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The First Crystal Structure of a Mimosoideae Lectin Reveals a Novel Quaternary Arrangement of a Widespread Domain
doi:10.1016/j.jmb.2005.08.055
J. Mol. Biol. (2005) 353, 574–583
The First Crystal Structure of a Mimosoideae Lectin Reveals a Novel Quaternary Arrangement of a Widespread Domain Francisca Gallego del Sol1, Celso Nagano1,2, Benildo S. Cavada2 and Juan J. Calvete1* 1
Instituto de Biomedicina de Valencia, C.S.I.C., E-46010 Valencia, Spain 2
BioMol-Laboratory, Departamento de Bioquı´mica e Biologia Molecular, Universidade Federal do Ceara´, Fortaleza, CE 60451-970, Brazil
The crystal structures of the apo and mannose-bound Parkia platycephala seed lectin represent the first structure of a Mimosoideae lectin and a novel circular arrangement of b-prism domains, and highlight the adaptability of the b-prism fold as a building block in the evolution of plant lectins. The P. platycephala lectin is a dimer both in solution and in the crystals. Mannose binding to each of the three homologous carbohydrate-recognition domains of the lectin occurs through different modes, and restrains the flexibility of surface-exposed loops and residues involved in carbohydrate recognition. The planar array of carbohydrate-binding sites on the rim of the toroidshaped structure of the P. platycephala lectin dimer immediately suggests a mechanism to promote multivalent interactions leading to cross-linking of carbohydrate ligands as part of the host strategy against phytopredators and pathogens. The cyclic structure of the P. platycephala lectin points to the convergent evolution of a structural principle for the construction of lectins involved in host defense or in attacking other organisms. q 2005 Elsevier Ltd. All rights reserved.
*Corresponding author
Keywords: Parkia platycephala lectin; Mimosoideae lectin; crystal structure; quaternary structure; b-prism domain
Protein–carbohydrate interactions play biological roles in cellular processes, such as cell communication, host defense, fertilization, development, parasitic infection, tumor metastasis, and plant defense against herbivores and pathogens. Lectins, the carbohydrate-binding proteins that decipher the glycocodes encoded in the structure of glycans,1 are ubiquitous in animals, plants and microorganisms. Mechanisms for sugar recognition have evolved independently in diverse protein frameworks.2 In addition, lectins sharing a common structural fold may differ in their carbohydrate-binding specificities.3 Multivalence is used frequently by lectins for increasing their glycan binding avidity and specificity.4 Hence, most lectins are oligomeric or multidomain proteins. Quaternary structure variation is also observed among lectins with the same tertiary fold and belonging to the same protein family3–7
and constitutes a further level of structural divergence. As the number of lectin structure–function correlation studies increases, more parallels between plant and animal lectins become apparent. Specifically, a small number of protein folds (e.g. jelly-roll domain, C-type lectin fold, b-propeller, b-trefoil motif, b-prism domain, monocot fold; consult also the 3D Lectin Database† appear to have been widely used by evolution for the generation of carbohydrate specificity and variability in subunit association3,5–11 that enables homologous lectins to adapt to the different spacings and orientations of the lectin’s carbohydrate ligands on soluble and cell surface-exposed glycoproteins. The b-prism fold, a w135–150 amino acid residue domain first reported in the structure of jacalin,12 the mannose-binding lectin of jack fruit (Artocarpus integrifolia) seeds, defines Family PD000864 of the ProDom database,‡ and consists of 12 b-strands arranged in three four-stranded Greek key motifs
Abbreviation used: PPL, P. platycephala seed lectin. E-mail address of the corresponding author: [email protected]
† http://www.cermav.cnrs.fr/lectines/ ‡ http://protein.toulouse.inra.fr/prodom/current/ html/form.php
Introduction
0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
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First Crystal Structure of a Mimosoideae Lectin
that possess an approximate internal 3-fold symmetry and form the faces of a triangular prism structure. Mannose-binding jacalin-related lectins are widespread among plants of taxonomically distant families, including Spermatophyta, Angiosperm, monocots: banana (Musa acuminata, Musaceae),13 rice (Oryza sativa, Poaceae);14 and dicots: jack fruit (Artocarpus integrifolia)12 and black mulberry (Morus nigra) (Moraceae);15 hedge bindweed (Calystegia sepium, Convolvulaceae);16 Jerusalem artichoke (Helianthus tuberosus, Asteraceae),17 Parkia platycephala (Mimosoideae),18 Japanese chestnut (Castanea crenata, Fagaceae);19 Spermatophyta, Gymnosperm Japanese cycad (Cycas revoluta Thunb.);20 and Pteridophyta: the true fern Phlebodium aureu (Polypodiaceae).21 This widespread distribution and the fact that mannose is an abundant building block of surface-exposed glycoconjugates of viruses, bacteria, and fungi, supports the view that the mannose-recognizing lectins fulfil a role in the plant defense against pathogens.2 Moreover, b-prism jacalin-related lectins shows also sequence similarity with proteins inducible by the plant hormone jasmonic acid, and with salt-stressinduced gene products of a number of different plants,14,18 suggesting a common ancestry for jacalinrelated lectins and inducible defense proteins.22 With the exceptions of the Japanese chestnut agglutinin and the P. platycephala seed lectin, whose primary structures consist, respectively, of two19 and three18 consecutive b-prism domains, all known mannose-binding jacalin-related lectin protomers are single b-prism domain proteins. The crystal structures of the seed lectin (apo and in complex with 5-bromo-4-chloro-3-indolyl-a-D-mannose) of P. platycephala reported here represent the first structures of both, a lectin from Mimosoideae, the most primitive group of Leguminosae plants, and
of a protein composed of three tandemly arranged b-prism domains. These structures reveal unique structural features among jacalin-related lectins: a novel quaternary arrangement of the b-prism; nonidentical mannose-binding modes between each of the three homologous carbohydrate recognition sites; and mannose-binding restraint of the conformational flexibility of surface-exposed loops and residues of the carbohydrate coordination sphere.
Results and Discussion A novel hexameric association of b-prism domains in the crystal structure of the P. platycephala seed lectin The Br atom of X-Man was used as an anomalous X-ray scatterer for solving the crystal structure of the P. platycephala seed lectin (PPL)-X-Man complex by multiple-wavelength anomalous dispersion (MAD). The final structures (Figure 1), refined to a ˚ , have good stereochemistry resolution of 2.5 A (Table 1). The PPL-X-Man monomer structure contains 441 amino acid residues (residues 3–444) and 202 water molecules. The coordinates of the PPL-X-Man complex were used to solve the structure of the apo lectin by molecular replacement. The apo lectin structure contains 441 amino acid residues and 178 water molecules, and was ˚ (Table 1). Both PPL solved to a resolution of 2.5 A structures are structurally very similar, and unless indicated otherwise, reference to the structure pertains to that of the X-Man complex. Local structural differences between the apo and the X-Man complexed lectin will be highlighted and discussed below.
Figure 1. The crystal structure of the P. platycephala seed lectin. Stereo drawing of the P. platycephala lectin dimer showing the six X-mannose residues located at the periphery of the dimeric assembly. The three tandemly arranged b-prism domains of a lectin subunit are color-coded, from the N to the C terminus, green (A1, B1), red (A2, B2) and blue (C3, B3). The Br atoms of X-Man used as anomalous scatterers for MAD phasing are represented by green spheres. The orphan cysteine residue 199 on b5 of b-prism 2 is depicted in yellow and in the space-filling model. The plot was made with MOLSCRIPT.44
576
First Crystal Structure of a Mimosoideae Lectin
Table 1. Summary of data collection and refinement statistics
A. Data collection statistics Space group Cell constants ˚) a (A ˚) b (A ˚) c (A a (deg.) b (deg.) g (deg.) ˚) Resolution (A Unique reflections Data coverage (%) Rmerge (%)a B. Refinement statistics ˚) Resolution range (A Reflections R-factor (%)b Rfree (%)c rms deviations ˚) Bond lengths (A Bond angles (deg.)
Apo P. platycephala lectin
X-Man-P. platycephala lectin complex
P212121
P21
63.60 68.51 208.53 90 90 90 2.5 31,540 96.6 6.2
80.24 114.18 80.30 90 119.88 90 2.5 43,451 99.9 5.3
30.0–2.5 29,482 21.2 24.4
30.0–2.5 39,474 20.9 24.4
0.016 1.687
0.017 3.152
a Rmerge Z jIKhIij=hIi, where I is the intensity and hIi is the average I for all observations of equivalent reflections. b R-factorZ jFobs jKjFcalc j=jFobs j. c Calculated with 5% of the reflections omitted from refinement.
The asymmetric units of the orthorhombic (apo PPL) and the monoclinic (PPL-X-Man complex) crystals were each occupied by two lectin molecules associated into the same flat, toroid-shaped struc˚ !82 A ˚ !20– ture with overall dimensions of 108 A ˚ and with a 16 A ˚ wide solvent channel at the centre 40 A (Figure 1). The fact that equilibrium sedimentation
analytical ultracentrifugation experiments showed that PPL behaves as a pH-independent dimer in dilute solutions,18 strongly indicates that the crystal structure may truly represent the quaternary conformation of the P. platycephala lectin in solution. The conformation of each PPL subunit is maintained by interactions between the b-prism domains 1 and 2 and 2 and 3, and the dimeric assembly is further stabilized by two distinct intersubunit interfaces involving contacts between the N-terminal (A1-B1) and the C-terminal (A3-B3) b-prism domains of the two PPL molecules (Figure 1). Each PPL subunit possesses a solventexposed cysteine residue (Cys199, Figure 2) located on b5 of b-prism 2 (Figure 1). Reactive sulfhydryl groups are rare in b-prism lectins, and generally in lectins. The only other known b-prism example is heltuba, which contains a buried orphan cysteine residue (Cys40) at the beginning of b4. In contrast, PPL Cys199 reacts in the native lectin with alkylating reagents,18 though it does not participate in any inter or intrasubunit contacts, either within the canonical dimer or between crystal mates. Intrasubunit interactions between adjacent PPL b-prism domains 1 and 2 are established essentially through contacts between residues from b-strands 1, 10 and 12, and between residues from the loop b10-b11 of both b-prism domains (Figures 2 and 3(a)). The contacting surfaces between b-prism domains 2 and 3 are formed by residues of the N-terminal stretches, b-strands 2, loops b2-b3 and b4-b5 of both domains, and residues from b12 of domain 2 (Figures 2 and 3(b)). A further intrasubunit contact is established at the central cavity between the N-terminal extension and the loop connecting the b-prism domains 2 and 3. On the other hand, intersubunit contacts at the A1-B1
Figure 2. Summary of structural features. Sequence alignment of the three b-prism domains of the P. platycephala seed lectin with the 12 bstrands indicated above the sequences and color-coded cyan, green and magenta for the Greek key motifs 1, 2, and 3 of each bprism domain, respectively. Invariant and conserved residues, which define the signature of the b-prism domain, are highlighted in red and blue background, respectively. Mannose-binding residues are in boldface and underlined. Residues buried at the intrasubunit interfaces between b-prisms 1-2 and 23 are identified by black and green circles, respectively. Residues at the intersubunit contact areas A1-B1 and A3-B3 are marked, respectively, by blue and yellow triangles.
577
First Crystal Structure of a Mimosoideae Lectin
Figure 3. Contact surfaces. Stick representation of the intrasubunit interfaces between (a) b-prism domains 1 and 2 and (b) 2 and 3, and (c) of the intersubunit contacts between the N-terminal (A1-B1) and (d) the C-terminal (A3-B3) b-prism domains of the P. platycephala lectin dimer rendered with MOLSCRIPT.44 The relative orientation and color of the interacting domains are as in Figure 1. Carbon, oxygen, and nitrogen atoms are colored gold, red, and blue, respectively. Broken lines represent hydrogen bonds.
interface involve amino acid residues from strands b2 and b5 of both b-prism domains, whereas the A3-B3 contact area is formed by interactions between b-strands 1 and 10 and the loop b10-b11 of both domains (Figures 2 and 3(c) and (d)). The buried surface areas on each b-prism domain are ˚ 2, 600 A ˚ 2, and 610 A ˚ 2 for the 1-2, 2-3, ˚ 2, 570 A 674 A A1-B1, and A3-B3 interfaces, respectively, and represent 68% (1-2), 42% (2-3), 55% (A1-B1) and 60% (A3-B3) of hydrophobic residues. For compari˚ 2 are son, in the octameric assembly of heltuba 800 A buried per subunit in each interface (54–57% of hydrophobic residues)17 and in the artocarpin tetramer (dimer of dimers) the surface contact areas between the two subunits of a dimer are ˚ 2 between dimers, of which 60% is ˚ 2, and 610 A 930 A contributed by hydrophobic residues.23 Jacalin-related lectins of the Moraceae family have been crystallized as a dimer (calsepa),16 tetramer (dimer of dimers) (jacalin,12 Maclura pomifera agglutinin (MPA),24 artocarpin23) and an octamer (tetramer of dimers) (heltuba).17 These assemblies of b-prism domains exhibit respectively two, four, and eight carbohydrate-binding sites oriented towards the periphery. The structure of the PPL dimer, exposing functional mannose-binding sites located at the outer faces of each of its six b-prism domains, adds further diversity to the mode b-prisms can associate into higher structural levels. Despite the high conservation of the overall b-prism fold in all the reported jacalin-related lectin structures, only a handful of residues out of about 150 are invariant,16,17 and these residues have a key role in maintaining the packing of the three Greek
key motifs of the domain (Figure 2). P. platycephala lectin amino acids involved in tertiary intrasubunit and quaternary intersubunit interactions fall within the category of non-conserved residues (Figure 2). In addition, PPL represents the first Mimosoideae lectin to have its structure solved, as well as the first example of a b-prism lectin in the Leguminosae family. All the other known lectin structures belong to the other two subfamilies of Leguminosae (Papilionoideae and Caesalpinoideae) and are made up by ConA-like jelly-roll domains. Hence, the PPL structure highlights a probably not yet fully recognized structural and functional adaptability of the b-prism fold as a building block in the evolution of plant lectins. Structural diversification of b-prism lectins The crystal structure of the PPL-X-Man complex unambiguously reveals the location and architecture of the mannose recognition site within each lectin subunit. The spatial relative orientation of these binding sites primarily determines the glycan crosslinking properties underlying the agglutinating capability, and thus the biological role, of the lectin. The average distances between the six mannose-binding sites in the PPL dimer (Figure 1) ˚ (A1-A2ZB1-B2), 42 A ˚ (A2-A3ZB2-B3), are 50 A ˚ ˚ 28 A (A1-B1), and 46 A (A3-B3). For comparison, in the calsepa dimer, the two carbohydrate-binding sites, which are located on the opposite face in each ˚ .16 The four protomer, are separated by 38 A carbohydrate-binding sites in the tetrameric lectins jacalin,12 MPA,24 and artocarpin23 contributed by
578
First Crystal Structure of a Mimosoideae Lectin
Figure 4. The X-mannose-binding sites. Close-up views of the X-mannose recognition sites of the b-prism domains (a), A1/B1 (b) A2/B2 and (c) A3/B3 of the P. platycephala seed lectin showing the hydrogen-bonding network (broken lines) established between the protein and the sugar (Table 2). Water molecules are rendered as blue spheres. The stick Figure was produced using MOLSCRIPT.44 Protein atoms are color-coded as in Figure 3.
each monomer occupy approximately tetrahedral ˚ from the common centre of the positions at 36 A tetrameric assembly, and are separated from each ˚ (A-BZC-D) and 59 A ˚ (B-DZA-C). other by 46 A The eight carbohydrate-binding sites of the heltuba donut-shaped octamer are separated from each ˚ in a dimer and 29 A ˚ between other by about 58 A 17 adjacent dimers. The P. platycephala lectin possesses three non-identical mannose-binding sites With the exception of a stacking interaction exclusively found in b-prism 2 between Phe239 (loop b7-b8) and the B-face of the mannose pyranose ring, the mannose recognition sites located at the top of each of the three b-prism domains of PPL are formed by residues from loops b1-b2 and b11-b12 and an aspartate residue from the surface-exposed strand b12. These three structural elements are contributed exclusively by Greek key motif 1 within each b-prism domain (Figures 2 and 4, Table 2). In all three b-prism domains of PPL the main-chain nitrogen of a conserved glycine residue within the b1-b2 loops establishes a hydrogen bond with the mannose O3 atom and van der Waals contacts with the O4 hydroxyl (Figure 4). In b-prism 2, a hydrogen bond between Asp165 and the sugar O3 atom further contributes to the mannose-recognition site (Table 2). However, the network of hydrogen bonds with the O4, O5, and O6 hydroxyl groups of the mannose is not conserved in the combining sites of the b-prism 1 (Gly136, Tyr137, Tyr138, Asp140), 2 (Tyr283, Tyr284, Asp286) and 3 (Asp432, Asp435) domains. In addition, the key mannose-binding residues of PPL A1 are conserved in the crystal structure of calsepa16 (Gly17, Gly140, Tyr141, Tyr142, Asp144) but depart from those of heltuba17 (Gly18, Gly135, Asp136, Val137, Asp139). On the other hand, the mannosebinding residues of PPL b-prism 3 appear to be conserved in the sugar-binding site of artocarpin23 (Gly15, Asp138, Asp141). Hence, in terms of combining site architecture, PPL can be regarded as a covalent “calsepa-pseudocalsepa-artocarpin”
construct. Further stacking interactions between the indol ring of X-Man and residues Tyr137 and Tyr283 are found in the X-mannose-binding cleft of the b-prism domains 1 and 3, respectively. The P. platycephala seed lectin exhibits specificity towards mannose and its epimer glucose,25 though isothermal titration calorimetry and surface plasmon resonance have shown that the lectin binds glucose with a fourfold lower affinity than mannose.26 Mannose and glucose differ in the conformation of their O2 hydroxyl groups, which is axial in mannose and equatorial in glucose. In the PPL mannose-bound structure, the protein establishes interactions with the X-Man O2 atom in b-prism 2 through a hydrogen bond with the main-chain nitrogen of Asp165 and a water-mediated contact with Gln206 (Figure 4(b), Table 2). Another water
Table 2. Protein–carbohydrate interactions in the three PPL mannose-binding sites PPL b-prism domain
Sugar atom
A1, B1
O3 O4 O5 O6
A2, B2
O2 O3 O4 O5 O6
A3, B3
O2 O3 O4 O5 O6
Amino acid residue N Gly16 OD2 Asp140 N Tyr137 N Gly136 N Tyr137 N Tyr138 OD1 Asp140 N Asp165 Gln 206.H2O 186 N Gly164 OD1 Asp165 OD1 Asp286 N Tyr283 N Tyr283 N Tyr284 O Tyr284 OD2 Asp286 Asp312.H2O 203 N Gly311 Asp312.H2O 203 OD1 Asp435 N Asp432 N Asp432 OD2 Asp435
Average distance ˚) (A 2.93 2.67 2.78 3.10 2.93 2.92 2.56 3.01 2.65 2.96 2.85 2.62 2.98 3.17 2.83 3.17 2.77 2.97 2.78 2.86 2.54 2.98 2.61 2.55
First Crystal Structure of a Mimosoideae Lectin
579
Figure 5. Structural comparisons. Superposition of the Ca traces of the three b-prism domains of the P. platycephala lectin, (a) apo and (b) with bound X-Man. The domains are color-coded as in Figure 1. The three-residue longer b7-b8 loop of the b-prism domain 3 is labeled. (c) Superposition of PPL b-prism domain 1 (blue) and that of heltuba structure 1C3K (red) showing the shifted positions of the loops b5-b6, b7-b8, and b-strand b5.
molecule bridges the O2 atom of X-Man to Asp312 in b-prism 3 (Figure 4(c), Table 2). These interactions would not be possible with the equatorial O2 hydroxyl of glucose, and thus the structural data provide a rationale for explaning the PPL’s preferred mannose binding, and strongly suggest the involvement of water molecules in enhancing the binding affinity for mannose over glucose. Restrained conformational flexibility of carbohydrate-binding residues in the X-Man-bound P. platycephala lectin versus the unliganded lectin Superposition of the three b-prism domains of the apo PPL molecule and the PPL-X-Man complex between themselves (Figure 5) shows average r.m.s. ˚ on equivalent Ca positions. deviations of 0.7–0.9 A The largest structural differences between the three PPL domains correspond to loops b1-b2 (one residue longer in b-prism 2), b5-b6 (two residues longer in b-prism 1), and b7-b8 (three residues longer in b-prism 3), and b-strand 10 (one residue longer in domain 1) (Figures 2 and 5(a)). These amino acid insertions/deletions do not affect regions involved in intra- and interdomain contacts or in mannose recognition. On the other hand, structural comparison of the PPL b-prism domains with the homologous domains of heltuba (PDB code 1C3K) and calsepa (1OUW) yielded r.m.s.
values for all equivalent Ca atom pairs of 0.99– ˚ (Figure 5(c)). Major structural differences 1.17 A between the b-prisms of heltuba (calsepa) and PPL are the different lengths of the b5 strand and the b5-b6 loop. In addition, loop b7-b8 of PPL b-prism 3 is three residues longer than the corresponding loop of heltuba/calsepa (Figure 5(c)). The distinct loop conformations between PPL b-prism domains 1 and 2 versus 3, and between the PPL domains and those of heltuba/calsepa explain the failure of molecular replacement methods to solve the crystal structure of the P. platycephala lectin using the coordinates 1C3K or 1OUW as search models.26,27 Though the overall structure of the apo and the sugar-bound P. platycephala lectins closely resemble each other (least-squares superposition between apo PPL and the PPL-X-Man complex yields a ˚ ), the global r.m.s. deviation in Ca positions of 0.4 A average B-factor value for the main chain of the ˚ 2) is significantly higher unliganded lectin (37.6 A ˚ 2). The than that of the PPL-X-Man complex (25.2 A major differences between both molecules correspond to external loops, including b3-b4, b5-b6, b7-b8, b9-b10 and b11-b12 of b-prism A1/B1, b1-b2, b5-b6, b7-b8, and b11-b12 of b-prism A2, and b3-b4 of b-prism 3 of subunit A, and most of the C-terminal half of b-prism 3 of the subunit B (Figure 6). It is worth noticing that among the residues exhibiting higher conformational flexibility in the apo PPL than in the lectin with mannose
Figure 6. B-factors. (a) Mapping of B-factors along the structure of the unliganded and (b) the mannose-bound P. platycephala lectin dimer structures. The molecules are oriented as in Figure 1. Bfactors are color-coded from 25 A2 (blue) to 60 A2 (red).
580
First Crystal Structure of a Mimosoideae Lectin
Figure 7. Cyclic oligomeric lectins. Models of (a) the P. platycephala lectin dimer, (b) the GNA tetramer, (c) the 5-fold tachylectin2 b-propeller, (d) the 6-fold b-propeller structure of the fungus Aleuria aurantia lectin monomer, (e) the pentameric human serum pentraxin, and (f ) the Crotalus atrox C-type lectin, illustrating the evolutionary convergence of planar arrays of carbohydratebinding sites on the rim of cyclic lectin structures. Models are rendered approximately to scale. Bound monosaccharides are depicted in the space-filling model and colored green. Figures were rendered with GRASP.45 Electrostatic surface potentials range from K15 (red) to C15 kT (blue).
bound are the carbohydrate-binding amino acid residues Gly163, Asp164, Tyr137, Tyr138, Tyr283 and Tyr284. This situation has not been reported in other b-prism structures, whose combining sites have been described as pre-formed to a remarkable extent.28 Our results, however, suggest that binding of mannose to the P. platycephala lectin restrains the conformational flexibility of solvent-exposed loops and of carbohydrate-binding residues. Concluding remarks: cyclic lectin oligomers, a convergent structural solution Many lectin types are multivalent either by clustering carbohydrate-binding sites on a single polypeptide or through oligomerization. The planar array of carbohydrate-binding sites on the rim of the cyclic structure of the P. platycephala lectin dimer (Figure 7(a)), and other b-prism oligomeric lectins, immediately suggests a mechanism to promote higher affinities through multivalent interactions leading to cross-linking of carbohydrate ligands as part of the host defense strategy against phytopredators and pathogens. Moreover, conceptually the same structural strategy has been adopted by a number of structurally unrelated oligomeric lectins. Hence, the structure of the Galanthus nivalis agglutinin (GNA) (b-barrel, mannose-specific; PDB code 1JPC) (Figure 7(b)), a potent inhibitor of retroviruses, and other Amaryllidaceae bulb lectins consist of a flattened crown-shaped association of two dimers of dimers with a solvent channel at the centre and 12 surface-exposed mannose-binding sites.29–31 Five and six-bladed b-propeller folds have been described in the crystal structures of tachylectin-2 (1TL2) (Figure 7(c)), a GalNAc/ GlcNAc-binding player of the innate immunity host defense system of the Japanese horseshoe crab Tachypleus tridentus,32 and in the fungus Aleuria aurantia fucose-specific lectin (1OFZ)33,34
(Figure 7(d)), which plays a role in the host recognition strategy against several pathogenic fungi (Aspergillus) and bacteria (Ralstonia solanacearum). The five subunits of the human serum amyloid P component (pentraxin) (1SAC), a lectin thought to participate too in innate host resistance to infection, display the tertiary fold of the legume lectins and are arranged into a donut-shaped ˚ in diameter at the structure with a hole 20 A 35 centre (Figure 7(e)). RSL, the galactose-specific C-type lectin from the western diamond rattlesnake (Crotalus atrox) venom (PDB accession code 1JZN) and a potent inducer of both erythrocyte agglutination and platelet aggregation, is composed of two 5-fold symmetric pentamers arranged in a staggered back-to-back orientation (Figure 7(f)).36 Despite having different tertiary structures and carbohydrate-binding specificities, these proteins represent examples of the convergent evolution of a structural principle for the construction of lectins involved in host defense or in attacking other organisms.
Materials and Methods Protein purification and crystallization The P. platycephala lectin was purified from mature seeds collected in the state of Ceara´ (Northeast of Brazil) by ammonium sulphate-precipitation followed by affinity chromatography on a Sephadex G100 column, as described.18 Homogeneity of the purified P. platycephala lectin was verified by SDS/polyacrylamide gel electrophoresis, N-terminal sequence analysis (using an Applied Biosystems Procise 492 sequencer), and MALDI-TOF mass spectrometry using an Applied Biosystems Voyager DE-PRO instrument operating at 25 kV accelerating voltage in the linear mode, and using 3,5-dimethoxy-4hydroxycinnamic acid (sinapinic acid) saturated in 70% acetonitrile and 0.1% trifluoroacetic acid as the matrix.
581
First Crystal Structure of a Mimosoideae Lectin
Orthorhombic crystals of apo P. platycephala lectin grew to maximal dimensions of 0.6 mm!0.3 mm!0.1 mm within four to five weeks at 22 8C by the vapor-diffusion method using hanging drops composed of equal volumes of protein solution (2–4 mg/ml in 50 mM Hepes, pH 8) and reservoir buffer (100 mM Hepes (pH 7.5), 10% (v/v) isopropanol and 20% (w/v) polyethylene glycol 4000).27 Monoclinic crystals of the P. platycephala lectin complexed with 5-bromo-4-chloro-3-indolyl-a-D-mannose (X-Man) (0.6 mm!0.4 mm!0.4 mm) were grown within five weeks at 22 8C in hanging drops composed of equal volumes of protein solution (5 mg/ml in 50 mM Hepes (pH 7.5) containing 3 mM X-Man) and reservoir buffer (2.0 M ammonium dihydrogen phosphate, 0.1 M Tris– HCl (pH 8.5), containing 3 mM X-mannose).26
Data collection and structure determination Diffraction data from crystals of both, the apo lectin and the lectin/X-Man derivative, were collected at cryogenic temperature (100 K) on beamline BM16 of ESRF (Grenoble, France). A complete MAD experiment was performed at three different wavelengths (edge, ˚ ; peak, 0.91932 A ˚ ; remote, 0.92191 A ˚ ) using the 0.91995 A bromide of X-Man as an anomalous scatterer. The data were processed with MOSFLM37 and scaled and merged with SCALA and TRUNCATE.38 Br atoms were located with SOLVE.39 After density modification using RESOLVE,40 a density map suitable for polypeptide tracing was obtained, and was skeletonized with MAPMAN.41 Refinement with REFMAC38 and manual model building resulted in a complete model for the protein sugar complex (Rfactor 21.2, Rfree to 24.4) (Table 1), which was subsequently used as search model to solve the structure of the apo lectin by molecular replacement using AMoRe.42 Residues 1–3 and 297–298 of both subunits of the PPL-X-Man complex were ill-defined and were not incorporated into the model. In addition, the side-chains of Glu61, Lys64 (both PPL subunits), Leu223 and Gln381 (subunit A) are poorly defined in the electron density map. Residues 1–3, 59–63, and 297–298 of subunit A and 1–3, 297–298, 328–332, 357–359, and 380–388 of the apo lectin structure were disordered. These residues, and the side-chains of Lys64, Asp165, Asp166, Leu223, Lys280 (subunit A) and Leu223, Leu334, Asp353, and Tyr433 (subunit B), were ill-defined or invisible in the electron density map, and were therefore not incorporated into the final model. The final models were checked with PROCHECK.43 Residue numbering is as used by Mann et al.18 (accession code P83304).† In both, the X-Man-bound and the apo lectins, 87.1% and 12.9% of the residues mapped to the most favored and the additionally allowed regions of the Ramachandran plot, respectively.
Accession numbers The coordinates of the apo lectin and the lectin-X-Man complex have been deposited with the RCSB Protein Data Bank‡ with accession codes 1ZGR and 1ZGS, respectively.
† http://us.expasy.org/sprot/ ‡ http://www.rcsb.org/pdb
Acknowledgements This work is part of F.G.S.’s PhD and has been financed, in part, by grant BFU2004-01432/BMC from the Ministerio de Educacio´ n y Ciencia, Madrid, Spain (to J.J.C.). F.G.S. is a recipient of an F.P.I. fellowship from the Ministerio de Educacio´n y Ciencia, Madrid (Spain). B.S.C. is a senior investigator of Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Brazil. The authors are grateful to Dr Antonio Romero (Centro de Investigaciones Bio´logicas, C.S.I.C., Madrid, Spain), Professor Pedro Alzari (Institute Pasteur, Paris, France), and Dr Pablo Fuentes-Prior and Professor Robert Huber (Max-Planck-Institut fu¨r Biochemie, Martinsried, Germany) for helping F.G.S. with data analysis during short stays in their laboratories.
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43. Laskowski, R. A., Moss, D. S. & Thornton, J. M. (1993). Main-chain bond lengths and bond angles in protein structures. J. Mol. Biol. 231, 1049–1067. 44. Kraulis, P. J. (1991). MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallog. 24, 946–950.
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Edited by R. Huber (Received 17 May 2005; received in revised form 22 August 2005; accepted 23 August 2005) Available online 9 September 2005
J. Mol. Biol. (2005) 353, 574–583
The First Crystal Structure of a Mimosoideae Lectin Reveals a Novel Quaternary Arrangement of a Widespread Domain Francisca Gallego del Sol1, Celso Nagano1,2, Benildo S. Cavada2 and Juan J. Calvete1* 1
Instituto de Biomedicina de Valencia, C.S.I.C., E-46010 Valencia, Spain 2
BioMol-Laboratory, Departamento de Bioquı´mica e Biologia Molecular, Universidade Federal do Ceara´, Fortaleza, CE 60451-970, Brazil
The crystal structures of the apo and mannose-bound Parkia platycephala seed lectin represent the first structure of a Mimosoideae lectin and a novel circular arrangement of b-prism domains, and highlight the adaptability of the b-prism fold as a building block in the evolution of plant lectins. The P. platycephala lectin is a dimer both in solution and in the crystals. Mannose binding to each of the three homologous carbohydrate-recognition domains of the lectin occurs through different modes, and restrains the flexibility of surface-exposed loops and residues involved in carbohydrate recognition. The planar array of carbohydrate-binding sites on the rim of the toroidshaped structure of the P. platycephala lectin dimer immediately suggests a mechanism to promote multivalent interactions leading to cross-linking of carbohydrate ligands as part of the host strategy against phytopredators and pathogens. The cyclic structure of the P. platycephala lectin points to the convergent evolution of a structural principle for the construction of lectins involved in host defense or in attacking other organisms. q 2005 Elsevier Ltd. All rights reserved.
*Corresponding author
Keywords: Parkia platycephala lectin; Mimosoideae lectin; crystal structure; quaternary structure; b-prism domain
Protein–carbohydrate interactions play biological roles in cellular processes, such as cell communication, host defense, fertilization, development, parasitic infection, tumor metastasis, and plant defense against herbivores and pathogens. Lectins, the carbohydrate-binding proteins that decipher the glycocodes encoded in the structure of glycans,1 are ubiquitous in animals, plants and microorganisms. Mechanisms for sugar recognition have evolved independently in diverse protein frameworks.2 In addition, lectins sharing a common structural fold may differ in their carbohydrate-binding specificities.3 Multivalence is used frequently by lectins for increasing their glycan binding avidity and specificity.4 Hence, most lectins are oligomeric or multidomain proteins. Quaternary structure variation is also observed among lectins with the same tertiary fold and belonging to the same protein family3–7
and constitutes a further level of structural divergence. As the number of lectin structure–function correlation studies increases, more parallels between plant and animal lectins become apparent. Specifically, a small number of protein folds (e.g. jelly-roll domain, C-type lectin fold, b-propeller, b-trefoil motif, b-prism domain, monocot fold; consult also the 3D Lectin Database† appear to have been widely used by evolution for the generation of carbohydrate specificity and variability in subunit association3,5–11 that enables homologous lectins to adapt to the different spacings and orientations of the lectin’s carbohydrate ligands on soluble and cell surface-exposed glycoproteins. The b-prism fold, a w135–150 amino acid residue domain first reported in the structure of jacalin,12 the mannose-binding lectin of jack fruit (Artocarpus integrifolia) seeds, defines Family PD000864 of the ProDom database,‡ and consists of 12 b-strands arranged in three four-stranded Greek key motifs
Abbreviation used: PPL, P. platycephala seed lectin. E-mail address of the corresponding author: [email protected]
† http://www.cermav.cnrs.fr/lectines/ ‡ http://protein.toulouse.inra.fr/prodom/current/ html/form.php
Introduction
0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
575
First Crystal Structure of a Mimosoideae Lectin
that possess an approximate internal 3-fold symmetry and form the faces of a triangular prism structure. Mannose-binding jacalin-related lectins are widespread among plants of taxonomically distant families, including Spermatophyta, Angiosperm, monocots: banana (Musa acuminata, Musaceae),13 rice (Oryza sativa, Poaceae);14 and dicots: jack fruit (Artocarpus integrifolia)12 and black mulberry (Morus nigra) (Moraceae);15 hedge bindweed (Calystegia sepium, Convolvulaceae);16 Jerusalem artichoke (Helianthus tuberosus, Asteraceae),17 Parkia platycephala (Mimosoideae),18 Japanese chestnut (Castanea crenata, Fagaceae);19 Spermatophyta, Gymnosperm Japanese cycad (Cycas revoluta Thunb.);20 and Pteridophyta: the true fern Phlebodium aureu (Polypodiaceae).21 This widespread distribution and the fact that mannose is an abundant building block of surface-exposed glycoconjugates of viruses, bacteria, and fungi, supports the view that the mannose-recognizing lectins fulfil a role in the plant defense against pathogens.2 Moreover, b-prism jacalin-related lectins shows also sequence similarity with proteins inducible by the plant hormone jasmonic acid, and with salt-stressinduced gene products of a number of different plants,14,18 suggesting a common ancestry for jacalinrelated lectins and inducible defense proteins.22 With the exceptions of the Japanese chestnut agglutinin and the P. platycephala seed lectin, whose primary structures consist, respectively, of two19 and three18 consecutive b-prism domains, all known mannose-binding jacalin-related lectin protomers are single b-prism domain proteins. The crystal structures of the seed lectin (apo and in complex with 5-bromo-4-chloro-3-indolyl-a-D-mannose) of P. platycephala reported here represent the first structures of both, a lectin from Mimosoideae, the most primitive group of Leguminosae plants, and
of a protein composed of three tandemly arranged b-prism domains. These structures reveal unique structural features among jacalin-related lectins: a novel quaternary arrangement of the b-prism; nonidentical mannose-binding modes between each of the three homologous carbohydrate recognition sites; and mannose-binding restraint of the conformational flexibility of surface-exposed loops and residues of the carbohydrate coordination sphere.
Results and Discussion A novel hexameric association of b-prism domains in the crystal structure of the P. platycephala seed lectin The Br atom of X-Man was used as an anomalous X-ray scatterer for solving the crystal structure of the P. platycephala seed lectin (PPL)-X-Man complex by multiple-wavelength anomalous dispersion (MAD). The final structures (Figure 1), refined to a ˚ , have good stereochemistry resolution of 2.5 A (Table 1). The PPL-X-Man monomer structure contains 441 amino acid residues (residues 3–444) and 202 water molecules. The coordinates of the PPL-X-Man complex were used to solve the structure of the apo lectin by molecular replacement. The apo lectin structure contains 441 amino acid residues and 178 water molecules, and was ˚ (Table 1). Both PPL solved to a resolution of 2.5 A structures are structurally very similar, and unless indicated otherwise, reference to the structure pertains to that of the X-Man complex. Local structural differences between the apo and the X-Man complexed lectin will be highlighted and discussed below.
Figure 1. The crystal structure of the P. platycephala seed lectin. Stereo drawing of the P. platycephala lectin dimer showing the six X-mannose residues located at the periphery of the dimeric assembly. The three tandemly arranged b-prism domains of a lectin subunit are color-coded, from the N to the C terminus, green (A1, B1), red (A2, B2) and blue (C3, B3). The Br atoms of X-Man used as anomalous scatterers for MAD phasing are represented by green spheres. The orphan cysteine residue 199 on b5 of b-prism 2 is depicted in yellow and in the space-filling model. The plot was made with MOLSCRIPT.44
576
First Crystal Structure of a Mimosoideae Lectin
Table 1. Summary of data collection and refinement statistics
A. Data collection statistics Space group Cell constants ˚) a (A ˚) b (A ˚) c (A a (deg.) b (deg.) g (deg.) ˚) Resolution (A Unique reflections Data coverage (%) Rmerge (%)a B. Refinement statistics ˚) Resolution range (A Reflections R-factor (%)b Rfree (%)c rms deviations ˚) Bond lengths (A Bond angles (deg.)
Apo P. platycephala lectin
X-Man-P. platycephala lectin complex
P212121
P21
63.60 68.51 208.53 90 90 90 2.5 31,540 96.6 6.2
80.24 114.18 80.30 90 119.88 90 2.5 43,451 99.9 5.3
30.0–2.5 29,482 21.2 24.4
30.0–2.5 39,474 20.9 24.4
0.016 1.687
0.017 3.152
a Rmerge Z jIKhIij=hIi, where I is the intensity and hIi is the average I for all observations of equivalent reflections. b R-factorZ jFobs jKjFcalc j=jFobs j. c Calculated with 5% of the reflections omitted from refinement.
The asymmetric units of the orthorhombic (apo PPL) and the monoclinic (PPL-X-Man complex) crystals were each occupied by two lectin molecules associated into the same flat, toroid-shaped struc˚ !82 A ˚ !20– ture with overall dimensions of 108 A ˚ and with a 16 A ˚ wide solvent channel at the centre 40 A (Figure 1). The fact that equilibrium sedimentation
analytical ultracentrifugation experiments showed that PPL behaves as a pH-independent dimer in dilute solutions,18 strongly indicates that the crystal structure may truly represent the quaternary conformation of the P. platycephala lectin in solution. The conformation of each PPL subunit is maintained by interactions between the b-prism domains 1 and 2 and 2 and 3, and the dimeric assembly is further stabilized by two distinct intersubunit interfaces involving contacts between the N-terminal (A1-B1) and the C-terminal (A3-B3) b-prism domains of the two PPL molecules (Figure 1). Each PPL subunit possesses a solventexposed cysteine residue (Cys199, Figure 2) located on b5 of b-prism 2 (Figure 1). Reactive sulfhydryl groups are rare in b-prism lectins, and generally in lectins. The only other known b-prism example is heltuba, which contains a buried orphan cysteine residue (Cys40) at the beginning of b4. In contrast, PPL Cys199 reacts in the native lectin with alkylating reagents,18 though it does not participate in any inter or intrasubunit contacts, either within the canonical dimer or between crystal mates. Intrasubunit interactions between adjacent PPL b-prism domains 1 and 2 are established essentially through contacts between residues from b-strands 1, 10 and 12, and between residues from the loop b10-b11 of both b-prism domains (Figures 2 and 3(a)). The contacting surfaces between b-prism domains 2 and 3 are formed by residues of the N-terminal stretches, b-strands 2, loops b2-b3 and b4-b5 of both domains, and residues from b12 of domain 2 (Figures 2 and 3(b)). A further intrasubunit contact is established at the central cavity between the N-terminal extension and the loop connecting the b-prism domains 2 and 3. On the other hand, intersubunit contacts at the A1-B1
Figure 2. Summary of structural features. Sequence alignment of the three b-prism domains of the P. platycephala seed lectin with the 12 bstrands indicated above the sequences and color-coded cyan, green and magenta for the Greek key motifs 1, 2, and 3 of each bprism domain, respectively. Invariant and conserved residues, which define the signature of the b-prism domain, are highlighted in red and blue background, respectively. Mannose-binding residues are in boldface and underlined. Residues buried at the intrasubunit interfaces between b-prisms 1-2 and 23 are identified by black and green circles, respectively. Residues at the intersubunit contact areas A1-B1 and A3-B3 are marked, respectively, by blue and yellow triangles.
577
First Crystal Structure of a Mimosoideae Lectin
Figure 3. Contact surfaces. Stick representation of the intrasubunit interfaces between (a) b-prism domains 1 and 2 and (b) 2 and 3, and (c) of the intersubunit contacts between the N-terminal (A1-B1) and (d) the C-terminal (A3-B3) b-prism domains of the P. platycephala lectin dimer rendered with MOLSCRIPT.44 The relative orientation and color of the interacting domains are as in Figure 1. Carbon, oxygen, and nitrogen atoms are colored gold, red, and blue, respectively. Broken lines represent hydrogen bonds.
interface involve amino acid residues from strands b2 and b5 of both b-prism domains, whereas the A3-B3 contact area is formed by interactions between b-strands 1 and 10 and the loop b10-b11 of both domains (Figures 2 and 3(c) and (d)). The buried surface areas on each b-prism domain are ˚ 2, 600 A ˚ 2, and 610 A ˚ 2 for the 1-2, 2-3, ˚ 2, 570 A 674 A A1-B1, and A3-B3 interfaces, respectively, and represent 68% (1-2), 42% (2-3), 55% (A1-B1) and 60% (A3-B3) of hydrophobic residues. For compari˚ 2 are son, in the octameric assembly of heltuba 800 A buried per subunit in each interface (54–57% of hydrophobic residues)17 and in the artocarpin tetramer (dimer of dimers) the surface contact areas between the two subunits of a dimer are ˚ 2 between dimers, of which 60% is ˚ 2, and 610 A 930 A contributed by hydrophobic residues.23 Jacalin-related lectins of the Moraceae family have been crystallized as a dimer (calsepa),16 tetramer (dimer of dimers) (jacalin,12 Maclura pomifera agglutinin (MPA),24 artocarpin23) and an octamer (tetramer of dimers) (heltuba).17 These assemblies of b-prism domains exhibit respectively two, four, and eight carbohydrate-binding sites oriented towards the periphery. The structure of the PPL dimer, exposing functional mannose-binding sites located at the outer faces of each of its six b-prism domains, adds further diversity to the mode b-prisms can associate into higher structural levels. Despite the high conservation of the overall b-prism fold in all the reported jacalin-related lectin structures, only a handful of residues out of about 150 are invariant,16,17 and these residues have a key role in maintaining the packing of the three Greek
key motifs of the domain (Figure 2). P. platycephala lectin amino acids involved in tertiary intrasubunit and quaternary intersubunit interactions fall within the category of non-conserved residues (Figure 2). In addition, PPL represents the first Mimosoideae lectin to have its structure solved, as well as the first example of a b-prism lectin in the Leguminosae family. All the other known lectin structures belong to the other two subfamilies of Leguminosae (Papilionoideae and Caesalpinoideae) and are made up by ConA-like jelly-roll domains. Hence, the PPL structure highlights a probably not yet fully recognized structural and functional adaptability of the b-prism fold as a building block in the evolution of plant lectins. Structural diversification of b-prism lectins The crystal structure of the PPL-X-Man complex unambiguously reveals the location and architecture of the mannose recognition site within each lectin subunit. The spatial relative orientation of these binding sites primarily determines the glycan crosslinking properties underlying the agglutinating capability, and thus the biological role, of the lectin. The average distances between the six mannose-binding sites in the PPL dimer (Figure 1) ˚ (A1-A2ZB1-B2), 42 A ˚ (A2-A3ZB2-B3), are 50 A ˚ ˚ 28 A (A1-B1), and 46 A (A3-B3). For comparison, in the calsepa dimer, the two carbohydrate-binding sites, which are located on the opposite face in each ˚ .16 The four protomer, are separated by 38 A carbohydrate-binding sites in the tetrameric lectins jacalin,12 MPA,24 and artocarpin23 contributed by
578
First Crystal Structure of a Mimosoideae Lectin
Figure 4. The X-mannose-binding sites. Close-up views of the X-mannose recognition sites of the b-prism domains (a), A1/B1 (b) A2/B2 and (c) A3/B3 of the P. platycephala seed lectin showing the hydrogen-bonding network (broken lines) established between the protein and the sugar (Table 2). Water molecules are rendered as blue spheres. The stick Figure was produced using MOLSCRIPT.44 Protein atoms are color-coded as in Figure 3.
each monomer occupy approximately tetrahedral ˚ from the common centre of the positions at 36 A tetrameric assembly, and are separated from each ˚ (A-BZC-D) and 59 A ˚ (B-DZA-C). other by 46 A The eight carbohydrate-binding sites of the heltuba donut-shaped octamer are separated from each ˚ in a dimer and 29 A ˚ between other by about 58 A 17 adjacent dimers. The P. platycephala lectin possesses three non-identical mannose-binding sites With the exception of a stacking interaction exclusively found in b-prism 2 between Phe239 (loop b7-b8) and the B-face of the mannose pyranose ring, the mannose recognition sites located at the top of each of the three b-prism domains of PPL are formed by residues from loops b1-b2 and b11-b12 and an aspartate residue from the surface-exposed strand b12. These three structural elements are contributed exclusively by Greek key motif 1 within each b-prism domain (Figures 2 and 4, Table 2). In all three b-prism domains of PPL the main-chain nitrogen of a conserved glycine residue within the b1-b2 loops establishes a hydrogen bond with the mannose O3 atom and van der Waals contacts with the O4 hydroxyl (Figure 4). In b-prism 2, a hydrogen bond between Asp165 and the sugar O3 atom further contributes to the mannose-recognition site (Table 2). However, the network of hydrogen bonds with the O4, O5, and O6 hydroxyl groups of the mannose is not conserved in the combining sites of the b-prism 1 (Gly136, Tyr137, Tyr138, Asp140), 2 (Tyr283, Tyr284, Asp286) and 3 (Asp432, Asp435) domains. In addition, the key mannose-binding residues of PPL A1 are conserved in the crystal structure of calsepa16 (Gly17, Gly140, Tyr141, Tyr142, Asp144) but depart from those of heltuba17 (Gly18, Gly135, Asp136, Val137, Asp139). On the other hand, the mannosebinding residues of PPL b-prism 3 appear to be conserved in the sugar-binding site of artocarpin23 (Gly15, Asp138, Asp141). Hence, in terms of combining site architecture, PPL can be regarded as a covalent “calsepa-pseudocalsepa-artocarpin”
construct. Further stacking interactions between the indol ring of X-Man and residues Tyr137 and Tyr283 are found in the X-mannose-binding cleft of the b-prism domains 1 and 3, respectively. The P. platycephala seed lectin exhibits specificity towards mannose and its epimer glucose,25 though isothermal titration calorimetry and surface plasmon resonance have shown that the lectin binds glucose with a fourfold lower affinity than mannose.26 Mannose and glucose differ in the conformation of their O2 hydroxyl groups, which is axial in mannose and equatorial in glucose. In the PPL mannose-bound structure, the protein establishes interactions with the X-Man O2 atom in b-prism 2 through a hydrogen bond with the main-chain nitrogen of Asp165 and a water-mediated contact with Gln206 (Figure 4(b), Table 2). Another water
Table 2. Protein–carbohydrate interactions in the three PPL mannose-binding sites PPL b-prism domain
Sugar atom
A1, B1
O3 O4 O5 O6
A2, B2
O2 O3 O4 O5 O6
A3, B3
O2 O3 O4 O5 O6
Amino acid residue N Gly16 OD2 Asp140 N Tyr137 N Gly136 N Tyr137 N Tyr138 OD1 Asp140 N Asp165 Gln 206.H2O 186 N Gly164 OD1 Asp165 OD1 Asp286 N Tyr283 N Tyr283 N Tyr284 O Tyr284 OD2 Asp286 Asp312.H2O 203 N Gly311 Asp312.H2O 203 OD1 Asp435 N Asp432 N Asp432 OD2 Asp435
Average distance ˚) (A 2.93 2.67 2.78 3.10 2.93 2.92 2.56 3.01 2.65 2.96 2.85 2.62 2.98 3.17 2.83 3.17 2.77 2.97 2.78 2.86 2.54 2.98 2.61 2.55
First Crystal Structure of a Mimosoideae Lectin
579
Figure 5. Structural comparisons. Superposition of the Ca traces of the three b-prism domains of the P. platycephala lectin, (a) apo and (b) with bound X-Man. The domains are color-coded as in Figure 1. The three-residue longer b7-b8 loop of the b-prism domain 3 is labeled. (c) Superposition of PPL b-prism domain 1 (blue) and that of heltuba structure 1C3K (red) showing the shifted positions of the loops b5-b6, b7-b8, and b-strand b5.
molecule bridges the O2 atom of X-Man to Asp312 in b-prism 3 (Figure 4(c), Table 2). These interactions would not be possible with the equatorial O2 hydroxyl of glucose, and thus the structural data provide a rationale for explaning the PPL’s preferred mannose binding, and strongly suggest the involvement of water molecules in enhancing the binding affinity for mannose over glucose. Restrained conformational flexibility of carbohydrate-binding residues in the X-Man-bound P. platycephala lectin versus the unliganded lectin Superposition of the three b-prism domains of the apo PPL molecule and the PPL-X-Man complex between themselves (Figure 5) shows average r.m.s. ˚ on equivalent Ca positions. deviations of 0.7–0.9 A The largest structural differences between the three PPL domains correspond to loops b1-b2 (one residue longer in b-prism 2), b5-b6 (two residues longer in b-prism 1), and b7-b8 (three residues longer in b-prism 3), and b-strand 10 (one residue longer in domain 1) (Figures 2 and 5(a)). These amino acid insertions/deletions do not affect regions involved in intra- and interdomain contacts or in mannose recognition. On the other hand, structural comparison of the PPL b-prism domains with the homologous domains of heltuba (PDB code 1C3K) and calsepa (1OUW) yielded r.m.s.
values for all equivalent Ca atom pairs of 0.99– ˚ (Figure 5(c)). Major structural differences 1.17 A between the b-prisms of heltuba (calsepa) and PPL are the different lengths of the b5 strand and the b5-b6 loop. In addition, loop b7-b8 of PPL b-prism 3 is three residues longer than the corresponding loop of heltuba/calsepa (Figure 5(c)). The distinct loop conformations between PPL b-prism domains 1 and 2 versus 3, and between the PPL domains and those of heltuba/calsepa explain the failure of molecular replacement methods to solve the crystal structure of the P. platycephala lectin using the coordinates 1C3K or 1OUW as search models.26,27 Though the overall structure of the apo and the sugar-bound P. platycephala lectins closely resemble each other (least-squares superposition between apo PPL and the PPL-X-Man complex yields a ˚ ), the global r.m.s. deviation in Ca positions of 0.4 A average B-factor value for the main chain of the ˚ 2) is significantly higher unliganded lectin (37.6 A ˚ 2). The than that of the PPL-X-Man complex (25.2 A major differences between both molecules correspond to external loops, including b3-b4, b5-b6, b7-b8, b9-b10 and b11-b12 of b-prism A1/B1, b1-b2, b5-b6, b7-b8, and b11-b12 of b-prism A2, and b3-b4 of b-prism 3 of subunit A, and most of the C-terminal half of b-prism 3 of the subunit B (Figure 6). It is worth noticing that among the residues exhibiting higher conformational flexibility in the apo PPL than in the lectin with mannose
Figure 6. B-factors. (a) Mapping of B-factors along the structure of the unliganded and (b) the mannose-bound P. platycephala lectin dimer structures. The molecules are oriented as in Figure 1. Bfactors are color-coded from 25 A2 (blue) to 60 A2 (red).
580
First Crystal Structure of a Mimosoideae Lectin
Figure 7. Cyclic oligomeric lectins. Models of (a) the P. platycephala lectin dimer, (b) the GNA tetramer, (c) the 5-fold tachylectin2 b-propeller, (d) the 6-fold b-propeller structure of the fungus Aleuria aurantia lectin monomer, (e) the pentameric human serum pentraxin, and (f ) the Crotalus atrox C-type lectin, illustrating the evolutionary convergence of planar arrays of carbohydratebinding sites on the rim of cyclic lectin structures. Models are rendered approximately to scale. Bound monosaccharides are depicted in the space-filling model and colored green. Figures were rendered with GRASP.45 Electrostatic surface potentials range from K15 (red) to C15 kT (blue).
bound are the carbohydrate-binding amino acid residues Gly163, Asp164, Tyr137, Tyr138, Tyr283 and Tyr284. This situation has not been reported in other b-prism structures, whose combining sites have been described as pre-formed to a remarkable extent.28 Our results, however, suggest that binding of mannose to the P. platycephala lectin restrains the conformational flexibility of solvent-exposed loops and of carbohydrate-binding residues. Concluding remarks: cyclic lectin oligomers, a convergent structural solution Many lectin types are multivalent either by clustering carbohydrate-binding sites on a single polypeptide or through oligomerization. The planar array of carbohydrate-binding sites on the rim of the cyclic structure of the P. platycephala lectin dimer (Figure 7(a)), and other b-prism oligomeric lectins, immediately suggests a mechanism to promote higher affinities through multivalent interactions leading to cross-linking of carbohydrate ligands as part of the host defense strategy against phytopredators and pathogens. Moreover, conceptually the same structural strategy has been adopted by a number of structurally unrelated oligomeric lectins. Hence, the structure of the Galanthus nivalis agglutinin (GNA) (b-barrel, mannose-specific; PDB code 1JPC) (Figure 7(b)), a potent inhibitor of retroviruses, and other Amaryllidaceae bulb lectins consist of a flattened crown-shaped association of two dimers of dimers with a solvent channel at the centre and 12 surface-exposed mannose-binding sites.29–31 Five and six-bladed b-propeller folds have been described in the crystal structures of tachylectin-2 (1TL2) (Figure 7(c)), a GalNAc/ GlcNAc-binding player of the innate immunity host defense system of the Japanese horseshoe crab Tachypleus tridentus,32 and in the fungus Aleuria aurantia fucose-specific lectin (1OFZ)33,34
(Figure 7(d)), which plays a role in the host recognition strategy against several pathogenic fungi (Aspergillus) and bacteria (Ralstonia solanacearum). The five subunits of the human serum amyloid P component (pentraxin) (1SAC), a lectin thought to participate too in innate host resistance to infection, display the tertiary fold of the legume lectins and are arranged into a donut-shaped ˚ in diameter at the structure with a hole 20 A 35 centre (Figure 7(e)). RSL, the galactose-specific C-type lectin from the western diamond rattlesnake (Crotalus atrox) venom (PDB accession code 1JZN) and a potent inducer of both erythrocyte agglutination and platelet aggregation, is composed of two 5-fold symmetric pentamers arranged in a staggered back-to-back orientation (Figure 7(f)).36 Despite having different tertiary structures and carbohydrate-binding specificities, these proteins represent examples of the convergent evolution of a structural principle for the construction of lectins involved in host defense or in attacking other organisms.
Materials and Methods Protein purification and crystallization The P. platycephala lectin was purified from mature seeds collected in the state of Ceara´ (Northeast of Brazil) by ammonium sulphate-precipitation followed by affinity chromatography on a Sephadex G100 column, as described.18 Homogeneity of the purified P. platycephala lectin was verified by SDS/polyacrylamide gel electrophoresis, N-terminal sequence analysis (using an Applied Biosystems Procise 492 sequencer), and MALDI-TOF mass spectrometry using an Applied Biosystems Voyager DE-PRO instrument operating at 25 kV accelerating voltage in the linear mode, and using 3,5-dimethoxy-4hydroxycinnamic acid (sinapinic acid) saturated in 70% acetonitrile and 0.1% trifluoroacetic acid as the matrix.
581
First Crystal Structure of a Mimosoideae Lectin
Orthorhombic crystals of apo P. platycephala lectin grew to maximal dimensions of 0.6 mm!0.3 mm!0.1 mm within four to five weeks at 22 8C by the vapor-diffusion method using hanging drops composed of equal volumes of protein solution (2–4 mg/ml in 50 mM Hepes, pH 8) and reservoir buffer (100 mM Hepes (pH 7.5), 10% (v/v) isopropanol and 20% (w/v) polyethylene glycol 4000).27 Monoclinic crystals of the P. platycephala lectin complexed with 5-bromo-4-chloro-3-indolyl-a-D-mannose (X-Man) (0.6 mm!0.4 mm!0.4 mm) were grown within five weeks at 22 8C in hanging drops composed of equal volumes of protein solution (5 mg/ml in 50 mM Hepes (pH 7.5) containing 3 mM X-Man) and reservoir buffer (2.0 M ammonium dihydrogen phosphate, 0.1 M Tris– HCl (pH 8.5), containing 3 mM X-mannose).26
Data collection and structure determination Diffraction data from crystals of both, the apo lectin and the lectin/X-Man derivative, were collected at cryogenic temperature (100 K) on beamline BM16 of ESRF (Grenoble, France). A complete MAD experiment was performed at three different wavelengths (edge, ˚ ; peak, 0.91932 A ˚ ; remote, 0.92191 A ˚ ) using the 0.91995 A bromide of X-Man as an anomalous scatterer. The data were processed with MOSFLM37 and scaled and merged with SCALA and TRUNCATE.38 Br atoms were located with SOLVE.39 After density modification using RESOLVE,40 a density map suitable for polypeptide tracing was obtained, and was skeletonized with MAPMAN.41 Refinement with REFMAC38 and manual model building resulted in a complete model for the protein sugar complex (Rfactor 21.2, Rfree to 24.4) (Table 1), which was subsequently used as search model to solve the structure of the apo lectin by molecular replacement using AMoRe.42 Residues 1–3 and 297–298 of both subunits of the PPL-X-Man complex were ill-defined and were not incorporated into the model. In addition, the side-chains of Glu61, Lys64 (both PPL subunits), Leu223 and Gln381 (subunit A) are poorly defined in the electron density map. Residues 1–3, 59–63, and 297–298 of subunit A and 1–3, 297–298, 328–332, 357–359, and 380–388 of the apo lectin structure were disordered. These residues, and the side-chains of Lys64, Asp165, Asp166, Leu223, Lys280 (subunit A) and Leu223, Leu334, Asp353, and Tyr433 (subunit B), were ill-defined or invisible in the electron density map, and were therefore not incorporated into the final model. The final models were checked with PROCHECK.43 Residue numbering is as used by Mann et al.18 (accession code P83304).† In both, the X-Man-bound and the apo lectins, 87.1% and 12.9% of the residues mapped to the most favored and the additionally allowed regions of the Ramachandran plot, respectively.
Accession numbers The coordinates of the apo lectin and the lectin-X-Man complex have been deposited with the RCSB Protein Data Bank‡ with accession codes 1ZGR and 1ZGS, respectively.
† http://us.expasy.org/sprot/ ‡ http://www.rcsb.org/pdb
Acknowledgements This work is part of F.G.S.’s PhD and has been financed, in part, by grant BFU2004-01432/BMC from the Ministerio de Educacio´ n y Ciencia, Madrid, Spain (to J.J.C.). F.G.S. is a recipient of an F.P.I. fellowship from the Ministerio de Educacio´n y Ciencia, Madrid (Spain). B.S.C. is a senior investigator of Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Brazil. The authors are grateful to Dr Antonio Romero (Centro de Investigaciones Bio´logicas, C.S.I.C., Madrid, Spain), Professor Pedro Alzari (Institute Pasteur, Paris, France), and Dr Pablo Fuentes-Prior and Professor Robert Huber (Max-Planck-Institut fu¨r Biochemie, Martinsried, Germany) for helping F.G.S. with data analysis during short stays in their laboratories.
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Edited by R. Huber (Received 17 May 2005; received in revised form 22 August 2005; accepted 23 August 2005) Available online 9 September 2005