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

Structural analysis of the jacalin-related lectin MornigaM from the black mulberry (Morus nigra) in complex with mannose Anja Rabijns 1 , Annick Barre 2 , Els J. M. Van Damme 3 , Willy J. Peumans 3 , Camiel J. De Ranter 1 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, Po ˆ le 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, Po ˆ le 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. FEBS Journal 272 (2005) 3725–3732 ª 2005 FEBS 3725 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. Parameter MornigaM MornigaM– Man complex Data collection: Space group P65 P65 Used wavelength (A ˚ ) 0.912 0.910 Used beam line X11 X11 Resolution limit (A ˚ ) 1.8 (1.83–1.80) 2.0 (2.03–2.00) Total observations 250868 211071 Unique reflections 100939 (5072) 74409 (3763) Completeness of all data (%) 98.4 (99.7) 99.1 (100.0) Completeness of data (%) (I >2r) 90.6 (70.4) 89.8 (71.7) Mean I ⁄ r 20.8 (5.0) 21.3 (4.9) R sym value (%) 4.2 (16.0) 4.5 (18.9) Refinement: R-work (%) 19.0 19.1 R-free (%) 21.0 21.0 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 r.m.s.d. bond angles (°) 1.45 1.42 Structure of a jacalin-related lectin complexed to mannose A. Rabijns et al. 3726 FEBS Journal 272 (2005) 3725–3732 ª 2005 FEBS 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 b1tob1 (loop 1), b7tob8 (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. A. Rabijns et al. Structure of a jacalin-related lectin complexed to mannose FEBS Journal 272 (2005) 3725–3732 ª 2005 FEBS 3727 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 AB CD EF 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 jacalin 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]. AB 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. Structure of a jacalin-related lectin complexed to mannose A. Rabijns et al. 3728 FEBS Journal 272 (2005) 3725–3732 ª 2005 FEBS 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 P6 5 with a ¼ 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 ˚ 3 ÆDa )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. Solvent molecules were progres- sively added when they met the following requirements: (a) a minimum 3 r peak had to be present in the |F obs |-|F calc | difference map; (b) a peak had to be visible in the 2|F obs |- |F calc | 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. Mannose A B C D O1 – – H 2 O21: 2.84 A ˚ H 2 O572: 2.67 A ˚ O2 – – – – O3 – Gly27N: 3.17 A ˚ Gly27N: 2.84 A ˚ Gly27N: 2.85 A ˚ O3 – – H 2 O475: 2.76 A ˚ – O4 – H 2 O257: 2.57 A ˚ H 2 O174: 2.57 A ˚ H 2 O325 : 2.55 A ˚ O4 – Asp153Od1: 2.47 A ˚ Asp153Od1: 2.58 A ˚ Asp153Od1: 2.51 A ˚ O5 – Phe150N: 3.00 A ˚ Phe150N:3.06 A ˚ Phe150N: 2.99 A ˚ O6 – Asp153Od2: 2.57 A ˚ Asp153Od2 : 2.70 A ˚ Asp153Od2: 2.72 A ˚ O6 – Val151O: 3.14 A ˚ Val151O:3.19 A ˚ Val151O: 3.19 A ˚ O6 – Val151N: 3.05 A ˚ Val151N:3.11 A ˚ Val151N: 2.97 A ˚ O6 – Phe150N: 2.90 A ˚ Phe150N:2.90 A ˚ Phe150N:2.79 A ˚ A. Rabijns et al. Structure of a jacalin-related lectin complexed to mannose FEBS Journal 272 (2005) 3725–3732 ª 2005 FEBS 3729 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). 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Structural analysis of the jacalin-related lectin MornigaM from the black mulberry (Morus nigra) in complex with mannose Anja Rabijns 1 , Annick Barre 2 ,. 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