Báo cáo khoa học: Structural basis for the recognition of complex-type biantennary oligosaccharides by Pterocarpus angolensis lectin ppt

The mannose on the 1–6 arm occupies the monosaccharide binding site while the GlcNAc residue on this arm occupies a subsite that is almost iden-tical to that of concanavalin A con A.. Th

biantennary oligosaccharides by Pterocarpus angolensis lectin

Lieven Buts1, Abel Garcia-Pino1, Anne Imberty2, Nicolas Amiot3, Geert-Jan Boons3,

Sonia Beeckmans4, Wim Verse´es1, Lode Wyns1and Remy Loris1

1 Laboratorium voor Ultrastructuur, Vrije Universiteit Brussel and Department of Molecular and Cellular Interactions, Vlaams Interuniversitair Instituut voor Biotechnologie, Belgium

2 Centre de Recherches sur les Macromole´cules Ve´ge´tales (CERMAV) – CNRS (affiliated with Joseph Fourier University), Grenoble, France

3 Complex Carbohydrate Research Center, The University of Georgia, Athens, GA, USA

4 Laboratorium voor Scheikunde der Proteı¨nen, Instituut voor Moleculaire Biologie, Vrije Universiteit Brussel, Belgium

Lectins (or lectin domains) represent a specific class of

carbohydrate binding proteins distinct from enzymes

and antibodies Different lectin families are found in

a wide range of organisms including viruses, bacteria,

plants and animals Their biological activities are

diverse and include roles in the innate immunity,

bac-terial and viral infection, sorting and trafficking of

gly-coproteins, development and differentiation as well as

defense mechanisms in plants [1]

The lectins from legume plants belong to one of the

best studied lectin families [2] Members of this family

were initially identified in the seeds of legume plants,

but an increasingly larger number is found in the

vegetative parts They show strong similarities on the level of their amino acid sequences and tertiary struc-tures, and exhibits a wide range of carbohydrate spe-cificities and quaternary structures Recently, family members were discovered in nonlegume plants [3] Fur-thermore, the ER-Golgi intermediate (ERGIC) pro-teins that in animals play a role in glycoprotein transport though the golgi apparatus (but are absent

in plants) belong to the legume lectin family [4] Concanavalin A (con A) was the first lectin for which the crystal structure was determined [5,6] Since this early work, the crystal structures of 28 members

of the legume lectin family have been presented either

Keywords

carbohydrate; lectin; legume lectin;

protein-carbohydrate recognition

Correspondence

R Loris, Laboratorium voor Ultrastructuur,

Vrije Universiteit Brussel and Department of

Molecular and Cellular Interactions,

Vlaams Interuniversitair Instituut voor

Biotechnologie, Pleinlaan 2,

B-1050 Brussels, Belgium

Fax: +32 2 6291963

Tel: +32 2 6291989

E-mail: reloris@vub.ac.be

(Received 5 January 2006, revised 21 March

2006, accepted 28 March 2006)

doi:10.1111/j.1742-4658.2006.05248.x

The crystal structure of Pterocarpus angolensis lectin is determined in its ligand-free state, in complex with the fucosylated biantennary complex type decasaccharide NA2F, and in complex with a series of smaller oligosaccha-ride constituents of NA2F These results together with thermodynamic binding data indicate that the complete oligosaccharide binding site of the lectin consists of five subsites allowing the specific recognition of the penta-saccharide GlcNAcb(1–2)Mana(1–3)[GlcNAcb(1–2)Mana(1–6)]Man The mannose on the 1–6 arm occupies the monosaccharide binding site while the GlcNAc residue on this arm occupies a subsite that is almost iden-tical to that of concanavalin A (con A) The core mannose and the GlcNAcb(1–2)Man moiety on the 1–3 arm on the other hand occupy a series of subsites distinct from those of con A

Abbreviations

Con A, concanavalin A; ITC, isothermal titration calorimetry; LOL, Lathyrus ochrus lectin.

in their unbound form or in complex with mono- or

oligosaccharides (http://www.cermav.cnrs.fr/lectines)

From these studies, a detailed picture of the

relation-ship between amino acid sequence and carbohydrate

specificity has emerged (7,8), which is still being refined

[9–22]

Apart from structural studies, the past decade has

also brought a large number of studies involving

iso-thermal titration calorimetry (ITC) (for a recent

review, see [23]) Especially con A and its relatives

from the Diocleinae subtribe have been studied in

detail by different research teams [23] These include

binding studies of mono- and oligosaccharides, binding

of deoxy sugar analogs, hydroxyethyl analogs and

con-formationally constrained sugars, analysis of solvent

isotope effects, osmotic stress strategies and analysis of

the cluster glycoside-effect This wealth of data stems

largely from the ease with which even gram quantities

of con A can be produced, which allows the design of

experiments that are not possible with most other

car-bohydrate binding proteins The body of structural

and thermodynamic data that is available for legume

lectins has also provided an impetus to predict

thermo-dynamic binding parameters using calculations based

on the crystal structures [24–27]

The seeds of the bloodwood tree (Pterocarpus

ango-lensis) are rich in a Man⁄ Glc specific lectin (PAL)

This lectin was recently isolated, its encoding gene

sequenced [21] and its crystal structure determined in

complex with nine mono-, di- and trisaccharides

[21,22] As in other legume lectins, the carbohydrate

binding site consists of five loops which have been

des-ignated A-E [7,15] These loops form a groove on the

surface of the protein in which an oligosaccharide can bind (Fig 1) Central in this groove is the monosac-charide binding site (M), which is flanked by a series

of subsites The subsites harboring additional sac-charide residues linked to O1 of the mannose in the monosaccharide binding site are designated the ‘down-stream’ subsites +1, +2, +3, while those linked to O2 form the ‘upstream’ subsite)1 (Fig 1)

Here, we present the crystal structures of PAL in its ligand-free state, in complex with the complex type decasaccharide NA2F and in complex with four fur-ther oligosaccharides that are fragments of NA2F The structural information is complemented by thermody-namic parameters of binding determined by means of isothermal titration calorimetry for the above-men-tioned complexes as well as other complexes for which the crystal structures were determined previously [21] Our results indicate that the complete oligosaccharide binding site of PAL consists of four extended sites surrounding the primary monosaccharide binding sites, conferring a maximal affinity for the pentasaccha-ride GlcNAcb(1–2)Mana(1–3)[GlcNAcb(1–2)Mana(1– 6)]Man Although this arrangement bears many simi-larities to what is observed in con A, there are clear differences in the conformational details and the subsite energetics between both lectins Thus for the first time a detailed combined thermodynamic and crystallographic study can be placed next to the body

of data published on con A From this we see how two evolutionarily related proteins can show the same oligosaccharide specificity but yet show a different pat-tern of subsite affinities and different interactions in those subsites

Fig 1 Architecture of the carbohydrate bin-ding site of a Man ⁄ Glc specific lectin (PAL) Shown is a stereo-view of a CPK (Corey Pauling Koltun) representation of the carbo-hydrate binding site The five different loops that make up the binding site are colored: loop A: light blue, loop B: orange, loop C: yellow, loop D: green, loop E: dark blue The monosaccharide binding site is indicated by

a ball-and stick model of mannose The relative positions of the upstream ( )1) and downstream (+1, +2, +3) subsites are indicated as well.

Structure of unliganded PAL and its complex

with mannose

A correct understanding of ligand binding not only

requires structural data on the protein-carbohydrate

complexes, but also knowledge of the structure of the

ligand-free protein As we were unable to obtain crystals

of ligand-free PAL directly, we opted to remove the

bound ligand from a PAL:Mana(1–3)Man complex by

reverse soaking (see Experimental procedures, and [28])

These crystals contain the PAL dimer in their

asymmet-ric unit, the two subunits of which are labeled A and B

The structure resulting from this experiment, refined at

1.8 A˚ to R¼ 0.185 and Rfree¼ 0.205 (Table 1) shows a

clear Mana(1–3)Man disaccharide bound in the binding

site of subunit A, which is involved in crystal lattice

interactions It is in all respects identical to subunit A of

the original Mana(1–3)Man complex described earlier

[21] The binding site of subunit B on the other hand,

which is not involved in crystal packing, is devoid of any

electron density that could be interpreted as

carbohy-drate Instead the monosaccharide binding site contains

four ordered water molecules (B-factors 22, 29, 49 and

40 A˚2) which are expelled upon binding of mannose

(Fig 2A) Three of them closely mimic the positions of

hydroxyls OH3, OH4 and OH6 of mannose, which

binds in a lock-and-key fashion without significant

changes in protein conformation

The binding of mannose to PAL is essentially identi-cal to what is observed in the ManaMe complex pre-sented before [21] and is summarized in Table 2 The anomeric oxygen is entirely in the a-configuration in the binding site of subunit A, but adopts an a⁄ b mix-ture in the binding site of subunit B Accommodation

of the b-configuration in the binding site of PAL requires a change in the rotamer conformation of Glu221 (Fig 2B) Steric hindrance is observed only if the b-anomeric oxygen contains an additional substitu-ent such as a methyl group The side chain of Glu221

is completely disordered in the ligand-free structure

Crystal structure of the NA2F decasaccharide complex

The decasaccharide Galb(1–4)GlcNAcb(1–2)Mana(1– 6)[Galb(1–4)GlcNAcb(1–2)Mana(1–3)]Manb(1–4)Glc-NAcb(1–4)[Fucb(1–6)]GlcNAc (NA2F) was only available in small quantities (20 lg) and could be used for a single soaking experiment While there is defin-itely not enough space to accommodate such a large carbohydrate in the binding site of subunit A in our PAL crystals, the space around binding site B is ample and by all means sufficient to accommodate a decasac-charide In order not to risk destroing the crystal dur-ing the experiment, the soak was performed on a previously ‘desoaked’ Mana(1–3)Man cocrystal, which

as described above still retains the disaccharide in the binding site of subunit A, but has an empty binding

Table 1 Data collection and refinement statistics.

GlcNAc (b1–2)Man

GlcNAc(b1–2)Man

Unit cell:

Resolution limits 15.0–1.80 A˚ 15.0–1.70 A˚ 15.0–1.80 A˚ 15.0–1.95 A˚ 15.0–1.80 A˚ 15.0–2.00

R merge 0.080 0.050 0.078 0.091 0.052 0.054

Ramachandran plot

site for subunit B The crystal structure indeed shows

the binding site of subunit A to be occupied with

Mana(1–3)Man

In the binding site of subunit B, electron density for

a heptasaccharide (GlcNAcb(1–2)Mana(1–6)[Galb(1–

4)GlcNAcb(1–2)Mana(1–3)]Manb(1–4)GlcNAcb) is

clearly visible (Fig 3A) Binding of this

heptasaccha-ride shields a total surface of 1045 A˚2(protein and

car-bohydrate) from the solvent The Mana(1–3)[Mana(1–

6)]Man moiety is bound as in the previously described

complex with the core trimannose [21], involving three

direct hydrogen bonds with the core mannose (+1

subsite) and two with the 1–3 linked mannose (+2

subsite) (Fig 3B and Table 2) The mannose on the

1–6 arm occupies the monosaccharide binding site

The GlcNAc residue on the 1–6 arm is bound in the

)1 subsite and makes two hydrogen bonds with the

protein The GlcNAc residue on the 1–3 arm binds in the +3 subsite, making two hydrogen bonds with the side chain of Asn83 as well as van der Waals contacts with the side chains of Leu81 and Gln222 The galac-tose on the 1–3 arm has defined electron density, but does not make any direct hydrogen bonds with the protein while the galactose on the 1–6 arm is com-pletely disordered Electron density is also visible for

an additional GlcNAc residue linked b1–4 to the core mannose, but this sugar residue does not contact the protein The electron density for this GlcNAc residue

is well defined, probably due to conformational restric-tions The N-linked GlcNAc and the fucose branch are completely disordered All observed glycosidic linkages are near their global low energy conformation with the exception of the GlcNAcb(1–2)Man linkage on the 1–6 arm, which occupies a secondary minimum (Fig 4)

Fig 2 The monosaccharide binding site of PAL (A) Stereo view of the monosaccharide binding site of PAL in absence of bound car-bohydrate The electron density for four water molecules that occupy the binding site is shown, together with the hydrogen bond network these waters make with the protein Superimposed in black are the equivalent residues of the mannose com-plex (subunit B in the asymmetric unit) The water molecules clearly mimic the oxygen atoms of bound mannose (black, thick lines) (B) Stereo view of the interactions of mannose in the monosaccharide binding site The beta-anomeric oxygen position of mannose is drawn in light green, as is the corresponding orientation of the side chain

of Glu221 The second conformation of the side chain of Glu221, which also adopts two conformations but not correlated with the anomeric form of the bound mannose is shown in dark green.

Structure of PAL in complex with

GlcNAcb(1–2)Man

GlcNAcb(1–2)Man binds with mannose in the

mono-saccharide binding site and with its nonreducing

GlcNAc in the upstream )1 subsite (Table 2 and Fig 5), however, using a binding mode that is distinct from the one observed in the NA2F complex In this binding mode, a total surface of 615 A˚2 is shiel-ded from the solvent, compared to 565 A˚2 for the

Table 2 Interactions between lectin and carbohydrates (all distances in A ˚ ) NP, not present The values for the binding sites of subunit A and subunit B are separated by ( ⁄ ).

Fig 3 (A) Electron density for NA2F in the binding site of subunit B of PAL The map shown is a simulated-annealing OMIT map calculated after removal of the carbohydrate from the model and contoured at 3 sigma Clear density is seen for seven out of 10 sugar residues The final model of the carbohydrate is superimposed (B) Stereo view of the interactions between NA2F and PAL The heptasaccharide moiety

of NA2F that is visible in the electron density map is drawn in green ball-and-stick Protein residues that are part of the )1, +1, +2 and +3 subsites are coloured accoring to atom type Hydrogen bonds are shown as dotted lines The amino acids that make up the monosaccharide binding site are omitted for clarity.

GlcNAcb-(1–2)Man moiety from NA2F The

confor-mation of the b(1–2) linkage is near the global energy

minimum Extensive van der Waals contacts are made

between GlcNAc and the backbone atoms of loop B (as defined by Sharma & Surolia, [7]) The conforma-tion of the GlcNAcb(1–2)Man disaccharide is stabil-ized by an intramolecular hydrogen bond from O3(Man) to O5(GlcNAc) GlcNAc makes a direct hydrogen bond with its O4 hydroxyl to the carbonyl group of Gly102 A second potential, but weaker hydrogen bond may be present between O7 of the N-acetyl group and the side chain hydroxyl of Ser45 O3 of GlcNAc is bridged via a water molecule (Wat116) to the backbone NH group of Gly104 while another water (Wat117) bridges O7 of GlcNAc to the backbone NH of Gly105 and the backbone carbonyl

of Gly219 Both waters are conserved in the sugar-free protein as well as in all structures where the )1 subsite

is not occupied [21,22]

Crystal structure of the M592 pentasaccharide complex

Clear electron density is seen for the complete penta-saccharide GlcNAcb(1–2)Mana(1–3)[GlcNAcb(1– 2)Mana(1–6)]Man (M592) in the binding site of sub-unit B (Fig 6A), which is adjacent to a large solvent channel in the crystal and far away from any lattice contact Therefore, it is assumed that the interactions observed in binding site B correspond to the situation

in solution Binding of the pentasaccharide shields a total surface of 1030 A˚2 from the solvent, a value almost identical to that of NA2F The tetrasaccharide moiety GlcNAcb(1–2)Mana(1–3))[Mana(1–6)]Man binds in an identical way as seen in the complex with NA2F (Fig 6B) The GlcNAc residue in the )1 sub-site, however, is oriented as is the GlcNAcb(1–2)Man complex (Fig 6B) All glycosidic bonds adopt confor-mations that correspond to energy minima in the cal-culated energy landscapes (Fig 4)

The same binding mode is not possible in binding site A due to steric overlap with a symmetry-related molecule of PAL As a consequence, only the )1 and primary sites are occupied by a GlcNAcb(1–2)Man moiety in the A subunit, with some ill-defined density indicating the disordered binding of the remaining three monosaccharides

Fig 4 Rigid conformational energy maps in function of Phi and Psi for the different linkages observed in the pentasaccharide M592 and NA2F bound to PAL, con A and LOL (upper) a(1–6) linkages (middle) a(1–3) linkages (lower) b(1–2) linkages In each case, energy levels are contoured at 5 kcal ⁄ mol intervals starting from the minimum energy The omega angle of Mana(1–6)Man was fixed in the gauche ⁄ trans (gt) conformation The conformations observed in the crystal structures are indicated.

Structure of PAL in complex with

GlcNAcb(1–2)Mana(1–3)ManaMe

GlcNAcb(1–2)Mana(1–3)ManaMe corresponds to the

1–3 arm of the pentasaccharide M592 In the M592

and NA2F complexes this trisaccharide moiety occu-pies the +1, +2 and +3 subsites, whereas by itself it adopts a different binding mode and occupies the )1 (GlcNAc), M (Man) and +1 (ManaMe) subsites (Fig 7) The binding of

GlcNAcb(1–2)Mana(1–3)Man-Fig 5 (A) Electron density for GlcNAcb(1–2)Man in the binding site of subunit A of PAL (B) Interactions of PAL with GlcNAcb(1–2)Man The protein is coloured according to atom type The disaccharide is colored green Selected residues as well as the sugar residues occupying subsites M and )1 are labeled The co-ordinates of the GlcNAcb(1–2)Man moiety on the 1–6 arm of the decasaccharide NA2F is superim-posed in thin black lines.

Fig 6 (A) Electron density for the pentasaccharide M592 in the binding site of subunit B of PAL The map is calculated and drawn as in Fig 3 (B) Interactions of M592 with PAL The pentasaccharide is shown in green, protein atoms are coloured accoring to atom type The equivalent pentasaccharide from NA2F is superimosed in thin black lines.

aMe to PAL is thus a linear combination of what

is observed for Mana(1–3)Man21 and GlcNAcb

(1–2)Man, shielding a total surface of 785 A˚2 from the

solvent This is in agreement with the general

observa-tion on lectins that there is a primary monosaccharide

binding site that needs to be occupied first before

bind-ing to adjacent subsites will occur

Thermodynamics of carbohydrate binding to PAL

To complement our structural studies, the

thermody-namic parameters for the binding to PAL for

man-nose, GlcNAcb(1–2)Man, the pentasaccharide M592 as

well a number of other mono-, di- and trisaccharide

constituents of M592 were measured by isothermal

titration calorimetry The results are summarized in

Table 3 As is usually observed for

protein:carbohy-drate interactions, the binding constants are in the

mil-limolar range

Binding of mannose in the primary binding site is

enthalpy-driven Also for most oligosaccharides, the

entropy contributions remain unfavorable Ligand

binding in the downstream +1 subsites is not mirrored

by a significantly enhanced affinity compared to

man-nose and the differences in their thermodynamic

parameters are close to or perhaps within the error

limits of the measurements This contrasts with the

well-defined carbohydrate conformations observed in

the crystal structures of the Mana(1–3)Man, Mana(1–

4)Man and Mana(1–6)Man complexes [21], a situation also observed in con A [29] (often carbohydrate resi-dues have a well defined conformation only if they contribute to affinity) In the case of the core trisac-charide Mana(1–3)[Mana(1–6)]Man there is a small increase in affinity and the additional carbohy-drate:protein contacts are mirrored by a more favora-ble enthalpy of binding

On the other hand, binding of N-acetylglucosamine

to the upstream)1 subsite contributes to a modest (15 fold) increase of affinity (from 1.9·103 m)1 to 26·103

m)1, Table 3) Surprisingly, for GlcNAcb(1–2)Man, occupation of the upstream binding site by GlcNAc is entropy-driven A loss of 2.7 kcalÆmol)1 in binding enthalpy is compensated by a 4.3 kcalÆmol)1 gain in TDS
Although odd, this result is most likely real and not a consequence of the correlation of fitting parame-ters often observed for weak binding events Evidence for this stems from the reproducibility of this result as well as from the c-value used in the titration (34.7) which allows a meaningful separation of DG
into DH
and TDS
terms

Discussion

Thermodynamics versus structure The most surprising observation in the present study

is the entropy-favorable binding in the )1 subsite of

Fig 7 (A) Electron density for GlcNAcb(1–2)Mana(1–3)ManaMe in the binding site of subunit A of PAL (B) Interactions of PAL with Glc-NAcb(1–2)Mana(1–3)ManaMe The trisaccharide is shown in green, protein atoms are coloured accoring to atom type The equivalent trisac-charide from the NA2F complex is superimosed in thin black lines.

PAL, which is only rarely seen in

protein-carbohy-drate recognition systems One potential explanation

for this unexpected observation would be a sliding

mechanism of binding Such a mechanism was

pro-posed to explain thermodynamic data for the binding

of Mana(1–2)Man and GlcNAcb(1–2)Man to con A

[30], and was later confirmed for Mana(1–2)Man by

X-ray crystallography [31] In the case of PAL such a

sliding binding mechanism seems unlikely GlcNAc is

always seen to occupy the )1 subsite and never the

monosaccharide binding site When GlcNAc is

mode-led in the monosaccharide binding site, severe steric

clashes are observed between the mannose residue and

the protein for all conformations due to the b(1–2)

linkage A second possibility is that the b(1–2) linkage

adopts two different conformations, both of which

result in favorable interactions between GlcNAc and

the protein Indeed, a secondary minimum is occupied

in the NA2F complex, but this is most likely due to

the presence of an additional galactose residue that

prevents binding in the global minimum

conforma-tion Neither in the GlcNAcb(1–2)Man complex, nor

in the complexes with M592 or GlcNAcb(1–2)

Mana(1–3)ManaMe is there any evidence for two

conformations despite that they are not prevented by

crystal lattice contacts Thus, if the ligand in the

con-formation corresponding to the secondary minimum

of the b(1–2) linkage binds to PAL in solution, it is

almost certainly a minority binding mode (< 10%)

and would not significantly affect the outcome of the

ITC titration experiments

The biantennary pentasaccharide GlcNAcb(1– 2)Mana(1–3)[GlcNAcb(1–2)Mana(1–6)]Man (M592) shows the highest affinity of all carbohydrates tested

It is potentially bivalent, but analysis of the ITC titra-tion data indicates a 1 : 1 stoichiometry, in agreement with crystallographic data Here, the formal possibility

of a backwards binding mode [with the a(1–3) arm occupying the )1 and M subsites as also observed for GlcNAcb(1–2)Mana(1–3)ManaMe] needs to be consid-ered Again, no evidence for this is seen in the crystal structure despite that this would not be sterically hin-dered by lattice interactions in the binding site of sub-unit B Thus, most likely, the backwards binding mode will again be a minority species in solution and not sig-nificantly influence the binding data

Comparison with related systems The complex of PAL with NA2F is one of the few complexes between a lectin and a large biantennary glycan that have been studied using X-ray crystallogra-phy The Man⁄ Glc-specific lectins from the Viciae tribe have their highest affinity for N-acetyllactosamine type N-glycans bearing a fucose a(1–6) linked to the Asn-linked GlcNAc such as NA2F [32,33] In contrast to PAL, the lectin from Lathyrus ochrus (LOL) binds with its a1–3 arm in the monosaccharide binding site [34] The conformation of the GlcNAcb(1–2)Man entity occupying the )1 and M subsites of LOL roughly resembles the conformation seen in the NA2F complex of PAL (Fig 4) LOL binds the fucosylated

Table 3 Thermodynamic parameters of saccharide binding to PAL.

Carbohydrate

No of expts c-value Stoichiometry*

Kass (10 3

M )1)

DG 0

(kcalÆmol)1)†

DH 0

(kcalÆmol)1)†

TDS 0

(kcalÆmol)1)†

(a1–6)]Man

a(1–3)[Mana(1–6)

]Mana(1–6)]Man

a(1–3)[GlcNAcb(1–2)

Mana(1–6)]Man

* Obtained from fitting the ITC data The reported values for Kass, DG 0 , DH 0 and TDS 0 are determined with n fixed at 1.0 These values do not differ significantly from those obtained when treating the number of binding sites as a variable † The errors on DG 0 and DH 0 are of the order of magnitude of 0.1 kcalÆmol)1while for TDS0they are 0.2 kcalÆmol)1.

chitobiose stem in its downstream subsites, while the

a1–6 arm points away from the protein towards the

solvent PAL apparently lacks the fucose-recognizing

subsite of LOL

Within NA2F, the pentasaccharide GlcNAcb(1–

2)Mana(1–3)[GlcNAcb(1–2)Mana(1–6)]Man (M592)

seems to be the largest entity that is specifically

recog-nized by PAL as evidenced from our combined

crystal-lographic and thermodynamic results The same

pentasaccharide is the largest monovalent determinant

specifically recognized by con A [30,35] Significant

dif-ferences are observed between PAL and con A from the

structural as well as the thermodynamic viewpoint Both

proteins have the a(1–6)-linked mannose in their

mono-saccharide binding site as well as a GlcNAc residue in

the same orientation in the)1 subsite The )1 subsite,

which mainly consists of loop B (as defined by Sharma

& Surolia [7]), is well-conserved between con A and

PAL The only relevant substitution concerns Gly104

of PAL which is Thr226 in con A The side chain of Thr226 occupies the space taken by the conserved waters 116 and 117 of PAL and makes a hydrogen bond

to O3 of GlcNAc (Fig 8A) Binding of GlcNAc in the )1 subsite of PAL is energetically favorable and contri-butes significantly to the higher affinity of PAL for the pentasaccharide M592 (Table 3) In the case of con A

on the other hand, binding of GlcNAc to the)1 subsite does not affect affinity [30] This was attributed to a strained conformation of the disaccharide in the com-plex of con A with pentasaccharide M592 [35] These conclusions were, however, based on older, less accurate energy maps [36], which later have been updated [37]

As can be seen in Fig 4, this linkage conformation observed in the con A complex is very close to those observed in all PAL complexes, except for the NA2F complex and corresponds to a low energy conformation

Fig 8 Comparison between PAL and con

A (A) Comparison between con A and PAL Stereo view of the binding of M592 (green)

to the )1 and M subsites of con A (colored according to atom type) The corresponding situation in PAL is superimposed as shown

in thin black lines (B) Downstream binding sites Stereo view of the binding of M592 (green) to the +1, +2 and +3 subsites of con A (colored according to atom type) The corresponding situation in PAL is super-imposed as shown in thin black lines.