structures of MornigaM and the MornigaM–mannose complex have been determined at 1.8 A˚ and 2.0 A˚ resolution, respectively Both struc-tures adopt the typical b-prism motif found in other jacalin-related lectins and their tetrameric assembly closely resembles that of jacalin The carbo-hydrate-binding cavity of MornigaM readily binds mannose No major structural rearrangements can be observed in MornigaM upon binding of mannose These results allow corroboration of the structure–function rela-tionships within the small group of Moraceae lectins

Abbreviations

BanLec, banana (Musa acuminata) lectin; Calsepa, Calystegia sepium (hedge bindweed) agglutinin; Conarva, Convolvulus arvensis

(bindweed) agglutinin; Heltuba, Helianthus tuberosus (Jerusalem artichoke) agglutinin; JRL, jacalin-related lectin; gJRL, galactose-specific jacalin-related lectin; mJRL, mannose-specific jacalin-related lectin; MPA, Maclura pomifera (Osage orange) agglutinin; MornigaG, Gal-specific Morus nigra (black mulberry) agglutinin; MornigaM, Man-specific Morus nigra (black mulberry) agglutinin; Orysata, rice (Oryza sativa) agglutinin.

the true fern Phlebodium aureum (Polypodiaceae) The

subdivision in gJRL and mJRL was initially proposed

on the basis of the nominal specificity towards

mono-saccharides but is also in perfect agreement with a

classification of the JRL according to the overall

struc-ture of their corresponding genes More recent

specific-ity studies indicated that at least within the family

Moraceae the differences in specificity are not so a

clear-cut It has been demonstrated, indeed, that the

presumed T-antigen⁄ GalNac-specific jacalin behaves as

a polyspecific lectin capable of interacting) albeit with

a (much) lower affinity) with many other sugars

including Man, Gluc, Neu5Ac or MurNAc [14,15] In

this respect, the mJRL isolated from other plants

families such as, e.g Calsepa from Calystegia sepium

(Convolvulaceae) and Heltuba from Helianthus

tubero-sus (Asteraceae), which exhibit an exclusive specificity

towards mannose and do not interact with unrelated

sugars like Gal or GalNAc [7,16] differ from jacalin

However, these available data on the specificity of JRL

offer no explanation for the dramatic differences in the

agglutination activity between the mJRL of different

origin All mJRL studied thus far are very weak

agglu-tinins as compared to the gJRL (more than three

orders of magnitude less potent) except MornigaM

which is nearly as potent as jacalin Because one can

reasonably assume that the exceptionally high

agglu-tinating activity of MornigaM is intimately linked to

its sugar binding properties the three-dimensional

structure of MornigaM in complex with a simple sugar

was determined to decipher the structural features

responsible for the unusual enhanced activity of this

Moraceae lectin

Results

Quality and overall view of the structure

The final model obtained for uncomplexed MornigaM

converged to an R-factor of 19.0% and an R-free

value of 21.0% (Table 1) It comprises four MornigaM

subunits with 154 out of the total 161 amino acid

resi-dues, 575 water molecules, three acetate molecules,

four glycerol molecules and one sulfate molecule The

first seven N-terminal residues are not seen in the

elec-tron density map and are not included in the model

The model has a good geometry and has r.m.s

devia-tions from ideal bond lengths and angles of 0.005 A˚

and 1.45
, respectively All residues were found in the

most favoured (85.7%) and generously allowed regions

(14.3%) of the Ramachandran plot None of the

resi-dues was found in the disallowed regions The average

temperature factor for the main-chain and side-chain

atoms are 17.6 A˚2 and 19.9 A˚2, respectively In total

12 cis-peptide bonds were found in the structure (three per monomer: Leu21, Pro85 and Pro123) Further refinement statistics are given in Table 1 for both the uncomplexed and Man-complexed MornigaM

The MornigaM protomer exhibits the b-prism fold typically found in the JRL family (Fig 1) It consists

of three four-stranded b-sheets forming three Greek keys motifs: 1 (b1,b2,b11,b12), 2 (b3, b4, b5,b6) and

3 (b7,b8,b9b10) (Fig 2A,B) Although the single-chain MornigaM protomer definitely differs from jacalin by the lack of a proteolytic cleavage of its protomer into

an a and a b chain, it nicely superimposes on the simi-larly folded two-chain jacalin protomer and other single-chain Man-specific protomers of Heltuba or artocarpin

MornigaM adopts a homotetrameric quaternary arrangement very similar to that of many other JRL like jacalin [1], MPA [17] and artocarpin [18] (Fig 2C,D) All four monomers within the tetramer superimpose well with r.m.s differences ranging between 0.20 A˚ and 0.47 A˚ The largest structural dif-ference observed between the four protomers is the different conformation of loop 99–107 (L2) in the A protomer as compared to its conformation in the B, C and D protomers of the tetramer This loop forms the roof of the sugar binding pocket of MornigaM and hence its different orientation might be related

to the observed lack of binding of mannose in the A

Table 1 Crystallographic data for MornigaM and the MornigaM– Man complex.

MornigaM– Man complex Data collection:

Resolution limit (A ˚ ) 1.8 (1.83–1.80) 2.0 (2.03–2.00)

Unique reflections 100939 (5072) 74409 (3763) Completeness of all data (%) 98.4 (99.7) 99.1 (100.0) Completeness of

data (%) (I > 2 r)

90.6 (70.4) 89.8 (71.7)

Refinement:

Atoms in protein ⁄ solvent 4640 ⁄ 616 4640 ⁄ 583 Mean B in protein⁄ solvent (A˚ 2 ) 18.7 ⁄ 27.6 20.4⁄ 29.0 r.m.s.d bond lengths (A ˚ ) 0.005 0.005

protomer (see below) Furthermore, a minor structural

heterogeneity can be observed in the N-terminal region

of the different protomers Like jacalin, the MornigaM

homotetramer exhibits a strong electronegative surface

(Fig 2E,F) The carbohydrate-binding cavities are

electronegatively charged due to the Asp153 residue

that occupies the centre of the cavity

The carbohydrate-binding site

Comparison of the backbone atoms of the

uncom-plexed and Man-complexed MornigaM structures

shows that no significant structural rearrangements

occur upon mannose binding The r.m.s deviation

between both structures is only 0.11 A˚, based on the

superposition of the Ca atoms Except for protomer

A, a clear density is seen for the bound mannose

mole-cule in the carbohydrate-binding site of protomers B,

C and D which is composed of three convergent loops

connecting strands b1 to b1 (loop 1), b7 to b8 (loop 2)

and b11 to b12 (loop 3), located at the top of the

pro-tomers (Fig 3A) Man is specifically anchored to

resi-dues Gly27 (loop 1), Phe150-Val151-Asp153 (loop 3)

by a network of 8 hydrogen bonds (Table 2) with

oxy-gens O3, O4, O5 and O6, respectively (Fig 3B)

Depending on the protomer, Gly27N interacts

(pro-tomers C and D) or does not interact (protomer B)

with O4 of Man An additional stacking between the

aromatic ring of Phe150 and the pyranose ring of Man

reinforces the interaction of MornigaM with the sugar

A very similar H-bond network was observed in the

previously X-ray solved MeMan–artocarpin [18] and

Man–Heltuba [19] complexes Superposing the Ca of

the amino acid residues forming the monosaccharide-binding site of MornigaM with those of Heltuba and artocarpin yielded r.m.s values of 0.263 A˚ and 0.142 A˚, respectively, thus indicating that all these resi-dues occupy very similar positions in all the mJRL In fact, rather close r.m.s of 0.432 A˚ and 0.362 A˚ were measured with the corresponding amino acid residues forming the Gal⁄ GalNAc-binding site of jacalin (PDB code 1JAC) and MPA (PDB code 1JOT), suggesting a similar topology for the monosaccharide-binding site

of the gJRL that apparently accounts for the promis-cuous character of this gJRL

The size and shape of the carbohydrate-binding site

of MornigaM and other JRL essentially depends on both the conformation of the three loops that delineate the binding cavity and the presence therein of amino acid residues with bulky side chains The carbohy-drate-binding cavity of MornigaM is largely widened between loops 2 and 3 but its opening on the other side, between loops 1 and 2, is strongly restricted by the side chain of Lys106 which protrudes from loop 2

to close up the cavity These structural discrepancies

of the carbohydrate-binding cavity should have a pro-found influence on the type of oligosaccharides that fit

in the site of the JRL

Discussion

Resolution of the three-dimensional structure of apo MornigM X-ray at 1.8 A˚ revealed a homotetrameric organization similar to those found for jacalin and all Moraceae JRL studied thus far but not for any other JRL Resolution of the structure of a MornigaM⁄ Man

Fig 1 Sequence and structure comparison of MornigaM to other jacalin-related lectins (A) Sequence alignment of MornigaM with jacalin as

a member of the gJRL group and Heltuba and artocarpin as members of the mJRL group Identical residues are coloured white with a black background and similar residues are coloured black and open boxed The amino acid residues forming the monosaccharide-binding site are indicated by stars b-Strands forming the Greek keys 1 (b1,b2,b11,b12), 2 (b3-b6) and 3 (b7-b10) of jacalin (upper arrows) and MornigaM (lower arrows) protomers are indicated.

complex at 2.0 A˚ further demonstrated that a network

of seven or eight hydrogen bonds anchors O3, O4, O5

and O6 of Man to residues Gly27, Phe150, Val151 and

Asp153 (all of which protrude from loops 1 and 2 that

delineate the carbohydrate-binding cavity of the lectin

protomer) A closer examination of the structure of

the binding sites indicates that specificity of the Mora-ceae lectins is primarily determined by the orientation

of the side chains of the four amino acid residues resi-dues that form the monosaccharide-binding site of the lectins An r.m.s of only 0.2–0.4 A˚ was measured when superimposing the Ca of these four residues from different Moraceae JRL, which implies that there

is very little structural tolerance at the level of their respective monosaccharide-binding sites This holds true particularly for the Gly residue, which is either free at the N-terminus of the large polypeptide of the two-chain gJRL (in casu jacalin, MPA and MornigaG)

or located around position 10–20 in the uncleaved pro-tomer of the mJRL (in casu MornigaM and artocar-pin) Due to this strictly conserved orientation the Gly residue of both types of Moraceae JRL can interact with both the equatorial (Gal⁄ GalNAc) and axial (Man⁄ Glc) O4 of monosaccharides It should be emphasized, however, that in spite of this particular structural feature jacalin exhibits a preferential specific-ity for Gal, GalNAc and the T antigen, and binds other sugars with a much lower affinity

As with most other plant lectins the reactivity of MornigaM and other JRL is not limited to simple sugars but also extends to disaccharides and more complex oligosaccharides [20–22] Structural analyses indicated that the carbohydrate-binding cavity of MornigaM is sufficiently extended to accommodate more bulky saccharides and that the atomic structure

of this cavity accounts for the oligosaccharide-bind-ing specificity of each of these lectins It is worth mentioning in this context that loop 2, which forms the roof of the carbohydrate-binding cavity in all the

N N

β5

β4 β2 β12

β11 β8 β3 β6

β9 β7 β10 β1

Fig 2 Structure and surface analysis of MornigaM and jacalin (A,

B) Ribbon diagrams showing the arrangement of the 12 b-strands

of the MornigaM protomer in three Greek keys 1, 2 and 3 coloured

orange (strands b1, b2, b11, b12), blue (strands b3-b6) and pink

(strands b7-b10), respectively N and C indicate the N- and

C-ter-mini of the MornigaM polypeptide, respectively, and the stars

indi-cate the location of the monosaccharide-binding site (C, D)

Tetrameric arrangement of the protomers of MornigaM (C) and

jac-alin (D) The b-chains of the jacjac-alin protomers are coloured cyan.

The carbohydrate-binding sites are indicated by stars (E, F)

Mole-cular surface of the MornigaM (E) and jacalin (F) tetramers showing

the distribution of the electrostatic potentials The negative

poten-tial is coloured red and displayed at )5 kT level, the positive

poten-tial is colored blue and displayed at +5 kT level (1 kT ¼ 0.6 kcals).

Neutral surfaces are white The electrostatic potentials were

calcu-lated and displayed with GRASP [37].

Fig 3 The carbohydrate-binding site of MornigaM and other jac-alin-related lectins (A) Ribbon diagram of the carbohydrate-binding site of MornigaM showing the three loops L1 (grey), L2 (pale grey) and L3 (mid grey) forming the carbohydrate-binding cavity Man-nose (in grey ball-and-sticks) occupies the monosaccharide-binding site (B) Network of hydrogen bonds (dashes) anchoring Man to the amino acid residues of the monosaccharide-binding site of MornigaM.

Moraceae JRL plays a key role in the delineation of

the size and shape of the binding site The presence

in loop 2 of bulky amino acid residues has

appar-ently no effect on the accessibility of

monosaccha-rides but could dramatically reduce the accessibility

of the carbohydrate-binding cavity for

oligosaccha-rides This observation is in good agreement with

the recent suggestion (based on a database analysis

of jacalin-like lectins) that loop 2 is a very important

structural feature in determining the

oligosaccharide-binding specificity of JRL [23]

Experimental procedures

Isolation of the lectin

MornigaM was isolated from mulberry (Morus nigra) bark

by affinity chromatography on Man-Sepharose 4B, as

pre-viously described [11] Three successive rounds of affinity

chromatography were performed to ensure the purity of the

Man-specific lectin The lectin preparation gave a single

protein band when checked by SDS⁄ PAGE

Crystallization and structure resolution

Crystals suitable for diffraction analysis were grown from a

55% saturated ammonium sulfate solution containing 0.1 m

imidazole buffer pH 7.0 as described elsewhere [24]

Subse-quently, crystals of the complex between MornigaM and

Man were prepared by soaking an uncomplexed MornigaM

crystal with a 50 mm Man solution for approximately 48 h

Both the uncomplexed and complexed crystals could be

cryoprotected by soaking the crystals in the original

crystal-lization condition with 25% glycerol for
30 s Soaking

experiments performed under similar conditions with Gal

remained unsuccessful

Data collection with cryo-cooling at 100 K was carried

out on the native and soaked crystals at the X11 beam line

of the DESY synchrotron in Hamburg All data processing

was done using denzo and scalepack [25] The space group was assigned to be P65 with a¼ b ¼ 110.7, c ¼ 159.2 A˚ Checking of the diffraction data at the twin server [26] showed that all measured crystals were partially mero-hedrally twinned with variable twin fractions ranging from 11% to 35% The twin fractions for the data sets used for final structure determination of MornigaM and of MornigaM–Man were 14.5% and 18.7%, respectively Both data sets were detwinned using the algorithm described by Yeates [26], yielding data sets in which all reflections had their twin component removed Data collection statistics for both data sets are given in Table 1

The phase problem for the MornigaM structure was solved by the molecular replacement technique in x-plor [27] using the detwinned data The coordinates of half a jacalin molecule (chains A and C, PDB code 1JAC) were used as a search model for the uncomplexed MornigaM structure Two dimers were easily found and combining them into a tetramer gave an R factor of 46% (R-free 47%) after initial rigid body refinement to 3 A˚ Based on a calculated Matthews coefficient of 2.35 A˚3ÆDa)1[28], it was assumed that the asymmetric unit consisted of eight mono-mers However, in the molecular replacement search only a single tetramer was found The corresponding solvent con-tent for the crystals is 73%

Refinement was performed by the cns package [29] using torsional angle dynamics and individual B-factor refine-ment A randomly selected 10% of the data sets were set aside for cross-validation using the R-free value Bulk sol-vent correction was used Solsol-vent molecules were progres-sively added when they met the following requirements: (a)

a minimum 3 r peak had to be present in the |Fobs|-|Fcalc| difference map; (b) a peak had to be visible in the 2|Fobs

|-|Fcalc| map; (c) during refinement the B-value for the water molecule should not exceed 60 A˚2; and (d) the water mole-cule had to be stabilized by hydrogen bonding Refinement was always performed against detwinned data; different twin fractions were tested and used to optimize the refine-ment Eventually twin fractions of 11.60% and 14.74%

Table 2 List of hydrogen bonds connecting the mannose to the different residues of the MornigaM tetramer.

gave the best result for the MornigaM and the MornigaM–

Man structure refinement, respectively Visual inspection

and model building were done in O [30] Electron and

dif-ference density maps were used to confirm the presence of

the mannose molecule in the structure of the MornigaM–

Man complex To accurately fit the electron density map

several amino acids in primary sequence deposited in the

SWISS-PROT database (accession number Q8LGR3) had

to be changed These changes include V17I, P100A and

V157F The observed differences are probably due to the

(documented) heterogeneity of MornigaM The presence of

the replaced amino acid residues, the water molecules and

the ions were all checked in simulated annealing omit maps

Assessment of the quality of the coordinates was done with

the programs procheck [31] and moleman [32] The

coordinates and structure factors of the MornigaM and

the MornigaM–Man structure have been deposited in the

Protein Data Bank [33] with the codes 1XXQ and 1XXR,

respectively Ribbon diagrams of MornigaM and other

JRL were drawn with molscript [34] and rendered with

bobscript[35] and raster3d [36]

Molecular surface analysis

Molecular surface and electrostatic potentials were

calcula-ted and displayed with grasp using the parse3 parameters

[37] The solvent probe radius used for molecular surfaces

was 1.4 A˚ and a standard 2.0 A˚ Stern layer was used to

exclude ions from the molecular surface [38] The inner and

outer dielectric constants applied to the protein and the

sol-vent were, respectively, fixed at 4.0 and 80.0, and the

cal-culations were performed keeping a salt concentration of

0.145 m No even distribution of the net negative charge of

the carboxylic group of negatively charged residues was

performed between their two oxygen atoms prior to the

cal-culations Surface topology of the carbohydrate-binding

sites was rendered and analysed with pymol (W.L DeLano,

http://www.pymol.org)

Sequence comparison and alignment

The program espript [39] was used to compare the amino

acid sequence of MornigaM to other JRL sequences

(Fig 1A) Multiple amino acid sequence alignments were

based on clustal x [40]

Acknowledgements

A.R is a Postdoctoral Research Fellows of the Fund

for Scientific Research-Flanders (Belgium) The

finan-cial support of CNRS is gratefully acknowledged (A.B

and P.R.) We thank the beam line scientists at DESY

for technical support and the European Union for

sup-port of the work at EMBL Hamburg through the

HCMP to Large Installations Project, contract no CHGE-CT93-0040

References

1 Sankaranarayanan R, Sekar K, Banerjee R, Sharma V, Surolia A & Vijayan M (1996) A novel mode of carbo-hydrate recognition in jacalin, a Moraceae plant lectin with a b-prism fold Nat Struct Biol 3, 596–603

2 Bausch JN & Poretz RD (1977) Purification and proper-ties of the hemagglutinin from Maclura pomifera seeds Biochemistry 16, 5790–5794

3 Moreira RA & Ainouz IL (1981) Lectins from seeds of jack fruit (Artocarpus integrifolia L.): isolation and puri-fication of two isolectins from the albumin fraction Biol Plant (Praha) 23, 186–192

4 Van Damme EJM, Barre A, Verhaert P, Rouge´ P & Peumans WJ (1996) Molecular cloning of the mitogenic mannose⁄ maltose-specific rhizome lectin from Calyste-gia sepium FEBS Lett 397, 352–356

5 Peumans WJ, Zhang W, Barre A, Houles-Astoul C, Balint-Kurti P, Rovira P, Rouge´ P, May GD, Van Leuven F, Truffa-Bachi P & Van Damme EJM (2000) Fruit-specific lectins from banana and plantain Planta

211, 546–554

6 Geshi N & Brandt A (1998) Two jasmonate-inducible myrosinase-binding proteins from Brassica napus L seedlings with homology to jacalin Planta 204, 295–304

7 Van Damme EJM, Barre A, Mazard A-M, Verhaert P, Horman A, Debray H, Rouge´ P & Peumans WJ (1999) Characterization and molecular cloning of the lectin from Helianthus tuberosus Eur J Biochem 259, 135–142

8 Zhang W, Peumans WJ, Barre A, Houles-Astoul C, Rovira P, Rouge´ P, Proost P, Truffa-Bachi P, Jalali HA

& Van Damme EJM (2000) Isolation and characteri-zation of a jacalin-related mannose-binding lectin from salt-stressed rice (Oryza sativa) Plants Planta 210, 970– 978

9 Nomura K, Nakamura S, Fujitake M & Nakanishi T (2000) Complete amino acid sequence of Japanese chest-nut agglutinin Biochem Biophys Res Commun 276, 23– 28

10 Mann K, Farias CMSA, Gallego Del Sol F, Santos CF, Grangeiro TB, Nagano CS, Cavada BS & Calvete JJ (2001) The amino-acid sequence of the glucose⁄ man-nose-specific lectin isolated from Parkia platycephala seeds reveals three tandemly arranged jacalin-related domains Eur J Biochem 268, 4414–4422

11 Van Damme EJM, Hu J, Barre A, Houle`s-Astoul C, Rouge´ P, Proost P & Peumans WJ (2002) Two distinct jacalin-related lectins with a different specificity and subcellular location are major vegetative storage pro-teins in the bark of the mulberry tree Plant Physiol

130, 757–769

12 Yagi F, Iwaya T, Haraguchi T & Goldstein IJ (2002)

The lectin from leaves of Japanese cycad, Cycas revoluta

Thunb (gymnosperm) is a member of the jacalin-related

family Eur J Biochem 269, 4335–4341

13 Tateno H, Winter HC, Petryniak J & Goldstein IJ (2003)

Purification, characterization, molecular cloning, and

expression of novel members of jacalin-related lectins

from rhizomes of the true fern Phlebodium aureum (L)

J Smith (Polypodiaceae) J Biol Chem 278, 10891–10899

14 Bourne Y, Houle`s Astoul C, Zamboni V, Peumans WJ,

Menu-Bouaouiche L, Van Damme EJM, Barre A &

Rouge´ P (2002) Structural basis for the unusual

carbo-hydrate-binding specificity of jacalin towards galactose

and mannose Biochem J 364, 173–179

15 Jeyaprakash AA, Jayashree G, Mahanta SK,

Swami-nathan CP, Sekar K, Surolia A & Vijayan M (2005)

Structural basis for the energetics of jacalin–sugar

interactions: promiscuity versus specificity J Mol Biol

347, 181–188

16 Peumans WJ, Winter HC, Bemer V, Van Leuven F,

Goldstein IJ, Truffa-Bachi P & Van Damme EJM

(1997) Isolation of a novel plant lectin with an unusual

specificity from Calystegia sepium Glycoconjugate J 14,

259–265

17 Lee X, Thompson A, Zhang Z, Ton-That H,

Biester-feldt J, Ogata C, Xu L, Johnston AZ & Young NM

(1998) Structure of the complex of Maclura pomifera

agglutinin and the T-antigen disaccharide,

Galb1,3Gal-NAc J Biol Chem 273, 6312–6318

18 Pratap JV, Arockia Jeyaprakash A, Geetha Rani P,

Se-kar K, Surolia A & Vijayan M (2002) Crystal structures

of artocarpin, a Moraceae lectin with mannose

specifi-city, and its complex with methyl-a-d-mannose:

implica-tions to the generation of carbohydrate specificity

J Mol Biol 317, 237–247

19 Bourne Y, Zamboni V, Barre A, Peumans WJ, Van

Damme EJM & Rouge´ P (1999) Helianthus tuberosus

lectin, the crystal structure reveals a widespread scaffold

for mannose-binding lectins Structure Fold Des 7,

1473–1482

20 Rani PG, Bachhawat K, Misquith S & Surolia A (1999)

Thermodynamic studies of saccharide binding to

arto-carpin, a B-cell mitogen, reveals the extended nature of

its interaction with mannotriose

[3,6-Di-O-(a-d-manno-pyranosyl)-d-mannose] J Biol Chem 274, 29694–29698

21 Rani PG, Bachhawat K, Reddy GB, Oscarson S &

Surolia A (2000) Isothermal titration calorimetric

studies on the binding of deoxytrimannose derivatives

with artocarpin: implications for a deep-seated

combining site in lectins Biochemistry 39, 10755–

10760

22 Wu AM, Wu JH, Singh T, Chu K-C, Peumans WJ,

Rouge´ P & Van Damme EJM (2004) A novel lectin

(MornigaM) from mulberry (Morus nigra) bark

recog-nizes oligomannosyl residues N-glycans J Biomed Sci

11, 874–885

23 Raval S, Gowda SB, Singh DD & Chandra NR (2004)

A database analysis of¢jacalin-like¢ lectins: sequence-structure-function relationships Glycobiology 14, 1247– 1263

24 Rabijns A, Verboven C, Novoa de Armas H, Van Damme EJM, Peumans WJ & De Ranter CJ (2001) The crystals of a mannose-specific jacalin-related lectin from Morus nigraare merohedrally twinned Acta Crystallogr D57, 609–611

25 Otwinowski Z & Minor W (1997) Processing of x-ray diffraction data collected in oscillation mode Methods Enzymol 276, 307–326

26 Yeates TO (1997) Detecting and overcoming crystal twinning Methods Enzymol 276, 344–358

27 Bru¨nger AT (1992) X-PLOR, Version 3.1 A Systemn for X-Ray Crystallography and NMR Yale University Press, New Haven, CT

28 Matthews BW (1974) Determination of molecular weight from protein crystals J Mol Biol 82, 513–526

29 Bru¨nger AT, Adams PD, Clore GM, Delano WL, Gros

P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nilges

M, Pannu NS, Read RJ, Rice LM, Simonson T & Warren GL (1998) Crystallography & NMR system: a new software suite for macromolecular structure deter-mination Acta Crystallogr D54, 905–921

30 Jones TA, Zhou JY, Cowtan S & Kjelgaard M (1991) Improved methods for building protein models in elec-tron density maps and the location of errors in these models Acta Crystallogr A47, 110–119

31 Laskowski RA, MacArthur MW, Moss DS & Thornton

JM (1993) PROCHECK: a program to check the stereo-chemical quality of protein structures J Appl Crystal-logr 26, 283–291

32 Kleywegt GJ & Jones TA (1997) Model building and refinement practice Methods Enzymol 277, 208–230

33 Bernstein FC, Koetzel TF, Williams GJB, Meyer EF, Brice MD, Rodgers JR, Kennard O, Shimanouchi T & Tasumi M (1977) The protein data bank: a computer based archival file for macromolecular structures J Mol Biol 112, 535–542

34 Kraulis PJ (1991) MolScript: a program to produce both detailed and schematic plots of protein structures

J Appl Crystallogr 24, 946–950

35 Esnouf RM (1997) An extensively modified version of Molscript that includes greatly enhanced coloring capa-bilities J Mol Graphics 15, 132–134

36 Merrit EA & Bacon DJ (1997) Raster3D photorealistic molecular graphics Methods Enzymol 277, 505–524

37 Nicholls A, Sharp KA & Honig B (1991) Protein fold-ing and association: Insights from the interfacial and thermodynamic properties of hydrocarbons Proteins Struc Func Genet 11, 281–296

38 Gilson MK & Honing BH (1987) Calculation of

electro-static potential in an enzyme active site Nature 330, 84–

86

39 Gouet P, Courcelle E, Stuart DI & Metoz F (1999)

ESPript: analysis of multiple sequence alignments in

PostScript Bioinformatics 15, 305–308

40 Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F

& Higgins DG (1997) The CLUSTAL–X windows inter-face: flexible strategies for multiple sequence alignment aided by quality analysis tool Nucleic Acids Res 15, 4876–4882