Structural and Biochemical Characterization of the Interaction of Tubulin with Potent Natural Analogues of Podophyllotoxin.

Four natural analogues of podophyllotoxin obtained from the Mexican medicinal plant Bursera fagaroides, namely, acetyl podophyllotoxin (2), 5'-desmethoxy-ß-peltatin A methyl ether (3), 7',8'-dehydro acetyl podophyllotoxin (4), and burseranin (5), have been characterized, and their interactions with tubulin have been investigated. Cytotoxic activity measurements, followed by immunofluorescence microscopy and flow cytometry studies, demonstrated that these compounds disrupt microtubule networks in cells and cause cell cycle arrest in the G2/M phase in the A549 cell line. A tubulin binding assay showed that compounds 1-4 were potent assembly inhibitors, displaying binding to the colchicine site with Kb values ranging from 11.75 to 185.0 × 10(5) M(-1). In contrast, burseranin (5) was not able to inhibit tubulin assembly. From the structural perspective, the ligand-binding epitopes of compounds 1-3 have been mapped using STD-NMR, showing that B and E rings are the major points for interaction with the protein. The obtained results indicate that the inhibition of tubulin assembly of this family of compounds is more effective when there are at least two methoxyl groups at the E ring, along with a trans configuration of the lactone ring in the aryltetralin lignan core.

15-Deoxy-Delta 12,14-prostaglandin J2 inhibition of NF-kappaB-DNA binding through covalent modification of the p50 subunit.

Cyclopentenone prostaglandins display anti-inflammatory activities and interfere with the signaling pathway that leads to activation of transcription factor NF-kappaB. Here we explore the possibility that the NF-kappaB subunit p50 may be a target for the cyclopentenone 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)). This prostaglandin inhibited the DNA binding ability of recombinant p50 in a dose-dependent manner. The inhibition required the cyclopentenone moiety and could be prevented but not reverted by glutathione and dithiothreitol. Moreover, a p50 mutant with a C62S mutation was resistant to inhibition, indicating that the effect of 15d-PGJ(2) was probably due to its interaction with cysteine 62 in p50. The covalent modification of p50 by 15d-PGJ(2) was demonstrated by reverse-phase high pressure liquid chromatography and mass spectrometry analysis that showed an increase in retention time and in the molecular mass of 15d-PGJ(2)-treated p50, respectively. The interaction between p50 and 15d-PGJ(2) was relevant in intact cells. 15d-PGJ(2) effectively inhibited cytokine-elicited NF-kappaB activity in HeLa without reducing IkappaBalpha degradation or nuclear translocation of NF-kappaB subunits. 15d-PGJ(2) reduced NF-kappaB DNA binding activity in isolated nuclear extracts, suggesting a direct effect on NF-kappaB proteins. Finally, treatment of HeLa with biotinylated-15d-PGJ(2) resulted in the formation of a 15d-PGJ(2)-p50 adduct as demonstrated by neutravidin binding and immunoprecipitation. These results clearly show that p50 is a target for covalent modification by 15d-PGJ(2) that results in inhibition of DNA binding.

NMR investigations of protein-carbohydrate interactions: insights into the topology of the bound conformation of a lactose isomer and beta-galactosyl xyloses to mistletoe lectin and galectin-1.

A hallmark of oligosaccharides is their often limited spatial flexibility, allowing them to access a distinct set of conformers in solution. Viewing each individual or even the complete ensemble of conformations as potential binding partner(s) for lectins in protein-carbohydrate interactions, it is pertinent to address the question on the characteristics of bound state conformation(s) in solution. Also, it is possible that entering the lectin's binding site distorts the low-energy topology of a glycosidic linkage. As a step to delineate the strategy of ligand selection for galactosides, a common physiological docking point, we have performed a NMR study on two non-homologous lectins showing identical monosaccharide specificity. Thus, the conformation of lactose analogues bound to bovine heart galectin-1 and to mistletoe lectin in solution has been determined by transferred nuclear Overhauser effect measurements. It is demonstrated that the lectins select the syn conformation of lactose and various structural analogues (Galbeta(1-->4)Xyl, Galbeta(1-->3)Xyl, Galbeta(1-->2)Xyl, and Galbeta(1-->3)Glc) from the ensemble of presented conformations. No evidence for conformational distortion was obtained. Docking of the analogues to the modeled binding sites furnishes explanations, in structural terms, for exclusive recognition of the syn conformer despite the non-homologous design of the binding sites.

Differential mechanism-based labeling and unequivocal activity assignment of the two active sites of intestinal lactase/phlorizin hydrolase.

Milk lactose is hydrolysed to galactose and glucose in the small intestine of mammals by the lactase/phlorizin hydrolase complex (LPH; EC 3.2.1.108/62). The two enzymatic activities, lactase and phlorizin hydrolase, are located in the same polypeptide chain. According to sequence homology, mature LPH contains two different regions (III and IV), each of them homologous to family 1 glycosidases and each with a putative active site. There has been some discrepancy with regard to the assignment of enzymatic activity to the two active sites. Here we show differential reactivity of the two active sites with mechanism-based glycosidase inhibitors. When LPH is treated with 2',4'-dinitrophenyl 2-deoxy-2-fluoro-beta-D-glucopyranoside (1) and 2', 4'-dinitrophenyl-2-deoxy-2-fluoro-beta-D-galactopyranoside (2), known mechanism-based inhibitors of glycosidases, it is observed that compound 1 preferentially inactivates the phlorizin hydrolase activity whereas compound 2 is selective for the lactase active site. On the other hand, glycals (D-glucal and D-galactal) competitively inhibit lactase activity but not phlorizin hydrolase activity. This allows labeling of the phlorizin site with compound 1 by protection with a glycal. By differential labeling of each active site using 1 and 2 followed by proteolysis and MS analysis of the labeled fragments, we confirm that the phlorizin hydrolysis occurs mainly at the active site located at region III of LPH and that the active site located at region IV is responsible for the lactase activity. This assignment is coincident with that proposed from the results of recent active-site mutagenesis studies [Zecca, L., Mesonero, J.E., Stutz, A., Poiree, J.C., Giudicelli, J., Cursio, R., Gloor, S.M. & Semenza, G. (1998) FEBS Lett. 435, 225-228] and opposite to that based on data from early affinity labeling with conduritol B epoxide [Wacker, W., Keller, P., Falchetto, R., Legler, G. & Semenza, G. (1992) J. Biol. Chem. 267, 18744-18752].

Structural basis for chitin recognition by defense proteins: GlcNAc residues are bound in a multivalent fashion by extended binding sites in hevein domains.

BACKGROUND: Many plants respond to pathogenic attack by producing defense proteins that are capable of reversible binding to chitin, a polysaccharide present in the cell wall of fungi and the exoskeleton of insects. Most of these chitin-binding proteins include a common structural motif of 30 to 43 residues organized around a conserved four-disulfide core, known as the 'hevein domain' or 'chitin-binding' motif. Although a number of structural and thermodynamic studies on hevein-type domains have been reported, these studies do not clarify how chitin recognition is achieved. RESULTS: The specific interaction of hevein with several (GlcNAc)(n) oligomers has been studied using nuclear magnetic resonance (NMR), analytical ultracentrifugation and isothermal titration microcalorimetry (ITC). The data demonstrate that hevein binds (GlcNAc)(2-4) in 1:1 stoichiometry with millimolar affinity. In contrast, for (GlcNAc)(5), a significant increase in binding affinity is observed. Analytical ultracentrifugation studies on the hevein-(GlcNAc)(5,8) interaction allowed detection of protein-carbohydrate complexes with a ratio of 2:1 in solution. NMR structural studies on the hevein-(GlcNAc)(5) complex showed the existence of an extended binding site with at least five GlcNAc units directly involved in protein-sugar contacts. CONCLUSIONS: The first detailed structural model for the hevein-chitin complex is presented on the basis of the analysis of NMR data. The resulting model, in combination with ITC and analytical ultracentrifugation data, conclusively shows that recognition of chitin by hevein domains is a dynamic process, which is not exclusively restricted to the binding of the nonreducing end of the polymer as previously thought. This allows chitin to bind with high affinity to a variable number of protein molecules, depending on the polysaccharide chain length. The biological process is multivalent.

NMR investigations of protein-carbohydrate interactions binding studies and refined three-dimensional solution structure of the complex between the B domain of wheat germ agglutinin and N,N', N"-triacetylchitotriose.

The specific interaction of the isolated B domain of wheat germ agglutinin (WGA-B) with N,N',N"-triacetylchitotriose has been analyzed by 1H-NMR spectroscopy. The association constants for the binding of WGA-B to this trisaccharide have been determined from both 1H-NMR titration experiments and microcalorimetry methods. Entropy and enthalpy of binding have been obtained. The driving force for the binding process is provided by a negative DeltaH which is partially compensated by negative DeltaS. These negative signs indicate that hydrogen bonding and van der Waals forces are the major interactions stabilizing the complex. NOESY NMR experiments in water solution provided 327 protein proton-proton distance constraints. All the experimental constraints were used in a refinement protocol including restrained molecular dynamics in order to determine the refined solution conformation of this protein/carbohydrate complex. With regard to the NMR structure of the free protein, no important changes in the protein NOEs were observed, indicating that carbohydrate-induced conformational changes are small. The average backbone rmsd of the 35 refined structures was 1.05 A, while the heavy atom rmsd was 2.10 A. Focusing on the bound ligand, two different orientations of the trisaccharide within WGA-B binding site are possible. It can be deduced that both hydrogen bonds and van der Waals contacts confer stability to both complexes. A comparison of the three-dimensional structure of WGA-B in solution to that reported in the solid state and to those deduced for hevein and pseudohevein in solution has also been performed.

NMR investigations of protein-carbohydrate interactions: studies on the relevance of Trp/Tyr variations in lectin binding sites as deduced from titration microcalorimetry and NMR studies on hevein domains. Determination of the NMR structure of the complex between pseudohevein and N,N',N"-triacetylchitotriose.

Model studies on lectins and their interactions with carbohydrate ligands in solution are essential to gain insights into the driving forces for complex formation and to optimize programs for computer simulations. The specific interaction of pseudohevein with N,N', N"-triacetylchitotriose has been analyzed by (1)H-NMR spectroscopy. Because of its small size, with a chain length of 45 amino acids, this lectin is a prime target to solution-structure determination by NOESY NMR experiments in water. The NMR-analysis was extended to assessment of the topology of the complex between pseudohevein and N, N',N"-triacetylchitotriose. NOESY experiments in water solution provided 342 protein proton-proton distance constraints. Binding of the ligand did not affect the pattern of the protein nuclear Overhauser effect signal noticeably, what would otherwise be indicative of a ligand-induced conformational change. The average backbone (residues 3-41) RMSD of the 20 refined structures was 1.14 A, whereas the heavy atom RMSD was 2.18 A. Two different orientations of the trisaccharide within the pseudohevein binding site are suggested, furnishing an explanation in structural terms for the lectin's capacity to target chitin. In both cases, hydrogen bonds and van der Waals contacts confer stability to the complexes. This conclusion is corroborated by the thermodynamic parameters of binding determined by NMR and isothermal titration calorimetry. The association process was enthalpically driven. In relation to hevein, the Trp/Tyr-substitution in the binding pocket has only a small effect on the free energy of binding in contrast to engineered galectin-1 and a mammalian C-type lectin. A comparison of the three-dimensional structure of pseudohevein in solution to those reported for wheat germ agglutinin (WGA) in the solid state and for hevein and WGA-B in solution has been performed, providing a data source about structural variability of the hevein domains. The experimentally derived structures and the values of the solvent accessibilities for several key residues have also been compared with conformations obtained by molecular dynamics simulations, pointing to the necessity to further refine the programs to enhance their predictive reliability and, thus, underscoring the importance of this kind of combined analysis in model systems.

Generalized anomeric effect in action: synthesis and evaluation of stable reducing indolizidine glycomimetics as glycosidase inhibitors.

A series of aminoketalic castanospermine analogues incorporating a stereoelectronically anchored axial hydroxy group at the pseudoanomeric stereocenter (C-5) have been synthesized to satisfy the need for glucosidase inhibitors that are highly selective for alpha-glucosidases. The polyhydroxylated bicyclic system was built from readily available hexofuranose derivatives through a synthetic scheme that involved (i) the construction of a five-membered cyclic (thio)carbamate or (thio)urea moiety at the nonreducing end and (ii) the intramolecular nucleophilic addition of the heterocyclic thiocarbamic nitrogen atom to the masked aldehyde group of the monosaccharide. A biological screening of the resulting reducing 2-oxa- and 2-azaindolizidines against several glycosidase enzymes is reported.

Conformational differences between O- and C-glycosides: the alpha-O-man-(1-->1)-beta-Gal/alpha-C-Man-(1-->1)-beta-Gal case--a decisive demonstration of the importance of the exo-anomeric effect on the conformation of glycosides.

The conformational behavior of alpha-O-Man-(1-->1)-beta-Gal (1) and its C-analogue (2) has been studied using J/NOE NMR data, molecular mechanics, molecular dynamics, and ab initio calculations. The population distribution around the glycosidic linkages of 1 and 2 is rather different, especially for the alpha-Man linkage. A lower limit for the exo-anomeric effect in water has been experimentally determined.

Dietary flavonoid and isoflavone glycosides are hydrolysed by the lactase site of lactase phlorizin hydrolase.

Lactase phlorizin hydrolase (LPH; EC 3.2.1.62) is a membrane-bound, family 1 beta-glycosidase found on the brush border of the mammalian small intestine. LPH, purified from sheep small intestine, was capable of hydrolysing a range of flavonol and isoflavone glycosides. The catalytic efficiency (k(cat)/K(m)) for the hydrolysis of quercetin-4'-glucoside, quercetin-3-glucoside, genistein-7-glucoside and daidzein-7-glucoside was 170, 137, 77 and 14 (mM(-1) s(-1)) respectively. The majority of the activity occurred at the lactase and not phlorizin hydrolase site. The ability of LPH to deglycosylate dietary (iso)flavonoid glycosides suggests a possible role for this enzyme in the metabolism of these biologically active compounds.

A new combined computational and NMR-spectroscopical strategy for the identification of additional conformational constraints of the bound ligand in an aprotic solvent.

This study documents the feasibility of switching to an aprotic medium in sugar receptor research. The solvent change offers additional insights into mechanistic details of receptor--carbohydrate ligand interactions. If a receptor retained binding capacity in an aprotic medium, solvent-exchangeable protons of the ligand would not undergo transfer and could act as additional sensors, thus improving the level of reliability in conformational analysis. To probe this possibility, we first focused on hevein, the smallest lectin found in nature. The NMR-spectroscopic measurements verified complexation, albeit with progressively reduced affinity by more than 1.5 orders of magnitude, in mixtures of up to 50% dimethyl sulfoxide (DMSO). Since hevein lacks the compact beta-strand arrangement of other sugar receptors, such a structural motif may confer enhanced resistance to solvent exchange. Two settings of solid-phase activity assays proved this assumption for three types of alpha- and/or beta-galactoside-binding proteins, that is, a human immunoglobulin G (IgG) subfraction, the mistletoe lectin, and a member of the galectin family of animal lectins. Computer-assisted calculations and NMR experiments also revealed no conspicuous impact of the solvent on the conformational properties of the tested ligands. To define all possible nuclear Overhauser effect (NOE) contacts in a certain conformation and to predict involvement of exchangeable protons, we established a new screening protocol applicable during a given molecular dynamics (MD) trajectory and calculated population densities of distinct contacts. Experimentally, transferred NOE (tr-NOE) experiments with IgG molecules and the disaccharide Gal'alpha1-3Galbeta1-R in DMSO as solvent disclosed that such an additional crosspeak, that is, Gal'OH2--GalOH4, was even detectable for the bound ligand under conditions in which spin diffusion effects are suppressed. Further measurements with the plant lectin and galectins confirmed line broadening of ligand signals and gave access to characteristic crosspeaks in the aprotic solvent and its mixtures with water. Our combined biochemical, computational, and NMR-spectroscopical strategy is expected to contribute notably to the precise elucidation of the geometry of ligands bound to compactly folded sugar receptors and of the role of water molecules in protein--ligand (carbohydrate) recognition, with relevance to areas beyond the glycosciences.

Free and protein-bound carbohydrate structures.

Several areas of research in the study of the structure and dynamics of free and protein-bound carbohydrates have experienced considerable advances during the past year. These include the application of state-of-the-art NMR techniques using (13)C-labeled sugars to obtain conformational information, the full structural characterization of several saccharides that either form part of glycoproteins or form noncovalent complexes, both in solution and in the solid state, the description of several enzyme-carbohydrate complexes at the atomic level and last, but not least, the development and analysis of calculation protocols to predict the dynamical and conformational behavior of oligosaccharides.

NMR investigations of protein-carbohydrate interactions: refined three-dimensional structure of the complex between hevein and methyl beta-chitobioside.

The specific interaction of hevein with GlcNAc-containing oligosaccharides has been analyzed by1H-NMR spectroscopy. The association constants for the binding of hevein to a variety of ligands have been estimated from1H-NMR titration experiments. The association constants increase in the order GlcNAc-alpha(1-->6)-Man < GlcNAc < benzyl-beta-GlcNAc < p-nitrophenyl-beta-GlcNAc < chitobiose < p-nitrophenyl-beta-chitobioside < methyl-beta-chitobioside < chitotriose. Entropy and enthalpy of binding for different complexes have been obtained from van't Hoff analysis. The driving force for the binding process is provided by a negative DeltaH0which is partially compensated by negative DeltaS0. These negative signs indicate that hydrogen bonding and van der Waals forces are the major interactions stabilizing the complex. NOESY NMR experiments in water solution provided 475 accurate protein proton-proton distance constraints after employing the MARDIGRAS program. In addition, 15 unambiguous protein/carbohydrate NOEs were detected. All the experimental constraints were used in a refinement protocol including restrained molecular dynamics in order to determine the highly refined solution conformation of this protein-carbohydrate complex. With regard to the NMR structure of the free protein, no important changes in the protein nOe's were observed, indicating that carbohydrate-induced conformational changes are small. The average backbone rmsd of the 20 refined structures was 0.055 nm, while the heavy atom rmsd was 0.116 nm. It can be deduced that both hydrogen bonds and van der Waals contacts confer stability to the complex. A comparison of the three-dimensional structure of hevein in solution to those reported for wheat germ agglutinin (WGA) and hevein itself in the solid state has also been performed. The polypeptide conformation has also been compared to the NMR-derived structure of a smaller antifungical peptide, Ac-AMP2.

Analogs of farnesylcysteine induce apoptosis in HL-60 cells.

S-Farnesyl-thioacetic acid (FTA), a competitive inhibitor of isoprenylated protein methyltransferase, potently suppressed the growth of HL-60 cells and induced apoptosis, as evidenced by the development of increased annexin-V binding, decreased binding of DNA dyes and internucleosomal DNA degradation. FTA did not impair the membrane association of ras proteins, conversely, it brought about a decrease in the proportion of ras present in the cytosolic fraction. Farnesylated molecules which are weak inhibitors of the methyltransferase also induced DNA laddering and reduced the proportion of cytosolic ras. These findings suggest that neither inhibition of isoprenylated protein methylation nor impairment of ras membrane association are essential for apoptosis induced by farnesylcysteine analogs.

NMR studies of the conformation of thiocellobiose bound to a beta-glucosidase from Streptomyces sp.

The conformation of 4-thiocellobiose bound to beta-glucosidase from Streptomyces sp. has been studied by 1H-NMR transferred nuclear Overhauser effect spectroscopy (TR-NOE). Thiocellobiose behaves as an inhibitor of this glucosidase when cellobiose is used as substrate. NOE measurements and molecular mechanics calculations have also been performed to estimate the probability distribution of conformers of thiocellobiose when free in solution. Experimental data show that, in contrast with the natural O-analogue, thiocellobiose presents three conformational families in the free state, namely syn, anti-psi and anti-phi, whilst only one of them (syn) is recognized by the enzyme.

Regioselectivity of the enzymatic transgalactosidation of D- and L-xylose catalysed by beta-galactosidases.

The regioselectivity of enzymatic transgalactosidation depends on the source of the beta-galactosidase used. When the galactosyl acceptor only contains secondary hydroxyl groups, e.g., D- or L-xylose, it is possible to find an enzyme that catalyses preferentially the synthesis of any of the three regioisomers 4-, 3- and 2-O-beta-D-galactopyranosyl-D-xylose (1, 2 and 3, respectively) or 4-, 3- and 2-O-beta-D-galactopyranosyl-L-xylose (4, 5 and 6, respectively). Enriched mixtures in 1, 2 or 3 were obtained using beta-galactosidases from Escherichia coli, bovine testes or Aspergillus oryzae, respectively, by transgalactosidation reaction of O-nitrophenyl-beta-D-galactopyranoside and D-xylose, and enriched mixtures in 4, 5 or 6 were obtained in a similar way using beta-galactosidases from Aspergillus oryzae, lamb small-intestine (intestinal lactase-phloridzin hydrolase) or Saccharomyces fragilis, respectively, using L-xylose as acceptor.

A direct enzymatic synthesis of beta-D-galactopyranosyl-D-xylopyranosides and their use to evaluate rat intestinal lactase activity in vivo.

By enzymatic beta-D-galactosylation of D-xylose a mixture of 4-, 3-, and 2-O-beta-D-galactopyranosyl-D-xyloses (1, 4, and 7, respectively) was obtained in 50% isolated yield. Disaccharides 1, 4, and 7 are substrates of intestinal lactase isolated from lamb small intestine with K(m) values of 250.0, 4.5, and 14.0 mM, respectively. The mixture was used to monitor the normal decline in lactase activity in rats that takes place after weaning. The data obtained by this method correlated with the levels of intestinal lactase activity in the same animals after being sacrificed.

Role of tetrahydrobiopterin availability in the regulation of nitric-oxide synthase expression in human mesangial cells.

Human mesangial cells express an inducible form of nitric-oxide synthase (iNOS) after treatment with cytokines. Tetrahydrobiopterin (BH4), an essential cofactor for NOS, is required for cytokine-induced NO generation. We report here that BH4 is necessary not only for the activity but also for the expression of iNOS in human mesangial cells. Inhibition of de novo BH4 synthesis with 2,4-diamino-6-hydroxypyrimidine (DAHP) significantly attenuated iNOS activity as well as mRNA and protein expression in response to interleukin 1beta plus tumor necrosis factor alpha (IL-1beta/TNF-alpha). In contrast, sepiapterin, which provides BH4 through the pterin salvage pathway, strongly potentiated IL-1beta/TNF-alpha-induced iNOS expression and abrogated the inhibitory effect of DAHP. Inhibition of the pterin salvage pathway with methotrexate abolished sepiapterin potentiation of iNOS induction but did not alter the effect of IL-1beta/TNF-alpha. Determination of intracellular pteridines confirmed that sepiapterin markedly raised BH4 content, an effect that was blocked by methotrexate. These results suggest that BH4 availability plays an important role in the regulation of iNOS expression. The effect of BH4 appears to be mediated, at least in part, by an increase in mRNA stability, as indicated by the observation that DAHP shortened, whereas sepiapterin prolonged the half-life of IL-1beta/TNF-alpha-induced iNOS mRNA. Taken together, our results suggest that the biosynthesis of BH4 contributes to cytokine induction of iNOS expression in human mesangial cells through the stabilization of iNOS mRNA.

Studies of the bound conformations of methyl alpha-lactoside and methyl beta-allolactoside to ricin B chain using transferred NOE experiments in the laboratory and rotating frames, assisted by molecular mechanics and dynamics calculations.

The conformation in solution of methyl beta-galactopyranosyl-(1-->4)-alpha-glucopyranoside (methyl alpha-lactoside) and methyl beta-galactopyranosyl-(1-->6)-beta-glucopyranoside (methyl beta-allolactoside) has been studied through NMR spectroscopy and molecular mechanics calculations. NOE measurements both in the laboratory and rotating frames, have been interpreted in terms of an ensemble average distribution of conformers. Molecular mechanics calculations have been performed to estimate the probability distribution of conformers from the steric energy maps. The experimental results indicate that methyl alpha-lactoside spends about 90% of its time in a broad low-energy region close to the global minimum, while methyl beta-allolactoside presents much higher flexibility. The conformational changes that occur when both disaccharides are bound to the ricin B chain in aqueous solution have been studied using transferred NOE experiments at several protein/ligand ratios. The observed data indicate that the protein causes a conformational variation in the torsion angles of methyl alpha-lactoside changing towards smaller angle values (phi/psi approximately -20/-20), although the recognized conformer is still within the lowest energy region. In particular, the torsional changes separate Gal H1 from Glc H3 and Glc H6 protons, with a noticeable decrease in the intensities of the corresponding NOE cross-peaks, which were clearly observed for the free disaccharide. On the other hand, different conformations around the phi, psi, and omega glycosidic bonds of methyl beta-allolactoside are recognized by the lectin. In fact, for the methyl-beta-allolactoside-ricin-B complex, only the NOESY cross-peaks corresponding to the protons of the galactose residue are negative, as expected for a molecule in the slow motion regime. In contrast, the corresponding cross peaks for the glucose residue were about zero, as expected for a molecule whose motion is practically independent of the protein. However, for the methyl-alpha-lactoside-ricin-B complex, all the NOESY cross-peaks for both the galactose and glucose moieties were clearly negative. From the NMR experimental point of view, it is demonstrated that the comparison of longitudinal and transversal transferred NOEs allows one to clearly differentiate direct enhancements from spin diffusion effects, which are of major concern when analysing NOE spectra of macromolecules.(ABSTRACT TRUNCATED AT 250 WORDS)