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

Structural basis for the recognition of complex-type biantennary oligosaccharides by Pterocarpus angolensis lectin Lieven Buts 1 , Abel Garcia-Pino 1 , Anne Imberty 2 , Nicolas Amiot 3 , Geert-Jan Boons 3 , Sonia Beeckmans 4 , Wim Verse ´ es 1 , Lode Wyns 1 and Remy Loris 1 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. FEBS Journal 273 (2006) 2407–2420 ª 2006 The Authors Journal compilation ª 2006 FEBS 2407 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. Carbohydrate recognition by PAL L. Buts et al. 2408 FEBS Journal 273 (2006) 2407–2420 ª 2006 The Authors Journal compilation ª 2006 FEBS Results 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 R ¼ 0.185 and R free ¼ 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. Ligand- removed Mannose GlcNAc (b1–2)Man GlcNAc(b1–2)Man (a1–3)ManOMe M592 NA2F Space group P2 1 2 1 2 1 P2 1 2 1 2 1 P2 1 2 1 2 1 P2 1 2 1 2 1 P2 1 2 1 2 1 P2 1 2 1 2 1 Beamline X11 X11 X11 X11 X11 ID14-1 Unit cell: a ¼ 56.86 A ˚ 56.73 A ˚ 57.28 A ˚ 56.80 A ˚ 56.71 A ˚ 56.78 A ˚ b ¼ 83.21 A ˚ 83.36 A ˚ 82.68 A ˚ 83.14 A ˚ 83.59 A ˚ 83.32 A ˚ c ¼ 123.23 A ˚ 122.94 A ˚ 125.00 A ˚ 123.02 A ˚ 122.37 A ˚ 122.38 A ˚ 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 Number of measured reflections 681254 305125 214056 170746 285333 123639 Number of unique reflections 54780 53278 54204 43030 54417 34590 Completeness 99.7% 98.1% 98.4% 97.9% 98.8% 85.8% R merge 0.080 0.050 0.078 0.091 0.052 0.054 <I⁄ r(I) > 24.2 22.0 13.2 11.7 12.0 12.0 R-factor 0.185 0.170 0.179 0.179 0.176 0.176 R free -factor 0.205 0.204 0.212 0.199 0.201 0.216 Ramachandran plot core 86.9% 88.0% 88.8% 87.1% 87.2% 86.2% additionally allowed 12.9% 12.0% 11.2% 12.9% 12.1% 13.8% disallowed 0.2% 0.0% 0.0% 0.0% 0.0% 0.0% PDB code 1S1A 2ARE 2ARB 2AUY 2AR6 2ARX L. Buts et al. Carbohydrate recognition by PAL FEBS Journal 273 (2006) 2407–2420 ª 2006 The Authors Journal compilation ª 2006 FEBS 2409 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. Carbohydrate recognition by PAL L. Buts et al. 2410 FEBS Journal 273 (2006) 2407–2420 ª 2006 The Authors Journal compilation ª 2006 FEBS 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 ( ⁄ ). H-bond Mannose GlcNAcb(1–2)Man M592 NA2F Site )1 Gly102(O) GlcNAc(O4) NP 2.81 ⁄ 2.76 2.77 ⁄ 2.65 NP Ser45(OG) GlcNAc(O7) NP 3.48 ⁄ 3.45 3.28 ⁄ 3.38 NP Gly104(O) GlcNAc(N2) NP NP NP NP ⁄ 3.46 Glu221(OE2) GlcNAc(O6) NP NP NP NP ⁄ 3.22 Site M Asp86(OD1) Man(O4) 2.60 ⁄ 2.58 2.60 ⁄ 2.54 2.56 ⁄ 2.50 2.56 ⁄ 2.78 Asp86(OD2) Man(O6) 2.85 ⁄ 2.78 2.85 ⁄ 2.75 2.78 ⁄ 2.77 2.75 ⁄ 2.84 Gly106(N) Man(O3) 2.86 ⁄ 2.94 2.97 ⁄ 2.99 2.83 ⁄ 2.80 2.96 ⁄ 2.84 Asn138(ND2) Man(O4) 3.00 ⁄ 2.92 2.93 ⁄ 2.93 2.95 ⁄ 2.93 3.15 ⁄ 2.98 Glu221(N) Man(O5) 3.00 ⁄ 3.01 3.01 ⁄ 3.01 3.12 ⁄ 3.05 3.18 ⁄ 2.99 Gln222(N) Man(O6) 2.99 ⁄ 3.06 3.01 ⁄ 3.07 3.03 ⁄ 3.01 3.01 ⁄ 3.05 Site +1 Asp136(OD2) Man(O2) NP NP NP ⁄ 2.58 NP ⁄ 2.62 Ser137(OG) Man(O2) NP NP NP ⁄ 2.74 NP ⁄ 2.64 Gln222(NE2) Man(O4) NP NP NP ⁄ 2.62 NP ⁄ 2.46 Site +2 Asn83(ND2) Man(O3) NP NP NP ⁄ 2.84 NP ⁄ 3.02 Asp136(OD1) Man(O6) NP NP NP ⁄ 2.69 NP ⁄ 2.75 Site +3 Asn83(OD1) GlcNAc(O6) NP NP NP ⁄ 2.71 NP ⁄ 2.81 Asn83(ND2) GlcNAc(O5) NP NP NP ⁄ 2.84 NP ⁄ 2.88 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. L. Buts et al. Carbohydrate recognition by PAL FEBS Journal 273 (2006) 2407–2420 ª 2006 The Authors Journal compilation ª 2006 FEBS 2411 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 GlcNAc b(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. Carbohydrate recognition by PAL L. Buts et al. 2412 FEBS Journal 273 (2006) 2407–2420 ª 2006 The Authors Journal compilation ª 2006 FEBS 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. L. Buts et al. Carbohydrate recognition by PAL FEBS Journal 273 (2006) 2407–2420 ª 2006 The Authors Journal compilation ª 2006 FEBS 2413 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 1.9·10 3 m )1 to 26·10 3 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. Carbohydrate recognition by PAL L. Buts et al. 2414 FEBS Journal 273 (2006) 2407–2420 ª 2006 The Authors Journal compilation ª 2006 FEBS 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* K ass (10 3 M )1 ) DG 0 (kcalÆmol )1 )† DH 0 (kcalÆmol )1 )† TDS 0 (kcalÆmol )1 )† Mannose 4 3.8 0.90 1.9 )4.4 )6.3 )1.9 ManaMe 3 5.4 0.95 3.4 )4.8 )6.5 )1.7 Mana(1–2)Man 3 13.0 0.92 15.4 )5.7 )7.0 )1.3 Mana(1–3)Man 4 2.1 0.97 2.3 )4.6 )5.7 )1.1 Mana(1–4)Man 3 2.1 0.92 3.4 )4.8 )6.4 )1.6 Mana(1–6)Man 3 1.6 1.04 2.0 )4.5 )6.5 )2.0 Mana(1–3)[Man 3 1.4 0.95 5.6 )5.1 )7.1 )2.0 (a1–6)]Man Mana(1–3)[Man 3 2.3 0.95 6.6 )5.2 )8.7 )3.5 a(1–3)[Mana(1–6) ]Mana(1–6)]Man GlcNAc(b1–2)Man 3 34.7 0.90 26.0 )6.0 )3.6 2.4 GlcNAcb(1–2)Man 3 59.0 1.10 63.0 )6.5 )7.9 )1.4 a(1–3)[GlcNAcb(1–2) Mana(1–6)]Man * Obtained from fitting the ITC data. The reported values for K ass , 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 )1 while for TDS 0 they are 0.2 kcalÆmol )1 . L. Buts et al. Carbohydrate recognition by PAL FEBS Journal 273 (2006) 2407–2420 ª 2006 The Authors Journal compilation ª 2006 FEBS 2415 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. Carbohydrate recognition by PAL L. Buts et al. 2416 FEBS Journal 273 (2006) 2407–2420 ª 2006 The Authors Journal compilation ª 2006 FEBS [...]... Buts et al In the latter complex this conformation is made impossible by the additional galactose substituted on O4 of GlcNAc, as is also observed in LOL :biantennary glycans complexes [34] Therefore, we conclude that the reason for the difference in energetics of the )1 subsite between PAL and con A must be due to details in the interaction between the protein and the carbohydrate and not to the carbohydrate... DEXTRA laboratories and are claimed to be 98% pure by the manufacturer Solutions for ITC titrations were prepared by weighting the total amount of sugar on a microbalance prior to dissolving it into the buffer used for the final dialysis of the protein The errors on the sugar concentrations should be smaller than 2% for the oligosaccharides and negligible for mannose and methyl-a-d-mannopyrannoside Crystal... fresh solution every week The crystal did not show any visible signs of damage after this procedure To obtain data for the complexes of PAL with different sugars, crystals of the Mana(1–3)Man complex [21] were soaked for 1 h in artificial mother liquor enriched with 100 mm of the desired sugar as described [28] In the case of the decasaccharide, the soaking procedure was performed on a previously ‘desoaked’... affinity of M592 to the lectin For con A, a small improvement in interaction energy is also observed for GlcNAc binding in the +3 subsite [30], while for the lectin: ligand complexes of PAL this is neutral In conclusion, the combination of structural biology and thermodynamics has allowed us to obtain new insights in the mechanisms that govern protein–oligosaccharide interactions To explain thermodynamics... from the Vlaams Interuniversitair Instituut voor Biotechnologie (VIB), the Fonds voor Wetenschappelijk Onderzoek Vlaanderen (FWO) and the Onderzoeksraad of the Vrije Universiteit Brussel The authors acknowledge the use of synchrotron beamtime at the EMBL beamlines at the DORIS storage ring (Hamburg, Germany) and the ESRF (Grenoble France) GJB and NA were supported by the NIH Research Resource Center for. .. the mannose residue bound in the +1 subsite does not contribute to affinity but functions as a hinge For con A, interactions of mannose in the +2 subsite contribute dominantly to its higher affinity for the pentasaccharide M592 compared to methyl-a-d-mannopyrannoside [30] For PAL on the other hand, binding of mannose this subsite, although favorable, contributes only moderate to the overall affinity of. .. DHb À TDSb Errors on the thermodynamic parameters of binding were derived from a statistical analysis of at least three repetitions for each carbohydrate The initial protein concentration [C]0, the carbohydrate concentrations in the syringe [S]0 and the volume V of the calorimeter cell were considered to be known constants The experiments were carried out under conditions where the c-value (n.Ka.[C]0)... protein and ligand (highly conservative estimates for the errors on these concentrations) This led to variations in those two parameters that were within 0.1 kcalÆmol)1 Calculation of conformational energy maps Energy maps are calculated using the tripos force-field [47] together with the PIM parameterization [37] developed for carbohydrates as a function of the F and Y dihedral angles 2418 1 Taylor ME &... Isolectins I-A and I-B of Griffonia (Bandeiria) simplici- Carbohydrate recognition by PAL 21 22 23 24 25 26 27 28 29 30 31 32 folia: crystal structure of metal-free GSI-B4 and molecular bases for metal binding and monosaccharide specificity J Biol Chem 277, 6608–6614 Loris R, Van Walle I, De Greve H, Beeckmans S, Deboeck F, Wyns L & Bouckaert J (2004) Structural basis of oligomannose recognition by the. .. recognition by the Pterocarpus angolensis seed lectin J Mol Biol 335, 1227–1240 Loris R, Imberty A, Beeckmans S, Van Driessche E, Read JS, Bouckaert J, De Greve H, Buts L & Wyns L (2003) Crystal structure of Pterocarpus angolensis lectin in complex with glucose, sucrose and turanose J Biol Chem 278, 16297–16303 Dam TK & Brewer CF (2002) Thermodynamic studies of lectin carbohydrate interactions by isothermal titration . Structural basis for the recognition of complex-type biantennary oligosaccharides by Pterocarpus angolensis lectin Lieven Buts 1 ,. fur- ther oligosaccharides that are fragments of NA2F. The structural information is complemented by thermody- namic parameters of binding determined by