The IL-4-inducing principle from Schistosoma mansoni eggs (IPSE/α-1), the major secretory product of eggs from the parasitic worm S. mansoni, efficiently triggers basophils to release the immunomodulatory key cytokine interleukin-4. Activation by IPSE/α-1 requires the presence of IgE on the basophils, but the detailed molecular mechanism underlying activation is unknown. NMR and crystallographic analysis of IPSEΔNLS, a monomeric IPSE/α-1 mutant, revealed that IPSE/α-1 is a new member of the βγ-crystallin superfamily. We demonstrate that this molecule is a general immunoglobulin-binding factor with highest affinity for IgE. NMR binding studies of IPSEΔNLS with the 180-kDa molecule IgE identified a large positively charged binding surface that includes a flexible loop, which is unique to the IPSE/α-1 crystallin fold. Mutational analysis of amino acids in the binding interface showed that residues contributing to IgE binding are important for IgE-dependent activation of basophils. As IPSE/α-1 is unable to cross-link IgE, we propose that this molecule, by taking advantage of its unique IgE-binding crystallin fold, activates basophils by a novel, cross-linking-independent mechanism.

Non-structural protein 9 (Nsp9) of coronaviruses is believed to bind single-stranded RNA in the viral replication complex. The crystal structure of Nsp9 of human coronavirus (HCoV) 229E reveals a novel disulfide-linked homodimer, which is very different from the previously reported Nsp9 dimer of SARS coronavirus. In contrast, the structure of the Cys69Ala mutant of HCoV-229E Nsp9 shows the same dimer organization as the SARS-CoV protein. In the crystal, the wild-type HCoV-229E protein forms a trimer of dimers, whereas the mutant and SARS-CoV Nsp9 are organized in rod-like polymers. Chemical cross-linking suggests similar modes of aggregation in solution. In zone-interference gel electrophoresis assays and surface plasmon resonance experiments, the HCoV-229E wild-type protein is found to bind oligonucleotides with relatively high affinity, whereas binding by the Cys69Ala and Cys69Ser mutants is observed only for the longest oligonucleotides. The corresponding mutations in SARS-CoV Nsp9 do not hamper nucleic acid binding. From the crystal structures, a model for single-stranded RNA binding by Nsp9 is deduced. We propose that both forms of the Nsp9 dimer are biologically relevant; the occurrence of the disulfide-bonded form may be correlated with oxidative stress induced in the host cell by the viral infection.

Here, we describe an activity assay for sialyltransferases based on surface plasmon resonance (SPR). Different natural and synthetic oligosaccharides serving as acceptor substrates for the sialyltransferase ST3Gal-III (EC 2.4.99.6) were immobilized or synthesized on SPR chips. The chip was then exposed to different concentrations of a reaction mixture of ST3Gal-III and CMP-Neu5Ac either by injection or by external application of the reaction mixture to the chip surface. The binding of two lectins, one that specifically recognizes the unmodified acceptor, the other the sialylated oligosaccharide, was utilized to determine the extent of enzymatic turnover. In order to obtain enzymatic activities, the SPR data were correlated to data obtained from a classical radio assay. After regeneration, that is, cleavage of the sialic acid residues by using a sialidase, the chip is available for new experiments. The technique allows the rapid determination of sialyltransferase activity with only nanomolar quantities of acceptor substrates and should be of particular value in cases in which a large variety of samples, including cell lysates, have to be screened for their enzymatic activities.

The tRNAPhe‐bound conformation of the aminoglycoside neamine, a member of the neomycin B family, has been investigated by transferred NOE experiments in aqueous solution. This is the first time that the bioactive conformation of an RNA‐bound aminoglycoside has been determined by this method. In buffers without divalent Mg2+ ions, a high degree of electrostatically driven unspecific binding of aminoglycosides to the RNA was observed. Careful optimization of experimental conditions yielded buffer conditions optimized for cryo‐probe NMR experiments. In particular, addition of Mg2+ ions to the solutions was necessary to reduce the amount of unspecific binding as monitored by one‐dimensional NMR and surface plasmon resonance experiments. CD spectroscopy was used to probe the effect of aminoglycosides and buffer conditions on the double helical content of tRNAPhe. Finally the tRNAPhe‐bound conformation of neamine was determined by trNOE build‐up curves and compared with the previously reported crystal structure of neomycin B complexed to this RNA. Although the aminoglycoside in the crystal structure contains several configurational errors, the overall shape of the crystallographically determined RNA‐bound structure is identical to the RNA‐bound conformation defined by the NMR experiments. Therefore, the crystal structure has been refined by trNOE data. This is particularly important in the context of aminoglycosides being discussed as lead structures for the development of new anti‐RNA drugs.

Galectin‐1 is a member of a protein family historically characterized by its ability to bind carbohydrates containing a terminal galactosyl residue. Galectin‐1 is found in a variety of mammalian tissues as a homodimer of 14.5‐kDa subunits. A number of developmental and regulatory processes have been attributed to the ability of galectin‐1 to bind a variety of oligosaccharides containing the Gal‐β‐(1,4)‐GlcNAc (LacNAcII) sequence. To probe the origin of this permissive binding, solvated molecular dynamics (MD) simulations of several representative galectin‐1‐ligand complexes have been performed. Simulations of structurally defined complexes have validated the computational approach and expanded upon data obtained from X‐ray crystallography and surface plasmon resonance measurements. The MD results indicate that a set of anchoring interactions between the galectin‐1 carbohydrate recognition domain (CRD) and the LacNAc core are maintained for a diverse set of ligands and that substituents at the nonreducing terminus of the oligosaccharide extend into the remainder of a characteristic surface groove. The anionic nature of ligands exhibiting relatively high affinities for galectin‐1 implicates electrostatic interactions in ligand selectivity, which is confirmed by a generalized Born analysis of the complexes. The results suggest that the search for a single endogenous ligand or function for this lectin may be inappropriate and instead support a more general role for galectin‐1, in which the lectin is able to crosslink heterogeneous oligosaccharides displayed on a variety of cell surfaces. Such binding promiscuity provides an explanation for the variety of adhesion phenomena mediated by galectin‐1. Proteins 2003. © 2003 Wiley‐Liss, Inc.

This chapter contains sections titled: Introduction Oligosaccharide Modeling: Background and Theory Force Fields Molecular Dynamics Simulation versus Monte Carlo Sampling Explicit Solvation: Water Models Boundary Conditions MD Simulation of Oligosaccharides NMR Spectroscopy of Oligosaccharides 1H-1H NOE Experiments on Oligosaccharides ROE Experiments Relaxation Matrix Approach One- and Two-Dimensional NOE Experiments Example Application I: Conformational Analysis of a Disaccharide Employing NOE Curves in Conjunction with Monte Carlo and Molecular Dynamics Simulations Example Application II: PBC Simulation of the Oligomannoside Man9GlcNAc2 Conclusions

The interaction of sialyl Lewis(x), Lewis(x), and alpha-L-Fuc-(1-->3)-beta-D-GlcNAc with isolectin A from Lotus tetragonolobus (LTL-A), and with Aleuria aurantia agglutinin (AAA) was studied using NMR experiments and surface plasmon resonance. Both lectins are specific for fucose residues. From NMR experiments it was concluded that alpha-L-Fuc-(1-->3)-beta-D-GlcNAc and Lewis(x) bound to both lectins, whereas sialyl Lewis(x) only bound to AAA. Increased line broadening of 1H NMR signals of the carbohydrate ligands upon binding to AAA and LTL-A suggested that AAA bound to the ligands more tightly. Further comparison of line widths showed that for both lectins binding strengths decreased from alpha-L-Fuc-(1-->3)-beta-D-GlcNAc to Lewis(x) and were lowest for sialyl Lewis(x). Surface plasmon resonance measurements were then employed to yield accurate dissociation constants. TrNOESY, QUIET-trNOESY, and trROESY experiments delivered bioactive conformations of the carbohydrate ligands, and STD NMR experiments allowed a precise epitope mapping of the carbohydrates bound to the lectins. The bioactive conformation of Lewis(x) bound to LTL-A, or AAA revealed an unusual orientation of the fucose residue, with negative values for both dihedral angles, phi and psi, at the alpha(1-->3)-glycosidic linkage. A similar distortion of the fucose orientation was also observed for sialyl Lewis(x) bound to AAA. From STD NMR experiments it followed that only the L-fucose residues are in intimate contact with the protein. Presumably steric interactions are responsible for locking the sialic acid residue of sialyl Lewis(x) in one out of many orientations that are present in aqueous solution. The sialic acid residue of sialyl Lewis(x) bound to AAA adopts an orientation similar to that in the corresponding sialyl Lewis(x)/E-selectin complex.

We report an improved synthesis of the T-antigen disaccharide [beta -D-Gal-(1 -->3)-alpha -D-GalNAc-OMe], incorporating recycling of the undesired beta -glycosyl acceptor [methyl 2-azido-4,6-benzylidene-2-deoxy-beta -D-galactopyranoside (9 beta)] through anomerization by treatment with FeCl3. The conformational analysis of the disaccharide made use of high quality NOE data in combination with extensive Metropolis Monte-Carlo (MMC) and molecular dynamic (MD) simulations. To sample the conformational space sufficiently, 9.5(.)10(6) Monte-Carlo steps were collected for the MMC simulations, while the fully solvated MD simulations were performed for 10 ns for comparison. In general, the MMC and MD simulations agreed very well. Comparison of theoretical NOE curves from both MMC and MD simulations with the experimental curves showed that the disaccharide populates two regions of conformational space, with a population of about 95% for the global minimum energy region and about 5% for a local minimum energy region.

The determination of the bound solution conformation of D-gluco-dihydroacarbose (GAC), a tight-binding inhibitor of several glycosidase and amylase enzymes, by glucoamylase is described. Transferred NOE NMR experiments and line-broadening effects indicate that GAC is bound in a conformation resembling that observed in the crystal structure. This contrasts with the predominant conformation of GAC when free in solution. The NMR results also suggest regions on the carbohydrate that are in close contact with the protein. The determination of the bound solution conformation of GAC by glucoamylase using transferred NOE (trNOE) measurements is a significant achievement given the high affinity constant (Ka = 3 x 10(7) M(-1)) for this receptor-ligand pair. It is striking that the off-rate for complexation is still sufficiently high to permit observation of trNOEs.

Die fortschreitende Miniaturisierung und die sich daraus ergebende Steigerung der Empfindlichkeit von Oberflächen-Plasmonen-Resonanz- und Resonanzspiegel-Biosensoren sind ein wesentlicher Grund dafür, dass diese immer häufiger zur Charakterisierung biomolekularer Wechselwirkungen eingesetzt werden (siehe schematische Darstellung). Das Einsatzspektrum dieser Biosensoren erweitert sich zusätzlich durch neue Entwicklungen wie den Aufbau artifizieller Membransysteme auf den Sensoroberflächen.

Ohne Vorkenntnisse der Identität der einzelnen Komponenten konnte ein bioaktiver Ligand aus einer Mischung aus 15 Kohlenhydraten NMR-spektroskopisch vollständig charakterisiert werden. Ein 3D-TOCSY-Transfer(tr)-NOESY-Experiment lieferte die entscheidende Information über den Typ der glycosidischen Verknüpfung des bioaktiven Disaccharids (siehe Schema).

The recognition of carbohydrates by proteins is an important element in many biological events like cell targeting, tumor invasion, immune response or bacterial and viral adhesion to host cells.1,2 Besides structural data obtained from X-ray crystallography or NMR spectroscopy, the analysis of carbohydrate protein interactions should also take into consideration other biophysical parameters such as dissociation constants or kinetic rate constants of the complex. However, these parameters are often difficult to determine due to the rather low affinity of the systems under investigation.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Bioaffinity NMR is based on the observation of transferred NOE (trNOE) effects which stem from bioactive components in a mixture containing a library of small compounds and a protein target. Zero-quantum coherence signals can interfere with the trNOE cross peaks and, in the worst case, prevent the detection of a bioactive compound in a library. The dephasing of these zero-quantum coherence signals during the experiment significantly improves the bioaffinity NMR spectra obtained and greatly reduces the risk of misinterpretations. Copyright (C) 2000 John Wiley & Sons, Ltd.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

The complex of Maclura pomifera agglutinin with the T-antigen disaccharide (beta-d-Gal-(1-->3)-alpha-d-GalNAc-(1-->O)-Me) was investigated by NMR spectroscopy in aqueous solution. Intramolecular transferred nuclear Overhauser enhancement (NOE) effects between the monosaccharide moieties were used to derive the ligand conformation in the lectin-bound state. Ligand protons in contact with the protein were identified by saturation transfer difference experiments and intermolecular transferred NOE effects. It is demonstrated that structural differences exist for the ligand-lectin complex in aqueous solution as compared with the previously published crystal structure (Lee, X., Thompson, A., Zhiming, Z., Ton-that, H., Biesterfeldt, J., Ogata, C., Xu, L., Johnston, R. A. Z. , and Young, N. M. (1998) J. Biol. Chem. 273, 6312-6318). In order to accommodate the O-methyl group of the disaccharide, the amino acid side chain of Tyr-122 has to rotate from its position in the crystal. The NMR data are in accord with two conformational families at the beta-(1-->3)glycosidic linkage in the solution complex with interglycosidic angles phi/psi = 45/-65 degrees and -65/-18 degrees. These differ from the bound conformation of the ligand in the crystal (phi/psi = 39/-8 degrees ) and are not highly populated by the ligand in the free state. The reason for the structural differences at the beta-(1-->3)glycosidic linkage are hydrogen bonds that stabilize the relative orientation of the monosaccharide units in the crystal. Our results demonstrate that the crystallization of a protein-carbohydrate complex can interfere with the delicate process of carbohydrate recognition in solution.

Transferred nuclear Overhauser effect (trNOE) experiments have been performed to investigate the conformations of the competitive inhibitors, methyl 5'-thio-4-N-alpha-maltoside 3a and methyl 5'-thio-4-S-alpha-maltoside 4 when bound to the catalytic subunit of the enzyme glucoamylase. These NMR data suggest that, although each of the free ligands populates two conformational families, both heteroanalogues are bound by the enzyme in conformations in the area of the global energy minimum. These conformations have been used as initial points for docking into the active site of the enzyme taken from a X-ray crystal structure of the related glucoamylase-D-gluco-dihydroacarbose 2 complex. Minimization of the resulting complexes has yielded structures for the bound complexes. Corroboration of the structures is provided by fast T(1)(rho)-relaxation effects for certain ligand protons as a result of close contacts with protons in the enzyme active site. The results auger well for the combined use of transferred NOE spectroscopy and molecular modeling based on X-ray crystal structures of complexes of suitable congeners for the rapid analysis of ligand-receptor interactions.

The conformational analysis of three maltoside heteroanalogues containing sulfur in the nonreducing ring and either oxygen 1, sulfur 2 or selenium 3 atoms in the interglycosidic linkage is performed using high-quality NOE data for 1-3 and molecular mechanics calculations using the program PIMM91 for the derivatives 1 and 2. The compounds are substrate analogues of glucosidases and inhibit glucoamylase 2. Theoretical NOE data, obtained from Boltzmann averaging of potential energy maps from a grid search, are compared with the experimental data. The gross conformational features of all three compounds are similar in that they populate mainly two conformational regions of the potential energy maps. These two regions are equivalent to the ones found for maltose and interconvert through a rotation of the dihedral angle Psi from similar to - 30 degrees to similar to 180 degrees. Experimental NOE data and theoretical energy differences and population distributions show that the substitution of oxygen with sulfur or selenium results in an increase in the flexibility of the interglycosidic linkage in the latter compounds. Thus, the population of the conformational family with a dihedral angle Psi of similar to 180 degrees increases from similar to 1% (1) to similar to 10% (2). (C) 1999 Elsevier Science Ltd. All rights reserved.

The lipopolysaccharide from Klebsiella oxytoca rough mutant R29 (O1(-)/K29(-)) has been isolated and its complete structure has been elucidated by compositional analyses, NMR spectroscopy, and laser-desorption mass spectrometry. The carbohydrate backbone has the structure[GRAPHICS]of which the GlcN residues (the lipid A backbone) are acylated by 14:(3-OH) (amide-linked) and 12:0, 14:0(3-OH)(ester-linked) fatty acids. (C) 1998 Elsevier Science Ltd. All rights reserved.