Structural differences in the two major wheat germ agglutinin isolectins

We have combined amino acid sequence data with x-ray diffraction results to determine differences in structure of wheat germ agglutinin isolectin 1 (WGA1) relative to the known structure of wheat germ agglutinin isolectin 2 (WGA2). Electron density difference maps computed at 2.2 A resolution with coefficients [2F(WGA1) - F(WGA2)] and [F(WGA1) - F(WGA2)] and based on refined model phases of the WGA2 structure have revealed that the largest differences in the two isolectin structures are localized in the B-domain of the molecule. Amino acid sequence studies of tryptic and thermolytic peptides of WGA1 confirm the strong homology between the two isolectins and suggest variability at only four sequence positions. Three of these are closely spaced in domain B. The two histidines in WGA2, His59 and His66, are substituted by Gln and Tyr, respectively, and Pro56, by Thr in WGA1. The fourth difference at position 93 in domain C was identified as a change from Ser (WGA2) to Ala (WGA1). With these substitutions WGA1 exhibits a slightly higher degree of internal homology than does WGA2. In addition, we have carried out fluorescence studies on tryptic peptide T-3 to confirm the presence of a second Trp residue in the wheat germ agglutinin molecule, recently predicted at position 41 during the course of high resolution crystal structure refinement of WGA2.     

2.2 A resolution structure analysis of two refined N-acetylneuraminyl-lactose--wheat germ agglutinin isolectin complexes

The crystal structures of complexes of isolectins 1 and 2 of wheat germ agglutinin (WGA1 and WGA2) with N-acetylneuraminyl-lactose (NeuNAc-alpha(2-3)-Gal-beta(1-4)-Glc) have been refined on the basis of data in the 8 to 2.2 A resolution range to final crystallographic R-factors of 17.2% and 15.3% (Fo greater than 1 sigma), respectively. Specific binding interactions and water association, as well as changes in conformation and mobility of the structure upon ligand binding, were compared in the two complexes. The temperature factors (B = 16.3 A2 and 18.4 A2) were found to be much lower compared with those of their respective native structures (19 to 22 A2). Residues involved in sugar binding, dimerization and in lattice contacts exhibit the largest decreases in B-value, suggesting that sugar binding reduces the overall mobility of the protein molecules in the crystal lattice. The binding mode of this sialyl-trisaccharide, an important cell receptor analogue, has been compared in the two isolectins. Only one of the two unique binding sites (4 per dimer), located in the subunit/subunit interface, is occupied in the crystals. This site, termed the "primary" binding site, contains one of the five amino acid substitutions that differentiate WGA1 and WGA2. Superposition of the refined models in each of the independent crystallographic environments indicates a close match only of the terminal non-reducing NeuNAc residue (root-mean-square delta r of 0.5 to 0.6 A). The Gal-Glc portion was found to superimpose poorly, lack electron density, and possess high atomic thermal factors. In both complexes NeuNAc is stabilized through contact with six amino acid side-chains (Ser114 and Glu115 of subunit 1 and Ser62, Tyr64, Tyr(His)66 and Tyr73 of subunit 2), involving all NeuNAc ring substituents. Refinement has allowed accurate assessment of the contact distances for four hydrogen bonds, a strong buried non-polar contact with the acetamido CH3 group and a large number of van der Waals' interactions with the three aromatic side-chains. The higher affinity of N-acetylneuraminyl-lactose observed by nuclear magnetic resonance studies for WGA1 can be explained by the more favorable binding interactions that occur when residue 66 is a Tyr. The tyrosyl side-chain provides a larger surface for van der Waals' stacking against the NeuNAc pyranose ring than His66 and a hydrogen bond contact with Gal (C2-OH), not possible in WGA2.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interactions of wheat-germ agglutinin with GlcNAc beta 1,6Gal sequence

The interactions of wheat-germ agglutinin (WGA) with the GlcNAc beta 1,6Gal sequence, a characteristic component of branched poly-N-acetyllactosaminoglycans, were investigated using isothermal titration calorimetry and X-ray crystallography. GlcNAc beta 1,6Gal exhibited an affinity greater than GlcNAc beta 1,4GlcNAc to all WGA isolectins, whereas Gal beta 1,6GlcNAc showed much less affinity than GlcNAc beta 1,4GlcNAc. X-ray structural analyses of the glutaraldehyde-crosslinked WGA isolectin 3 crystals in complex with GlcNAc beta 1,6Gal, GlcNAc beta 1,4GlcNAc and GlcNAc beta 1,6Gal beta 1,4Glc were performed at 2.4, 2.2 and 2.2 A resolution, respectively. In spite of different glycosidic linkages, GlcNAc beta 1,6Gal and GlcNAc beta 1,4GlcNAc exhibited basically similar binding modes to each other, in contact with side chains of two aromatic residues, Tyr64 and His66. However, the conformations of the ligands in the two primary binding sites were not always identical. GlcNAc beta 1,6Gal showed more extensive variation in the parameters defining the glycosidic linkage structure compared to GlcNAc beta 1,4GlcNAc, demonstrating large conformational flexibility of the former ligand in the interaction with WGA. The difference in the ligand binding conformation was accompanied by alterations of the side chain conformation of the amino acid residues involved in the interactions. The hydrogen bond between Ser62 and the non-reducing end GlcNAc was always observed regardless of the ligand type, indicating the key role of this interaction. In addition to the hydrogen bonding and van der Waals interactions, CH--pi interactions involving Tyr64, His66 and Tyr73 are suggested to play an essential role in determining the ligand binding conformation in all complexes. One of the GlcNAc beta 1,6Gal ligands had no crystal packing contact with another WGA molecule, therefore the conformation might be more relevant to the interaction mode in solution.     

Preliminary X-ray diffraction results on co-crystals of wheat germ agglutinin with a sialoglycopeptide from the red cell receptor glycophorin A

Diffraction-quality crystals have been obtained for complexes of each of the major wheat germ agglutinin (WGA) isolectins with the tryptic sialoglycopeptide T-5 from the WGA red cell receptor glycophorin A. This octa-glycopeptide possesses a Thr-linked carbohydrate moiety (GalNAc(NeuNAc)-Gal-NeuNAc) with specificity for the WGA binding site. The crystals belong to the orthorhombic space group P2(1)2(1)2 and have unit cell dimensions: a = 112.2 A, b = 51.0 A, c = 63.5 A (isolectin 1); a = 109.0 A, b = 52.3 A, c = 62.4 A (isolectin 2). There are two monomer complexes in each asymmetric unit.     

Histidine determination in wheat germ agglutinin isolectin by X-ray diffraction analysis

Electron density maps based on 2·4 Å and 2·2 Å X-ray diffraction data for crystals of two isolectins of wheat germ agglutinin (designated isolectins 1 and 2) were compared in terms of side-chain identities. While the primary structure of wheat germ agglutinin is not available, a partial amino acid sequence for isolectin 2 has been deduced by inspection of the electron density map and through model building. The positions of the two histidines predicted from amino acid composition studies to be present in isolectin 2 but not in isolectin 1, were located by difference Fourier techniques and analysis of the heavy-atom binding properties of these two isolectins. Both histidines were found to reside in the B-domain of the multi-domain wheat germ agglutinin protomer (A, B, C, D). Histidine 57 lies in the contact region between the two subunits near the molecular dimer axis. The side-chain of histidine 64 forms part of the primary saccharide binding site at the interface where B and C-domains of opposite protomers make contact. In addition, this histidine serves as a major target for heavy-atom binding by platinum and mercury compounds.     

Sequence variability in three wheat germ agglutinin isolectins: Products of multiple genes in polyploid wheat

Summary Three highly homologous wheat germ isolectins (95–97%) are distinct gene products in hexaploid wheat. The amino acid sequences of two of these [wheat germ agglutinin 1 (WGA1) and 2 (WGA2)] are compared with sequence date derived from a complementary DNA (cDNA) clone for the third isolection (WGA3). This comparison includes three corrections to earlier amino acid sequences data of both WGA1 and WGA2 at positions 109 (from Ser to Phe), 134 (from Gly to Lys), and 150 (from Gly to Trp). These reassignments are based on new results from crystal structure refinement and amino acid sequence data of WGA1, as well as the recently determined nucleotide sequence of WGA3. In addition, the C-terminal residue of WGA1 has been revised to Gly 171 and now differs from WGA2 (Ala 171). Four other positions, Asn9, Ala53, Gly119, and Ser 123, at which WGA1 and WGA2 are identical but differ from the DNA sequence of WGA3, were also reinvestigated by amino acid sequencing techniques and confirmed.Variability among the three isolectins is observed at a total of 10 sequence positions: 9, 53, 56, 59, 66, 93, 109, 119, 123, and 171. Pairwise comparisons indicate that WGA3 deviates to a much larger extent from WGA1 (at eight positions) and from WGA2 (at seven positions) than the latter from one another (at five positions). Eight of the 10 mutations are equally distributed between domians B and C, the two intrior and more highly conserved of the four WGA domains (A, B, C, D). Correlation of the variable residues with the three-dimensional structure indicates that all except the two previously described B-domain residues, 56 and 59 (Wright and Olafsdottir 1986), are easily accommodated at the dimer surface.WGA3 displays a higher degree of inter-domain similarity than found in WGA1 and WGA2. Of the seven variable positions that are located in the domain core (residues 3–31), five are in perfect agreement with our earlier predicted domain ancestor sequence. This suggests that of the three isolectins WGA3 is most closely related to the common ancestral molecule.     

Molecular cloning, functional expression, and characterization of isolectin genes of hemolytic lectin CEL-III from the marine invertebrate Cucumaria echinata

CEL-III is a hemolytic lectin purified from the marine invertebrate Cucumaria echinata. Previous research has indictated that CEL-III is composed of several isoforms. Here we identified five CEL-III isolectin genes, designated CEL-III-L1, CEL-III-L2, CEL-III-S1, CEL-III-S2, and CEL-III-LS1, by cDNA cloning. The deduced amino acid sequences suggested they shared 94.0-99.8% identical residues. Among the amino acid residues involved in carbohydrate binding, the His residue, which contributes to stacking with sugar, in subdomain 1α was replaced by Tyr in CEL-III-L2. The recombinant proteins were expressed in Escherichia coli or insect cells. rCEL-III-L2 showed higher hemolytic activity than those of the other isolectins. Furthermore, an apparent oligomer band of rCEL-III-L2 was detected on erythrocyte membranes, although the other isolectins showed smear bands. These results suggest that Tyr36 of CEL-III-L2 is important for the expression of hemolytic activity and oligomerization.     

Crystal structure of a wheat germ agglutinin/glycophorin-sialoglycopeptide receptor complex. Structural basis for cooperative lectin-cell binding.

The crystal structure of wheat germ agglutinin isolectin 1 (WGA1) complexed with a tryptic sialoglycopeptide fragment (T-5) from its erythrocyte receptor glycophorin A, which contains the O-linked tetrasaccharide NeuNAc-alpha 2,3-Gal-beta 1,3-(alpha 2,6-NeuNAc) Gal-NAc-alpha 1-O-Thr, has been determined by molecular replacement techniques and refined at 2.0-A resolution (R = 18.1%). The structure reveals that association between WGA1 dimers, composed of two identical four-domain (A-D) monomers, and T-5 is asymmetric and involves sialic acid binding at three nonequivalent aromatic residue-rich sites. Two independent binding modes are observed. In the dominant ("major") binding mode, the two highest affinity sites are utilized to cross-link neighboring crystallographically related WGA1 dimers. The branched tetrasaccharide has an extended rigid conformation, and its terminal alpha 2,6-NeuNAc and alpha 2,3-NeuNAc residues occupy specificity sites in domains B1 (monomer 1) and C2 (monomer 2) on opposing dimers, respectively. This asymmetric selection of binding sites leads to infinite open-ended arrays of interlinked lectin molecules. In the subsidiary "minor" binding mode, only the terminal alpha 2,6-NeuNAc, anchored to the aromatic residue-rich binding site in domain A2, is clearly visible. The remaining portion of T-5 is disordered. This structure presents the first evidence for NeuNAc binding in the aromatic residue-rich sites of domains A and C and suggests a preference of WGA for alpha 2,6-linked NeuNAc. Moreover, the unusual asymmetric WGA1-tetrasaccharide association, involving domain binding sites that differ in their binding affinities for NeuNAc, offers explanations for the widely observed cooperative cell binding behavior of WGA.     

Phaseolus vulgaris isolectin binding to human erythrocytes.

The Phaseolus vulgaris isolectins L4,L3E1, L2E2, L1E3, and E4 were isolated by affinity and ion exchange chromatography. Pure isolectins were radiolabeled by the chloramine-T method with Na125IO4 and their binding to human erythrocytes was studied. A normal erythrocyte has approximately 8 times 10(5) receptor sites for each isolectin; however, the association constants (Ka) of binding increased from 1.1 times 10(7) M-1 to 3.8 times 10(8) M-1, with increasing number of E subunits per tetrameric isolectin molecule. Isolectin to erythrocyte binding reached equilibrium rapidly and was reversed by fetuin. All isolectins competed with 125I-E4 for erythrocyte binding sites, with a constant (KI) similar to the Ka calculated for each respective radiolabeled isolectin. When isolectin binding at 0 degrees C, 4 degrees C, or 8 degrees C was compared to that at 25 degrees C, there was no reduction in the number of binding sites per cell, but the Ka of E4 was reduced to 3 times 10(7) M-1. Fixed erythrocytes displayed similar isolectin binding characteristics.     

Facile preparation of the alpha-Gal-recognizing Griffonia simplicifolia I-B4 isolectin

The B4 isolectin from Griffonia simplicifolia is of great utility as a reagent for the identification of alpha-D-galactopyranosyl end groups. Its separation from isolectins containing A subunits has been greatly improved by a simple, rapid procedure using a column of N-acetylgalactosamine coupled to vinyl sulfone-activated Sepharose 4B to selectively retain the A subunit-containing isolectins. The procedure has the advantages over previous affinity procedures of speed (the isolation of B4 isolectin can be achieved in one day), simplicity, and high degree of resolution of the B4 isolectin.     

Isolectins in Narcissus: complexity, inter- and intraspecies differences and developmental control

Using high resolution ion-exchange chromatography and isoelectric focusing the heterogeneity of the daffodil ( Narcissus sp.) lectin in terms of isolectin composition was analyzed. A survey of about 30 cultivars and species of Narcissus demonstrates (i) that they all contain over 50 different lectin polypeptides and (ii) that there are pronounced inter- and intraspecies differences in the isolectin patterns. Analyses of lectin preparations isolated from different tissues at different developmental stages further indicate that the isolectin composition is tissue specific and developmentally regulated. Finally, affinity chromatography experiments suggest differences in affinity for a mannose-Sepharose 4B column of different isolectins.     

Intermolecular nuclear Overhauser effect and atomic pair potential approaches to wheat germ agglutinin-sugar binding

The detailed binding mechanism of wheat germ agglutinin (WGA) with N-acetylglucosamine (GlcNAc) was investigated using intermolecular 1H-1H nuclear Overhauser effect (NOE) and atomic pair potential (APP) calculations. Negative NOE was observed on the 1H spectrum of 1-O-methyl derivative of GlcNAc in a solution containing WGA, when the aromatic region of the WGA spectrum was irradiated. Analyses of the time dependence of NOE revealed that H2 and the N-acetyl methyl protons of the sugar are in close proximity to the aromatic protons of WGA in the bound state. This was confirmed and further elucidated by the APP calculations. According to the calculation, the major binding force comes from a hydrogen-bonding between C3-OH of sugar and an acidic residue present in each of the two binding sites of WGA: Glu115 in site 1 and Asp29 in site 2. The binding is further assisted by the N-acetyl group which interacts with a few more polar amino acid residues in the binding sites. The optimized binding mode suggested by the APP calculations supports the NMR results in that H2 and a part of the N-acetyl methyl protons are within 4.5 A distance from protons of both Tyr64 and Tyr73 in site 1 and of Tyr159 in site 2.     

A genetic basis for the origin of six different isolectins in hexaploid wheat

Wheat (Triticum aestivum) germ agglutinin represents a complex mixture of multiple isolectin forms. Upon ion exchange chromatography at pH 3.8, three isolectins can be separated, each of which is composed of two identical subunits. At pH 5.0, however, three additional isolectins can be distinguished, which are built up of two different subunits (heteromeric lectins). Evidence is presented that these heterodimers are normal constituents of the wheat embryo cells. Analyses of the isolectin patterns in extracts from Triticum monococcum, Triticum turgidum dicoccum and Triticum aestivum, provide evidence that each genome, either in simple or complex (polyploid) genomes, directs the synthesis of a single lectin subunit species. In addition, a comparison of the isolectin pattern in these wheat species of increasing ploidy level, made it possible to determine unequivocally the genome by which the individual lectin subunits in polyploid species are coded for. The possible use of lectins in studies on the origin of individual genoms in polyploid species is discussed.Abbreviations CL cereal lectin - PBS phosphate buffered saline - SP Sephadex sulfopropyl Sephadex - WGA wheat germ agglutinin     

Probing the topography of lectins with site-specific spin-labeled glycosides

Three new spin-labeled glycosides, spin-label I [1-[4-(beta-D-galactopyranosyloxy)phenyl]-3-(2,2,6,6-tetramethyl-1 -oxypiperidin-4-yl)-2-thiourea], spin-label II (2,2,6,6-tetramethyl-1-oxypiperidin-4-yl alpha-D-galactopyranoside), and spin-label III [1-(methyl 2-deoxy-alpha-D-galactopyranosid-2-yl)-3-(2,2,6,6- tetramethyl-1-oxypiperidin-4-yl)-2-thiourea], were investigated as structural probes of Griffonia simplicifolia I isolectins (GS I) A4 and B4, respectively, by electron spin resonance (ESR) and inhibition of guaran isolectin precipitation. The p-aminophenyl beta-galactoside spin-label I was strongly immobilized by the B4 isolectin (Kd = 0.42 mM; 2T parallel = 54.0 +/- 0.3 G), while binding to the A4 isolectin was so weak (KI congruent to 2 mM) that binding was undetectable by ESR. The preference for the B4 isolectin was indicative of a more extended hydrophobic binding locus adjacent to the carbohydrate-specific binding site. The alpha-galactosyl spin-label II bound slightly more strongly to the A4 than to the B4 isolectin, as evidenced in both Kd values and particularly by differences in the degree of immobilization (2T parallel = 53.5 vs. 51.5 G, respectively). The 2-N-substituted methyl galactoside spin-label III was so poor an inhibitor of both isolectins (KI congruent to 1-2 mM) that ESR detection of the bound complex was not feasible. In all cases above, the spin-labels were displaced by specific monosaccharide haptens.     

Isolectin composition of individual clones of Urtica dioica: Evidence for phenotypic differences

Analysis of the isolectin composition of 102 individual nettle ( Urtica dioica L.) clones by ion-exchange chromatography revealed the occurrence of at least 11 different isolectins, which all had the same molecular structure and exhibited identical carbohydrate-binding specificity and agglutination properties. All 11 isolectins, however, did not occur simultaneously; 34 combinations of either 1, 2, 3, 4 or 5 isolectins were found. Since the occurrence of multiple molecular forms of the nettle agglutinin cannot be explained by the (partial) autotetraploid character of stinging nettle it is postulated to rely on the expression of a family of closely related lectin genes.     

Amino acid sequence differences in the alpha chains of pea seed isolectins: C-terminal processing

The complete amino acid sequence of the alpha chains of both isolectins found in pea seeds has been determined using automated Edman degradation. We show that the alpha chains of these two proteins differ only at their C-termini: isolectin B is two amino acids longer than isolectin A. Furthermore, the alpha chains of both isolectins are shorter than would be predicted from the nucleotide sequence of a cDNA clone for pea lectin. We suggest, therefore, that these proteins arise from differential C-terminal processing. Amino acid composition data and C-terminal analysis show that the beta chains have also been processed at their C-termini, but in this case identical chains for both isolectins are produced.     

Wheat germ agglutinin dimers bind sialyloligosaccharides at four sites in solution: proton nuclear magnetic resonance temperature studies at 360 MHz

Equilibrium binding studies have been performed over a range of temperatures from 25.4 to 47.3 degrees C between wheat germ agglutinin isolectin I (WGA I) and the alpha 2-3 isomer of (N-acetylneuraminyl)lactose (NeuNAc alpha 2-3Gal beta 1-4G1c). Proton nuclear magnetic resonance spectroscopy at 360 MHz has been used to monitor titrations in this system under conditions where the fraction of total ligand which is bound is small, yet the fractional occupation of sites covers a wide range. Several of the ligand resonances, including the N-acetyl methyl and the axial and equatorial hydrogens at carbon 3 of the NeuNAc residue, are shifted and broadened in the presence of WGA due to chemical exchange between the free and bound environments. The lifetime broadening of the N-acetyl resonance at room temperature of a series of related sialyloligosaccharides has been previously used by us to measure binding affinities to two WGA isolectins [Kronis, K.A., & Carver, J.P. (1982) Biochemistry 21, 3050-3057]. In this paper we report the temperature dependence of the apparent bound shifts and the apparent bound line widths of the N-acetyl, H3a, and H3e peaks. The true bound shifts for the three resonances have been obtained from these data by using the equations derived by Swift and Connick [Swift, T.J., & Connick, R.E. (1962) J. Chem. Phys. 37, 307-320]. The total bound shifts, per monomer, were found to be -1.98, -4.0, and -0.8 ppm for the N-acetyl, the H3a, and the H3e resonances, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding and precipitating activities of Lotus tetragonolobus isolectins with L-fucosyl oligosaccharides. Formation of unique homogeneous cross-linked lattices observed by electron microscopy

We have recently observed that certain asparagine-linked oligosaccharides are multivalent and capable of binding and precipitating with the D-mannose-specific lectin concanavalin A [cf. Bhattacharyya, L., & Brewer, C. F. (1989) Eur. J. Biochem. 178, 721-726] and with a variety of D-galactose-specific lectins [Bhattacharyya, L., Haraldsson, M., & Brewer, C. F. (1988) Biochemistry 27, 1034-1041]. In the present study, we have examined the binding and precipitating activities of a variety of mono- and biantennary L-fucosyl oligosaccharides with three L-fucose-specific isolectins from Lotus tetragonolobus, LTL-A, LTL-B, and LTL-C. The results show that certain difucosyl biantennary oligosaccharides are capable of cross-linking and precipitating with tetrameric isolectins, LTL-A and LTL-C, but not with dimeric isolectin, LTL-B. Quantitative precipitation analyses show that biantennary oligosaccharides containing the Lewis(x) antigen (or type 2 chain of Lewis(a)), Gal beta (1-4)[Fuc alpha (1-3)]GlcNAc, at the nonreducing terminus of each arm are bivalent ligands. However, a biantennary oligosaccharide containing a Lewis(x) determinant on one arm and a type 2 chain of blood group H(O) determinant, Fuc alpha (1-2)Gal beta (1-4)GlcNAc, on the other arm and a monoantennary oligosaccharide containing two fucose residues (analogue of the Lewis(y) antigen) bind but do not precipitate with the isolectins, indicating that the positions and linkage of fucose residues are critical for cross-linking.(ABSTRACT TRUNCATED AT 250 WORDS)     

Five alpha-D-galactopyranosyl-binding isolectins from Bandeiraea simplicifolia seeds.

The alpha-D-galactopyranosyl-binding lectin previously purified from Bandeiraea simplicifolia seeds (Hayes, C.H., and Goldstein, I.J. (1974) J. Biol. Chem. 249, 1904) is shown to consist of five isolectins separable on polyacrylamide gel electrophoresis at pH 9.5. The isolectins are tetrameric structures composed of various combinations of two different glycoprotein subunits designated A and B. The A and B subunits appear to be immunochemically indistinguishable against rabbit antisera prepared from the isolectin mixture. The A subunit contains no methionine, whereas the B subunit contains 1 residue. The subunits migrate differently on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and, although each subunit contains 1 residue of cysteine, they react differently toward 5,5'-dithiobis(2-nitrobenzoic acid). The carbohydrate binding specificity of the two subunits differs significantly: the A subunit exhibits a primary specificity for alpha-D-GalNAcp but also reacts with alpha-D-Galp units, whereas the B subunit shows a sharp specificity toward alpha-D-Galp residues. The differences in carbohydrate binding specificity were exploited in separating the isolectins. B. simplicifolia I isolectins (A4) and (A3B) were purified on a Bio-Gel melibionate column, and (A2B2), (AB3), and (B4) were separated on a column of insolubilized blood group A substance.     

X-ray crystal structure determination and refinement at 1.9 A resolution of isolectin I from the seeds of Lathyrus ochrus

Orthorhombic crystals of isolectin I (LOLI) from the seeds of Lathyrus ochrus were first obtained during the STS 29 space shuttle mission. Subsequently, isostructural crystals were also obtained in the laboratory. They belong to the space group P2(1)2(1)2, with cell dimensions a = 135.84 A, b = 63.12 A and c = 54.54 A with one functional entity, a dimer, in the asymmetric unit (Vm = 2.2 A3/Da). The three-dimensional structure of LOLI, which was solved by the molecular replacement method using a 3 A resolution model of pea lectin, has subsequently been refined by using crystallographic data between 8.0 A and 1.9 A resolution, coupled to molecular dynamics and energy minimization techniques. The conventional R-factor is 0.185 for Fo greater than 1 sigma(Fo). The final model includes 220 well-defined water molecules and has root-mean-square deviations from ideal bond distances and angles of 0.004 A and 3 degrees, respectively. Only slight conformation differences have been found between the two alpha beta monomers. The molecular structure of LOLI, the first to be determined from the genus Lathyrus, is very similar to those of concanavalin A, pea lectin and favin. Differences are confined to the loop regions and beta-chain termini. Comparison of equivalent C alpha atom positions between our final model and the pea lectin structure shows slight differences in the association of the two monomers, which are probably due to the different environments in the crystals. The root-mean-square deviation between C alpha atoms of LOLI and pea lectin is 0.40 A. The metal binding sites are very similar in pea lectin, concanavalin A and LOLI. The sugar-binding sites of LOLI are occupied by four well-ordered water molecules each. The cleavage site for one of the monomers is specially well defined in the final electron density map: the amino group of Glul (alpha) seems to form a salt bridge with the carboxylate group of the terminal Asn181 (beta). A detailed analysis of the difference in crystal packing contacts between pea lectin and LOLI shows that, as might be expected, several of the intermolecular interactions are mediated by residues that correspond to substitutions in the LOLI amino acid sequence.