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The Antineoplastic Lectin of the Common Edible Mushroom (Agaricus bisporus) Has Two Binding Sites, Each Specific for a Different Configuration at a Single Epimeric Hydroxyl*

  • Maria E. Carrizo
    Footnotes
    Affiliations
    Biocrystallography Laboratory, Department of Science and Technology, University of Verona, Strada Le Grazie 15, 37134 Verona, Italy and
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  • Stefano Capaldi
    Affiliations
    Biocrystallography Laboratory, Department of Science and Technology, University of Verona, Strada Le Grazie 15, 37134 Verona, Italy and
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  • Massimiliano Perduca
    Affiliations
    Biocrystallography Laboratory, Department of Science and Technology, University of Verona, Strada Le Grazie 15, 37134 Verona, Italy and
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  • Fernando J. Irazoqui
    Affiliations
    Department of Biological Chemistry, Faculty of Chemical Sciences, National University of Córdoba, Ciudad Universitaria, 5000 Córdoba, Argentina
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  • Gustavo A. Nores
    Affiliations
    Department of Biological Chemistry, Faculty of Chemical Sciences, National University of Córdoba, Ciudad Universitaria, 5000 Córdoba, Argentina
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  • Hugo L. Monaco
    Correspondence
    To whom correspondence should be addressed
    Affiliations
    Biocrystallography Laboratory, Department of Science and Technology, University of Verona, Strada Le Grazie 15, 37134 Verona, Italy and
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  • Author Footnotes
    * This work was supported in part by a grant from Fondazione Cassa di Risparmio di Verona Vicenza Belluno e Ancona. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    The on-line version of this article (available at http://www.jbc.org) contains Fig. 1S, Tables 1S and 2S, and supplemental data.
    § Recipient of a fellowship from the Argentine Consejo Nacional de Investigaciones Científicas y Técnicas.
Open AccessPublished:December 13, 2004DOI:https://doi.org/10.1074/jbc.M411989200
      The lectin from the common mushroom Agaricus bisporus, the most popular edible species in Western countries, has potent antiproliferative effects on human epithelial cancer cells, without any apparent cytotoxicity. This property confers to it an important therapeutic potential as an antineoplastic agent. The three-dimensional structure of the lectin was determined by x-ray diffraction. The protein is a tetramer with 222 symmetry, and each monomer presents a novel fold with two β sheets connected by a helix-loop-helix motif. Selectivity was studied by examining the binding of four monosaccharides and seven disaccharides in two different crystal forms. The T-antigen disaccharide, Galβ1–3GalNAc, mediator of the antiproliferative effects of the protein, binds at a shallow depression on the surface of the molecule. The binding of N-acetylgalactosamine overlaps with that moiety of the T antigen, but surprisingly, N-acetylglucosamine, which differs from N-acetylgalactosamine only in the configuration of epimeric hydroxyl 4, binds at a totally different site on the opposite side of the helix-loop-helix motif. The lectin thus has two distinct binding sites per monomer that recognize the different configuration of a single epimeric hydroxyl. The structure of the protein and its two carbohydrate-binding sites are described in detail in this study.
      Lectins are proteins that recognize specific carbohydrate structures and thereby participate in molecular recognition events of fundamental relevance in a variety of biological processes (
      • Lis H.
      • Sharon N.
      ,
      • Sacchettini J.C.
      • Baum L.G.
      • Brewer C.F.
      ,
      • Loris R.
      ). The lectin from the common edible mushroom Agaricus bisporus (ABL)
      The abbreviations used are: ABL, Agaricus bisporus lectin; PNA, peanut (Arachis hypogaea) agglutinin; r.m.s.d., root mean square deviation.
      1The abbreviations used are: ABL, Agaricus bisporus lectin; PNA, peanut (Arachis hypogaea) agglutinin; r.m.s.d., root mean square deviation.
      is a member of a remarkable group that shares the property of binding, selectively and with high affinity, the Thomsen Friedenreich antigen or T antigen. The T antigen is a disaccharide, Galβ1–3GalNAc, linked to either serines or threonines on cell surface glycoproteins and hidden in healthy cells but exposed in a high percentage of human carcinomas and other neoplastic tissues (
      • Springer G.F.
      ,
      • Springer G.F.
      ). The effect of the lectins that bind the T antigen on cell proliferation can be very pronounced and very different. For instance, the peanut agglutinin (Arachis hypogaea agglutinin) (PNA) stimulates the proliferation of human intestinal epithelial cells (
      • Ryder S.D.
      • Smith J.A.
      • Rhodes J.M.
      ), whereas jacalin has the opposite effect, i.e. strong inhibition of cell growth (
      • Yu L.-G.
      • Milton J.D.
      • Fernig D.G.
      • Rhodes J.M.
      ). ABL has the remarkable property of reversibly inhibiting the proliferation of malignant epithelial cell lines without any apparent cytotoxicity for normal cells (
      • Yu L.-G.
      • Fernig D.G.
      • Smith J.A.
      • Milton J.D.
      • Rhodes J.M.
      ). This effect is thought to be a consequence of the selective blocking by ABL of nuclear localization sequence-dependent protein import, which is essential for cell functioning (
      • Yu L.-G.
      • Fernig D.G.
      • White M.R.H.
      • Spiller D.G.
      • Appleton P.
      • Evans R.C.
      • Grierson I.
      • Smith J.A.
      • Davies H.
      • Gerasimenko O.V.
      • Petersen O.H.
      • Milton J.D.
      • Rhodes J.M.
      ,
      • Yu L.-G.
      • Fernig D.G.
      • Rhodes J.M.
      ). More recently, it was shown that the lectin binds in the cytoplasm to a truncated form of oxygen-regulated protein 150 (Orp 150) that expresses the sialylated form of the ABL ligand, sialyl-2,3-galactosyl-β1,3-N-aceltylgalactosamine, and appears to be essential for nuclear localization sequence-dependent nuclear protein import (
      • Yu L.-G.
      • Andrews N.
      • Weldon M.
      • Gerasimenko O.V.
      • Campbell B.J.
      • Singh R.
      • Grierson I.
      • Petersen O.H.
      • Rhodes J.M.
      ).
      ABL is a homotetramer with a molecular mass of 64,000 Da and has a sequence that does not show any significant similarities to any of the other lectins that bind the T antigen studied thus far by x-ray diffraction (
      • Crenshaw R.W.
      • Harper S.N.
      • Moyer M.
      • Privalle L.S.
      ). Four different isoforms have been isolated, but the structural differences among them are still unclear (
      • Sueyoshi S.
      • Tsuji T.
      • Osawa T.
      ). The structural requirements of carbohydrates and glycotopes to bind to what is believed to be a single site per monomer have also been extensively investigated, and there is evidence that ABL behaves differently from the other lectins that bind the T-antigen disaccharide (
      • Irazoqui F.J.
      • Vides M.A.
      • Nores G.A.
      ,
      • Wu A.M.
      • Wu J.H.
      • Herp A.
      • Liu J.H.
      ).
      The x-ray structure of the ABL molecule described here reveals that the protein has a new fold, predicted to be present in other proteins of fungal origin, that is quite different from those of the other lectins of known three-dimensional structure that bind the T antigen. It also shows that the quaternary structure of the tetramer presents 222 (D2) point group symmetry. A most unexpected result is the discovery of a second carbohydrate-binding site that specifically binds N-acetylglucosamine. These two binding sites present in each of the four monomers of the tetramer appear to be independent from each other and are able to distinguish two monosaccharides that differ only in the configuration of a single epimeric hydroxyl.

      MATERIALS AND METHODS

      Protein Purification and Crystallization—ABL was purified from A. bisporus fruiting bodies by affinity chromatography in a column of human erythrocytic stroma incorporated into a polyacrylamide gel (
      • Betail G.
      • Coulet M.
      • Genaud L.
      • Guillot J.
      • Scandariato M.
      ) as described elsewhere (
      • Irazoqui F.J.
      • Zalazar F.E.
      • Chiabrando G.A.
      • Romero O.
      • Vides M.A.
      ). The five isoforms were separated by preparative isoelectric focusing, and the most basic apoprotein was crystallized either by vapor diffusion in hanging drops or in microdialysis cells using as precipitant 4 m sodium formate buffered with 0.02 m Tris-HCl, pH 8.0. The crystals of the apoprotein are orthorhombic space group C2221 with unit cell parameters a = 91.9 Å, b = 96.7 Å, and c = 75.4 Å and contain two monomers in the asymmetric unit (
      • Carrizo M.E.
      • Irazoqui F.J.
      • Lardone R.D.
      • Nores G.A.
      • Curtino J.A.
      • Capaldi S.
      • Perduca M.
      • Monaco H.L.
      ). A second crystal form was grown in the presence of N-acetylglucosamine using the vapor diffusion method by mixing equal volumes of the protein solution and a solution of 15% polyethylene glycol 8000, 0.5 m lithium sulfate, and 5% isopropanol as the precipitant. These crystals belong to the tetragonal space group P43212 with unit cell parameters a = 85.5 Å and c = 257.2 Å and contain four monomers in the asymmetric unit.
      Ligand Binding in the Crystals—The crystals were soaked in mother liquor containing sufficiently high concentrations of the carbohydrates. The monosaccharides used in binding studies are galactose, glucose, N-acetylglucosamine, and N-acetylgalactosamine. The disaccharides used are T-antigen disaccharide (Galβ1–3GalNAc), benzyl T-antigen disaccharide (Galβ1–3GalNAc-α-O-Bn), p-nitrophenyl T-antigen disaccharide (Galβ1–3GalNAc-α-O-PNP), T antigen (Galβ1–3GalNAc-α-O-Ser), lacto-N-biose (Galβ1–3GlcNAc), lactose (Galβ1–4Glc), and N-acetyllactosamine (Galβ1–4GlcNAc).
      Data Collection—The diffraction data were collected from crystals frozen at 100 K after a brief immersion in a mixture of 70% of the mother liquor and 30% glycerol. The data were initially obtained using copper Kα radiation from a Rigaku RU-300 rotating anode x-ray generator with either a Rigaku R-axis II or a Mar345 imaging plate area detector. The data used for the final refinement of the apoprotein were collected at the XRD1 beamline of the Elettra synchrotron in Trieste (λ = 1.00 Å). Two data sets, at high and low resolution, were collected from the same crystal using a Mar charge-coupled device area detector. The two heavy atom derivatives were prepared by overnight soaking of a crystal in mother liquor with the addition of the two compounds at a final concentration of ∼1 mm. The co-crystals with the different monosaccharides and disaccharides were prepared by soaking crystals of the apoprotein in mother liquor saturated with the carbohydrates. The data for the tetragonal crystal form were collected at the ID23 beamline of the European Synchrotron Radiation Facility (Grenoble, France). The data were indexed, integrated, and reduced using the programs MOSFLM (
      • Leslie A.G.W.
      ), AUTOMAR, and Scala (
      • Number Collaborative Computational Project
      ). The diffraction data statistics of the main selected data sets are summarized in Table I.
      Table IStructure determination statistics The values in parentheses refer to the highest resolution shell, i.e. for the data collected 1.58 to 1.50 Å for the native apo data set, 2.64 to 2.5 Å for the heavy atom derivatives, and 1.9 to 1.8 for the complexes without GlcNAc. The highest resolution shells used in the refinements are 1.54 to 1.50 Å for the apoprotein and 1.95 to 1.90 and 1.85 to 1.80 Å for the complexes with the T-antigen and with lacto-N-biose, respectively. The values for the co-crystals with GlcNAc are 1.79 to 1.74 Å for the orthorhombic form and 2.44 to 2.36 Å for the tetragonal form.
      Data setNative apoK2OsO4KAuBr4Galβ1-3GalNAcα1-Ser (T)Galβ1-3GlcNAcT + GlcNAcT + GlcNAc
      Space groupC2221C2221C2221C2221P43212
      a (Å)91.993.193.391.991.691.985.7
      b (Å)96.798.197.796.296.196.885.7
      c (Å)75.475.376.475.175.175.2257.1
      Resolution range (Å)74.5-1.520.0-2.533.7-2.550.0-1.925.0-1.8023.0-1.7430.0-2.35
      Observed reflections345,70264,37562,592140,611150,817252,998316,651
      Independent reflections53,75712,38112,38626,12328,13734,26640,259
      Rsym (%)4.7 (8.7)4.8 (11.0)10.8 (40.5)6.1 (27.9)6.7 (35.0)5.9 (28.5)7.9 (16.7)
      I/σ7.9 (7.1)13.0 (6.5)6.5 (1.8)10.1 (2.7)9.7 (2.2)10.4 (2.6)5.5 (3.5)
      Completeness (%)99.5 (97.6)99.8 (100.0)99.8 (100.0)98.6 (96.2)98.6 (96.0)98.8 (90.5)99.7 (98.6)
      Sites24
      Rcullis (acentric/centric)0.545/0.4950.718/0.650
      Phasing power (acentric/centric)2.211/1.9071.486/1.323
      Reflections in refinement48,26424,77926,70032,51638,380
      Rcryst (%)18.7 (20.0)19.5 (21.9)20.2 (23.5)19.4 (24.3)22.8 (26.3)
      Rfree (%) (test set 10%)20.3 (20.6)21.2 (25.8)21.4 (23.7)21.8 (27.8)25.2 (27.9)
      Protein atoms2,2702,2702,2702,2704,540
      Ligand atoms0665281192
      Water molecules168909612962
      r.m.s.d. on bond lengths (Å)0.0050.0080.0060.0060.008
      r.m.s.d. on bond angles (°)0.9031.2550.9481.1361.215
      Planar groups (Å)0.0020.0070.0030.0050.006
      Chiral volume dev. (Å3)0.0610.1190.0650.1030.116
      Average B factor (Å2)12.524.123.315.626.2
      Protein atomsA: 11.5, B:12.5A:23.7, B:24.2A:22.9, B:23.3A:14.7, B:15.9A:25.6, B:25.0
      C:27.4, D:25.9
      Ligand atoms31.330.020.334.4
      Solvent atoms18.724.624.619.322.9
      Structure Determination—Initial phases to 2.5 Å resolution were determined by multiple isomorphous replacement with the two heavy atom derivatives of the orthorhombic form. The two osmium sites were located in a difference Patterson map (
      • Sheldrick G.M.
      ) and refined using the program MLPHARE (
      • Number Collaborative Computational Project
      ). The single isomorphous derivative phases were used to locate the most significant gold site in the difference Fourier map. These two major sites (one site from each of the two derivatives) were used as input for the program autoSHARP (
      • Bricogne G.
      • Vonrhein C.
      • Flensburg C.
      • Schiltz M.
      • Paciorek W.
      ), which was used to locate the minor sites of the two derivatives, and for density modification and final phasing. The electron density map thus produced was of excellent quality and could be readily interpreted. The initial model of the apoprotein was built in the high quality map at 2.5 Å resolution using the program O (
      • Jones T.A.
      • Zou J.Y.
      • Cowan S.W.
      • Kjeldgaard M.
      ). Model building proceeded without difficulty from Thr2 to Gly133 following the sequence from nucleic acid available at the ExPASy server (
      • Crenshaw R.W.
      • Harper S.N.
      • Moyer M.
      • Privalle L.S.
      ). At this point, two facts became evident: the first was that the electron density did not match the published sequence, and the second was that there was no electron density in the map beyond amino acid 143 (the published sequence is 154 amino acid long). Both facts could be easily explained as described below, and therefore a 142-amino acid polypeptide chain (from Thr2 to Gly143) was built for both monomers in the asymmetric unit. This model was initially refined using the program Crystallography and NMR System (
      • Brunger A.T.
      • Adams P.D.
      • Clore G.M.
      • DeLano W.L.
      • Gros P.
      • Grosse-Kunstleve R.W.
      • Jiang J.S.
      • Kuszewski J.
      • Nilges M.
      • Pannu N.S.
      • Read R.J.
      • Rice L.M.
      • Simonson T.
      • Warren G.L.
      ) without imposing non-crystallographic symmetry. Subsequent refinement using the high resolution synchrotron data was carried out with the program REFMAC (
      • Murshudov G.N.
      • Vagin A.A.
      • Dodson E.J.
      ). During the process of refinement and model building, the quality of the model was controlled with the program PROCHECK (
      • Laskowski R.A.
      • MacArthur M.W.
      • Moss D.S.
      • Thornton J.M.
      ). Solvent molecules were added to the model in the final stages of refinement according to hydrogen bond criteria and only if their B factors refined to reasonable values and if they improved the Rfree value. The final model of the apoprotein contains 2270 non-hydrogen protein atoms and has very reasonable geometry (see Table I), with 89.2% of the residues in the most favored regions of the Ramachandran plot, and the remaining 10.8% of the residues in the additionally allowed region. The monosaccharides and disaccharides in the co-crystals were modeled into difference Fourier maps phased by the refined, unliganded structure. The models of the complexes were refined with REFMAC using the same criteria followed in the refinement of the apoprotein. The final statistics of three selected orthorhombic co-crystals are given in Table I.
      The tetragonal crystal form, grown in the presence of N-acetylglucosamine, was solved by molecular replacement with the program MOLREP (
      • Vagin A.
      • Teplyakov A.
      ). The model of this form, as well as that of its co-crystals with the T antigen, was refined using the program REFMAC.

      RESULTS AND DISCUSSION

      Amino Acid Sequence—The 2.5 Å high quality electron density map of the orthorhombic crystals was very straightforward to interpret from Thr2 to Gly133 in terms of the translated sequence of the cDNA coding for the protein (
      • Crenshaw R.W.
      • Harper S.N.
      • Moyer M.
      • Privalle L.S.
      ). However, when this point in the chain trace was reached, it became evident that there was no longer correspondence between electron density and amino acid sequence and also that the chain appeared to be shorter than predicted by the published sequence. The C-terminal portion of the polypeptide chain, based on the cDNA sequence and starting with amino acid number 131, was reported to be the following: 130-TEGIISRPISSSDKCFIRLPSQKS-Stop. This amino acid sequence corresponds to the following nucleotide sequence: 390-acc-gaa-ggg-ata-atc-tca-agg-cca-atc-tca-tca-tcg-gat-aag-tgc-ttt-atc-cgc-cta-ccg-tct-cag-aaa-tca-tga-[Stop].
      If, after the first six nucleotides, at the point where the sequence presents three guanines, a fourth guanine is introduced, the new nucleotide sequence becomes 390-acc-gaa-ggg-gat-aat-ctc-aag-gcc-aat-ctc-atc-atc-gga-taa-[Stop], and, accordingly, the new amino acid sequence is 130-TEGDNLKANLIIG-Stop, which is 143 rather than 154 amino acids long and totally different from amino acids 134 to 143. The electron density of both chains in the asymmetric unit of the high resolution map of the apoprotein fits very well to this new sequence, and therefore, the model was built accordingly. The refinement at 1.5 Å resolution strengthened this interpretation, and the maps calculated with the phases from the final model confirmed the absence of electron density beyond amino acid 143. This result was further validated in all the co-crystals of the protein examined. The possibility that the nucleotide chromatogram should have been interpreted in terms of four rather than three guanines is not the only one that will yield the amino acid sequence compatible with the experimental electron density, but it is the most likely. Another point where there is a discrepancy is in amino acid number 64, an Ile (atc) according to the published sequence and a Ser (agc) as judged by the electron density in the maps. The predicted molecular mass of a monomer with the sequence used to fit the electron density maps from Thr2 to Gly143 is 16,053.8 Da, a value in very good agreement with the experimental results of mass spectrometry measurements (see the supplemental data). The sequence similarity with other members of the fungal saline soluble lectin family is also preserved in the C-terminal portion of the polypeptide chain used to fit the electron density maps (see Fig. 5).
      Figure thumbnail gr5
      Fig. 5Sequence comparison of fungal lectins. The sequences were aligned using the program CLUSTALW (
      • Thompson J.D.
      • Higgins D.G.
      • Gibson T.J.
      ) and correspond to the following lectins: XCL, X. chrysenteron lectin; PCL, P. cornucopiae lectin; AOL, A. oligospora lectin; PAL, P. anserina lectin; and NCL, N. crassa lectin. The residues involved in the binding of the T antigen to ABL are indicated with a T, and those that participate in the binding of N-acetylglucosamine are indicated with an N. The residues conserved in all the members of the group are represented in red.
      Overall Structure of the Monomer—The final model of the apoprotein comprises 142 amino acid residues for each of the two monomers present in the asymmetric unit of the orthorhombic crystal form (see Table I). The maps do not show electron density for Met1. The two molecules in the asymmetric unit are related by a non-crystallographic dyad with an r.m.s.d. of 0.25 Å calculated over 142 Cα pairs of equivalent residues. The most important difference between the monomers is the presence in monomer A of a 310 helix spanning residues 28–30, in a region where the other monomer presents a β turn. A monomer of ABL fits into a box with the approximate dimensions 45 × 40 × 25 Å. The maps do not show electron density other than that of the amino acid side chains in any of the three potential O-glycosylation sites or the N-glycosylation site that could be present according to the amino acid sequence.
      The ABL monomer is a single domain structure organized as a β sandwich (Fig. 1A) with six strands of β chain in the first sheet (strands H, I, J, A, D, and C) and four strands in the second sheet (strands B, E, F, and G). The first sheet is of the mixed type, and the second sheet is antiparallel. The topology of the first sheet is -2X 1 4 -1 -1, and that of the second sheet is -1 -1 -1 (Fig. 1B). A helix-loop-helix motif (spanned by residues 90–108), packed against the second sheet and found between strands G and H, connects the two sheets. The space between the two sheets is filled mostly with hydrophobic side chains. The external surface of the second β sheet and the helix-loop-helix motif form a pocket filled with the side chains of Trp26, Trp77, Tyr28, Tyr98, His72, Asn94, and Arg107. The T-antigen disaccharide binds at the edge of this pocket, interacting with the residues of the chain connecting the two sheets through the loops present between strands B and C and D and E and the β turn between strands F and G of the second sheet. A hairpin at the other edge of the first sheet, which is formed by strands C and D, is found protruding away from the body of the monomer and the second sheet. A very clear salt bridge is formed at the interface between the two sheets between Glu36 of strand C of the first sheet and Arg23 of strand B of the second sheet. The overall shape of the monomer is thus that of a central roughly cylindrical body with a hairpin protruding in one direction and the helix-loop-helix motif protruding in the opposite direction. The only Cys present in the sequence, Cys78, is found in strand G of the second sheet, and its side chain does not point toward the pocket but toward the space between the two sheets.
      Figure thumbnail gr1
      Fig. 1Overall structure and folding of ABL.A, ribbon representation of the ABL monomer. The six-stranded β sheet is shown in blue, and the four-stranded sheet is shown in red. The two short helices are yellow, and the connections are gray. N-Acetylgalactosamine is represented as a ball-and-stick model at the T-antigen binding site (top), and N-acetylglucosamine is represented as a ball-and-stick model at the other site (bottom). B, a topological diagram of the ABL monomer. β Strands are labeled in the order of their appearance from the N terminus to the C terminus using the letters A-J. The 10 strands span the following residues: A, 3–11; B, 19–25; C, 33–37; D, 40–45; E, 51–58; F, 63–72; G, 75–82; H, 113–117; I, 123–130; and J, 135–142. The two helices span residues 90–96 and 101–108. C, stereodiagram of the ABL tetramer viewed looking down one of the crystallographic dyads. The other two dyads are approximately in the plane of the figure. The two top (or bottom) monomers define the dimer present in the crystallographic asymmetric unit. D, the tetramer viewed looking down the other crystallographic dyad with the T antigen and N-acetylglucosamine represented as ball-and-stick models. Only two N-acetylglucosamine molecules are visible because the other two are behind the helices represented in the plane of the figure. The figures were prepared using the programs MOLSCRIPT (
      • Kraulis P.J.
      ) and Raster3D (
      • Merritt E.A.
      • Bacon D.J.
      ).
      Quaternary Structure—The asymmetric unit of the orthorhombic crystals of ABL is a dimer in which each monomer buries 1178 Å2 of solvent-accessible area, 16% of 7369 Å2, the total surface area of a monomer. This value is consistent with a physiological role for the dimer (
      • Janin J.
      • Rodier F.
      ,
      • Ponstingl H.
      • Henrick K.
      • Thornton J.M.
      ). The contacts between the two monomers in the crystallographic asymmetric unit are established through the two strands B present at the edge of the second β sheet. The two four-stranded sheets, although in contact with each other, do not form a single eight-stranded sheet in the dimer in the manner that has been observed quite frequently in legume lectins and many other oligomeric proteins (
      • Rini J.M.
      ). Two hydrogen bonds are established between the nitrogen and the carbonyl of Arg23 of one monomer and those of the other monomer related to it by the non-crystallographic dyad. In addition to these hydrogen bonds between the two strands, another contact is established between the side chains of Glu22 of one chain and Asn25 of the other. Another contact is established between strand B of one monomer and strand C of the other sheet in the other monomer: the side chains of Arg19 in one monomer and Asp35 of the other monomer are in contact with each other. These contacts between monomers force the two Arg23 residues into positions that are quite close to one another (and also very close to the non-crystallographic dyad), but the positive charges of the arginines are neutralized by those of the two Glu36 of the same monomer.
      All the biochemical evidence available is consistent with the existence of an ABL tetramer under physiological conditions (
      • Sage H.J.
      • Connett S.L.
      ). The solvent-accessible area of the asymmetric unit and those of the symmetry-related dimers was calculated and used to identify the other dimer in the physiological tetramer. The solvent-accessible area of the asymmetric unit is 12,381 Å2, which reduces to 10,587 Å2 upon tetramer formation. Thus, the area of each dimer that becomes buried upon tetramer formation is 1794 Å2, about 14.5% of the total surface and a value that is perfectly in line with those observed for other physiological oligomers (
      • Janin J.
      • Rodier F.
      ,
      • Ponstingl H.
      • Henrick K.
      • Thornton J.M.
      ). A tetrameric assembly with an open structure in which there are two dyads that form an angle of 73° and -73° has been described for PNA, another lectin that binds the T antigen (
      • Banerjee R.
      • Mande S.C.
      • Ganesh V.
      • Das K.
      • Dhanaraj V.
      • Mahanta S.K.
      • Suguna K.
      • Surolia A.
      • Vijayan M.
      ). As shown in Fig. 1, C and D, the ABL tetramer is an object with 222 (D2) symmetry.
      The interaction between dimers in the tetramer is characterized by the contacts established between each monomer and both members of the other dimer. This fact reinforces the contacts between dimers that are not particularly extensive in each of the two other interfaces.
      The point of closest contact at a second interface between monomers is found between the two chains A of the first β strand, more specifically, the nitrogen of Tyr10 is in contact with the carbonyl of Gln11 of the other monomer, and the nitrogen of Gln11 is in contact with the carbonyl of Tyr10 of the other chain. The corresponding side chains are, however, at almost 5 Å from each other. The other relevant contacts between monomers in this region are hydrophobic.
      The interface with the other monomer in the second dimer is at the point where all the hairpins formed by strands C and D of the first sheet are close to each other in the center of the molecule (Fig. 1C). In particular, the carbonyl of Glu36 is in contact with the nitrogen of Arg38 of the other monomer and vice versa. Another important contact is established between the side chains of Arg38 and Asp35 of the other monomer.
      Overall, these interactions appear to be less extensive than those observed within the dimer in the asymmetric unit (Fig. 1D), but they are strengthened by the fact that they occur repeatedly. There are no reports concerning the existence of the dimer as a stable unit.
      In conclusion, the quaternary structure of the ABL molecule can be described as a dimer of dimers with 222 symmetry. Fig. 1C is a stereodiagram of the tetramer viewed looking down one of the crystallographic dyads, and Fig. 1D shows it rotated 90°, i.e. looking down the other crystallographic dyad.
      T-antigen Binding Site—The remarkable antineoplastic properties of ABL are due to the selectivity with which it binds the T-antigen disaccharide moiety, i.e. Galβ1–3GalNAc. Therefore the T-antigen and benzyl T-antigen disaccharide were the first two molecules to be tested by soaking the orthorhombic crystals of the apoprotein in the original mother liquor (4.0 m sodium formate, 0.02 m Tris, pH 8.0) saturated with the disaccharides. The electron density maps revealed the position of the binding sites present in each of the two monomers of the asymmetric unit, but careful analysis of the maps showed the presence of extra electron density in one of the two binding sites that could not be ascribed to the ligands. Several alternatives to the original mother liquor as well as to the cryoprotectant were tested, and the extra density was identified as a Tris molecule. To eliminate the possible interference of one or more ingredients of the mother liquor (
      • Carrizo M.E.
      • Irazoqui F.J.
      • Lardone R.D.
      • Nores G.A.
      • Curtino J.A.
      • Capaldi S.
      • Perduca M.
      • Monaco H.L.
      ), for all subsequent soaking experiments, the orthorhombic crystals were first transferred to a solution of 30% polyethylene glycol 4000, 0.1 m sodium acetate buffered with 0.1 m borate, pH 8.5. A test with the two disaccharides mentioned above showed that ligand binding in this solution was identical to that observed in the original mother liquor, but the Tris molecule was not present in the electron density maps.
      Four monosaccharides and seven disaccharides were tested by preparing co-crystals and examining electron density maps of the binding site. The only monosaccharide that showed very clear electron density in the T-antigen binding site was N-acetylgalactosamine. Crystals soaked in galactose or glucose did not show any electron density anywhere in the maps, and crystals soaked in N-acetylglucosamine did not diffract at all after a soaking time of longer than ∼2 h, but shorter soaking times (of about 0.5 h) revealed that N-acetylglucosamine binds at a totally different site (see below). In addition to the T-antigen and benzyl T-antigen disaccharides, three other disaccharides displayed very clear electron densities at this binding site: the closely related p-nitrophenyl T-antigen disaccharide, the T antigen (Galβ1–3GalNAc-α-O-Ser), and lacto-N-biose (Galβ1–3GlcNAc). Lactose and N-acetyllactosamine did not appear to bind in the crystals under these conditions. The structure determination statistics of the co-crystals with the T antigen and lacto-N-biose (Galβ1–3GlcNAc), representative of the two classes of disaccharide that were found to bind at this site, are listed in Table I.
      The binding of the four variants of the T-antigen disaccharide was identical for the Galβ1–3GalNAc moiety present in all of them. The only monosaccharide that binds at this site: Gal-NAc does it in a position that overlaps completely with that moiety in the disaccharides and, the other disaccharide that binds in the crystals, Lacto-N-biose (Galβ1–3GlcNAc), binds overlapping with the T-antigen variants with the exception of the only different epimeric hydroxyl, OH4. Therefore the main interactions at this binding site can be illustrated by discussing the contacts established by the T antigen (Galβ1–3GalNAc-α-O-Ser) and the protein (Fig. 2A).
      Figure thumbnail gr2
      Fig. 2The T-antigen-binding site. Electron density of carbohydrates at the T-antigen-binding site in the orthorhombic crystal form. The 2 Fobs-Fc maps are contoured at the 1.5 σ level. The solvent-accessible surface of the protein is shown in light gray, and it was drawn using the programs DINO (www.dino3d.org) and MSMS (
      • Sanner M.F.
      • Olson A.J.
      • Spehner J.C.
      ). The two-dimensional plots on the right were prepared using the program LIGPLOT (
      • Wallace A.C.
      • Laskowski R.A.
      • Thornton J.M.
      ). The three parts of the figure show (A) T antigen (Galβ1–3GalNAc-α-O-Ser), (B) lacto-N-biose (Galβ1–3GlcNAc), and (C) N-acetylgalactosamine.
      The T-antigen binding site is a shallow depression delimited by the loops connecting strands B→C, D→E, and F→G on one side and the helix-loop-helix motif on the other. Following a standard convention, we will use the numbering of GalNAc, Gal, and Ser to describe the interactions of the T antigen with the protein. The two most important interactions of the disaccharide are with Ser48 and Asn73. The OG of Ser48 interacts with O7 of GalNAc and its carbonyl with O2 of Gal, whereas the OD of Asn73 interacts with N2 and its N with O7 of GalNAc. Single interactions are observed between the carbonyl of Gly49 and O4 of GalNAc and between the nitrogen of Ala29 and O4 of Gal.
      The ND1 of His72 and NH1 of Arg107, positioned at the end of strand F and in the middle of the first helix interact with O4 and O5 of GalNAc through a bridge with 2 water molecules; O2 and O3 of Gal are coordinated with another water molecule. Relevant hydrophobic contacts are established with Tyr28 and, to a lesser extent, with Tyr74 and Tyr98. The Ser moiety of the T antigen does not form any hydrogen bridges with the protein and is found protruding into the solvent. The side chains that are closest to it are those of His72 and Asn73. The distances for the contacts described above are all very similar in all the co-crystals examined. The most important of these interactions are illustrated in Fig. 2 for two disaccharides and for N-acetylgalactosamine.
      N-Acetylglucosamine Binding Site—When crystals of the orthorhombic form of ABL were soaked in mother liquor saturated with N-acetylglucosamine for periods of no longer than ∼30 min and frozen immediately after that at 100 K, they almost completely retained their integrity and isomorphism. Longer soaking times caused severe deterioration of the crystalline order, and after about 2 h, the diffraction patterns were totally abolished. Difference electron density maps revealed that, in the crystals soaked for 30 min, there was extra electron density present at a site that was totally different from the T-antigen binding site. The density could be clearly interpreted in terms of a single molecule of N-acetylglucosamine bound per dimer of ABL. Crystal breakdown is caused by the interference of N-acetylglucosamine with crystal packing contacts between the second ABL monomer and a symmetry-related protein molecule. This result was observed consistently, not only with crystals of the apoprotein but also with co-crystals of the T-antigen disaccharide and with N-acetylgalactosamine. Therefore, a crystal screen was set up with solutions of ABL that contained 20 mmN-acetylglucosamine, and two new crystal forms were found. The first diffracted only to a resolution of about 3.5 Å, but the second, a tetragonal form, diffracted to 2.4 Å resolution. This second form, which contains one tetramer of ABL in the asymmetric unit, was solved by molecular replacement, and it confirmed the presence of the N-acetylglucosamine binding site in all four monomers of the asymmetric unit. The site is identical to that identified in the orthorhombic form. Soaking of these crystals in solutions containing the T antigen revealed that the presence of N-acetylglucosamine did not interfere with the binding at the other site. The last two columns of Table I list the structure determination statistics of the two crystal forms with N-acetylglucosamine in this second carbohydrate-binding site.
      The N-acetylglucosamine binding site is delimited by residues from the last strand of the first β sheet (strand G), the first strand of the second β sheet (strand H), and the second of the two helices of the helix-loop-helix motif. The two sugar binding sites of the protein are thus situated on the two sides of the motif as shown in Fig. 1A. The main interactions of N-acetylglucosamine are with Thr82 and Asp79 that appear to be responsible for the specificity of the site for N-acetylglucosamine (Fig. 3A). Thr82 makes a hydrogen bond with O7 of the N-acetyl group, which explains why glucose does not bind at this site. The two carboxylate oxygens of Asp79 make contacts with O6 and O4 that point in the same direction in this sugar and in opposite directions in N-acetylgalactosamine, which explains why the latter does not bind at this site. Other contacts are established with Arg103, placed at the beginning of the second helix and Tyr114. The former is in contact with O5 and O6, and the OH of the latter is at ∼2.6 Å from O3 of the sugar and about 3.0 Å from O4. The carbonyl of Ile80 makes a hydrogen bond with O3 of the sugar. A water molecule is seen in contact with O4 in all the co-crystals of the orthorhombic form, but not in those of the tetragonal form (fewer water molecules were included in these models because of the lower resolution). The distances for all the contacts described above are very similar in all the co-crystals examined, and no conformational changes in the active site are detected between the free and ligand-bound protein. The main interactions of crystals that contain in every case N-acetylglucosamine in this site and selected ligands in the other site are illustrated in Fig. 3. Fig. 4 is a stereodiagram showing N-acetylglucosamine and N-acetylgalactosamine binding at the two sites of ABL.
      Figure thumbnail gr3
      Fig. 3The N-acetylglucosamine-binding site. The figure shows the binding of (A) N-acetylglucosamine and (B) N-acetylglucosamine and N-acetylgalactosamine in the orthorhombic crystal form and (C) N-acetylglucosamine and T antigen in the tetragonal crystal form. Electron density contour levels were as described in the legend, and the same programs were used to draw the figure.
      Figure thumbnail gr4
      Fig. 4The two binding sites in a monomer. Stereodiagram of a monomer of ABL with a molecule of N-acetylgalactosamine (NGA) bound at the T-antigen binding site (top) and a molecule of N-acetylglucosamine (NAG) bound at the second binding site (bottom). The electron density of the 2Fobs-Fc map corresponds to the ligands bound in the orthorhombic form, and it was contoured at the 1.5 σ level. The side chains of the main amino acids involved in the interactions are represented in the figure. The figure was prepared using the program DINO (www.dino3d.org).
      Comparison of Ligand Binding with Other Lectins—The structure of several proteins complexed with the T-antigen disaccharide has been determined by x-ray diffraction analysis of single crystals. The group includes the PNA (
      • Banerjee R.
      • Mande S.C.
      • Ganesh V.
      • Das K.
      • Dhanaraj V.
      • Mahanta S.K.
      • Suguna K.
      • Surolia A.
      • Vijayan M.
      ,
      • Adhikari P.
      • Bachhawat-Sikder K.
      • Thomas C.J.
      • Ravishankar R.
      • Jeyaprakash A.A.
      • Sharma V.
      • Vijayan M.
      • Surolia A.
      ), amaranthin (Amaranthus caudatus agglutinin, Ref.
      • Transue T.R.
      • Smith A.K.
      • Mo H.
      • Goldstein I.J.
      • Saper M.A.
      ), Maclura pomifera agglutinin (
      • Lee X.
      • Thompson A.
      • Zhang Z.
      • Ton-that H.
      • Biesterfeldt J.
      • Ogata C.
      • Xu L.
      • Johnston R.A.
      • Young N.M.
      ), jacalin (Artocarpus integrifolia lectin, Refs.
      • Sankaranarayanan R.
      • Sekar K.
      • Banerjee R.
      • Sharma V.
      • Surolia A.
      • Vijayan M.
      and
      • Jeyaprakash A.A.
      • Geetha Rani P.
      • Banuprakash Reddy G.
      • Banumathi S.
      • Betzel C.
      • Sekar K.
      • Surolia A.
      • Vijayan M.
      ), a heat-labile Escherichia coli enterotoxin (
      • van den Akker F.
      • Steensma E.
      • Hol W.G.
      ), and, more recently, the fungal galectin CGL2 (
      • Walser P.J.
      • Haebel P.W.
      • Kunzler M.
      • Sargent D.
      • Kues U.
      • Aebi M.
      • Ban N.
      ). The T-antigen binding lectins have been classified into two groups on the basis of the monosaccharide that occupies the primary binding site (
      • Jeyaprakash A.A.
      • Geetha Rani P.
      • Banuprakash Reddy G.
      • Banumathi S.
      • Betzel C.
      • Sekar K.
      • Surolia A.
      • Vijayan M.
      ). According to this classification, ABL belongs to the category that has GalNAc binding at the primary site. The group also includes M. pomifera agglutinin, jacalin, and amaranthin; the first two lectins bind the disaccharide with the Gal moiety completely exposed to the solvent and interact with the carbohydrate only through the GalNAc moiety. We tested experimentally the importance of the Gal moiety in the binding of disaccharides to ABL by studying the binding of lacto-N-biose (Galβ1–3GlcNAc) to the lectin. In the case of this disaccharide, it is the Gal moiety that determines the specificity of binding to the T-antigen binding site; GlcNAc alone binds at the other site. Therefore, binding of the T antigen to ABL appears to be more similar to the binding in amaranthin than that in the other lectins of the group, and ABL is thus the second example in which direct interactions exist between the Gal moiety of the T-antigen disaccharide and the protein. In the structure of a plant lectin complexed with the Tn determinant, GalNAc-α-O-Ser, determined to 2.7 Å resolution, it was observed that the Ser moiety is largely exposed to the solvent (
      • Babino A.
      • Tello D.
      • Rojas A.
      • Bay S.
      • Osinaga E.
      • Alzari P.M.
      ), as it is in the ABL co-crystals with the T antigen (Galβ1–3GalNAc-α-O-Ser).
      The structures of several nonspecific lectins in complex with N-acetylglucosamine are known from x-ray diffraction studies. Whereas the specificity of the second binding site of ABL has not yet been studied in detail, we have found that the site does not bind glucose, galactose, and N-acetylgalactosamine in the crystalline state. Therefore, the site appears to be rather specific for N-acetylglucosamine. The architecture of the nonspecific sites that bind N-acetylglucosamine has been discussed in detail recently (
      • Loris R.
      • De Greve H.
      • Dao-Thi M.H.
      • Messens J.
      • Imberty A.
      • Wyns L.
      ); overall, they are topologically quite different from that of the second binding site of ABL. The structure of Urtica dioica agglutinin, a small monomeric lectin that is specific for saccharides containing GlcNAc, was solved unliganded and in complexes with a trisaccharide and tetrasaccharide (
      • Saul F.A.
      • Rovira P.
      • Boulot G.
      • Van Damme E.J.
      • Peumans W.J.
      • Truffa-Bachi P.
      • Bentley G.A.
      ). In this case, a Ser and a Tyr are found to play central roles in binding the carbohydrates to the two slightly different sites, but there is also in both cases a strong aromatic component that seems to be totally absent in the second ABL binding site.
      Structural Similarity with Other Proteins—The ABL fold has no structural similarity to other T-antigen disaccharide-binding proteins. A comparison of the amino acid sequence of these proteins with the corrected ABL sequence using the program CLUSTALW (
      • Thompson J.D.
      • Higgins D.G.
      • Gibson T.J.
      ) reveals very low identity percentages: 18% for amaranthin (
      • Transue T.R.
      • Smith A.K.
      • Mo H.
      • Goldstein I.J.
      • Saper M.A.
      ) and M. pomifera agglutinin (
      • Young N.M.
      • Johnston R.A.
      • Watson D.C.
      ), 14% for PNA (
      • Young N.M.
      • Johnston R.A.
      • Watson D.C.
      ), 13% for jacalin (
      • Mahanta S.K.
      • Sanker S.
      • Rao N.V.
      • Swamy M.J.
      • Surolia A.
      ), and 16% for LT-1. Similar results are obtained using the program ALIGN (
      • Pearson W.R.
      • Wood T.
      • Zhang Z.
      • Miller W.
      ).
      A search for three-dimensional similarity with the ABL coordinates in the Dali server (
      • Holm L.
      • Sander C.
      ) identifies 11 proteins that score above the significance similarity threshold. They are mostly β sandwiches and are molecules with very diverse functions. The protein with the highest score, more than twice the value of the second in the list, is the soluble form of equinatoxin II, a pore-forming toxin from the sea anemone Actinia equina (
      • Athanasiadis A.
      • Anderluh G.
      • Macek P.
      • Turk D.
      ). The equinatoxin II fold (Protein Data Bank accession code 1LAZ) is also a single domain β sandwich, but it has 12 β sheet strands, and its topology is totally different from that of ABL. The r.m.s.d. for 110 of a total of 175 Cα of the toxin compared with ABL is 2.7 Å.
      A sequence similarity search in the ExPASy server identifies the members of a fungal saline-soluble lectin family as similar to ABL. The family includes the lectins from the parasitic nematode trapping fungus Arthrobotrys oligospora (
      • Nordbring-Hertz B.
      • Mattiasson B.
      ,
      • Rosen S.
      • Kata M.
      • Persson Y.
      • Lipniunas P.H.
      • Wikstrom M.
      • Van Den Hondel M.J.
      • Van Den Brink J.
      • Rask L.
      • Heden L.O.
      • Tunlid A.
      ,
      • Rosen S.
      • Bergstrom J.
      • Karlsson K.A.
      • Tunlid A.
      ), the two mushrooms Pleurotus cornucopiae (
      • Iijima N.
      • Yoshino H.
      • Ten L.C.
      • Ando A.
      • Watanabe K.
      • Nagata Y.
      ) and Xerocomus chrysenteron (
      • Trigueros V.
      • Lougarre A.
      • Ali-Ahmed D.
      • Rahbe Y.
      • Guillot J.
      • Chavant L.
      • Fournier D.
      • Paquereau L.
      ), and the filamentous fungi Podospora anserina and Neurospora crassa used in many studies of fundamental cell biology (
      • Hynes M.J.
      ).
      The A. oligospora lectin was identified in the fungus as responsible for the capture of the host that takes place through its interaction with a receptor bearing the specific carbohydrate present on the nematode surface (
      • Nordbring-Hertz B.
      • Mattiasson B.
      ). This mechanism of adhesion to host surfaces is believed to be present in many other both parasitic and symbiotic fungi. The lectin binds not only the T-antigen disaccharide but also sulfated glycoconjugates and phospholipids (
      • Rosen S.
      • Kata M.
      • Persson Y.
      • Lipniunas P.H.
      • Wikstrom M.
      • Van Den Hondel M.J.
      • Van Den Brink J.
      • Rask L.
      • Heden L.O.
      • Tunlid A.
      ).
      The gene sequence encoding two fruit body lectins of P. cornocupiae was reported recently (
      • Iijima N.
      • Yoshino H.
      • Ten L.C.
      • Ando A.
      • Watanabe K.
      • Nagata Y.
      ). They are very similar to one another, and the proteins they code for clearly belong to the saline-soluble lectin family. Although their physiological function and ligand binding specificity are not known, they are believed to be involved in the defense mechanism of the fungus.
      X. chrysenteron is an edible mushroom of the boletus family that possesses remarkable insecticidal properties. The lectin responsible for this property was suggested as a likely candidate to confer pest resistance to transgenic plants (
      • Trigueros V.
      • Lougarre A.
      • Ali-Ahmed D.
      • Rahbe Y.
      • Guillot J.
      • Chavant L.
      • Fournier D.
      • Paquereau L.
      ). The presence of genes coding for proteins of the saline-soluble lectin family in the filamentous fungi P. anserina and N. crassa gives a clear indication that this lectin family is very widely distributed among fungi (
      • Hynes M.J.
      ).
      Fig. 5 compares the sequences of ABL with those of five members of the fungal saline-soluble lectin family. The residues involved in T-antigen binding to ABL are indicated with a T, and those involved at the N-acetylglucosamine-binding site are indicated with an N. Only one of the two P. cornocupiae lectins was included in the comparison because the two forms characterized differ in only a few amino acids. The sequence similarities in the family are evident, as is the conservation (or substitution by acceptable alternatives) of the residues involved in T-antigen disaccharide and N-acetylglucosamine binding. Therefore, not only can the ABL fold be proposed as characteristic of the entire family, but the presence of two saccharide binding sites can also be postulated for the other members of the family.
      The crystal structure of ABL defines the fold for a new family of lectins and provides the basis for understanding their interactions with the family of the T-antigen-derived molecules. Although the existence of two distinct binding sites with different binding specificities was documented for another fungal lectin, Psathyrella velutina lectin (molecular mass of a monomer, 40,000 Da; specificity for heparin/pectin and N-acetylglucosamine/N-acetylneuraminic acid; Ref.
      • Ueda H.
      • Saitoh T.
      • Kojima K.
      • Ogawa H.
      ), the occurrence of two binding sites in a single domain, which can discriminate between two monosaccharides differing only in the configuration of a single epimeric hydroxyl, is reported here for the first time. The presence in ABL of the two types of sites with different specificities explains the conflicting evidence in the literature for the binding of different saccharides based on ligand competition studies. The complex carbohydrate binding specificity reported for many other lectins might have, in some cases, an equally simple explanation. The physiological role of different sites in lectins discriminating with this level of subtlety is unknown, as is the diffusion of this phenomenon in lectins from other living organisms.
      Note Added in Proof—A study with the determination of the three-dimensional structure of the X. chrysenteron lectin has been published recently (
      • Birck C.
      • Damian L.
      • Marty-Detraves C.
      • Lougarre A.
      • Schulze-Briese C.
      • Koehl P.
      • Fournier D.
      • Paquereau L.
      • Samama J.P.
      ).

      Acknowledgments

      We are grateful to the staff of Sincrotrone Elettra and the European Synchrotron Radiation Facility for assistance during data collection (Proposal MX 199). We thank Eleonora Perani and Monica Galliano for the mass spectra and L. Mario Amzel for critical review of the manuscript.

      Supplementary Material

      References

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