Thomas Weimar

Molecular Dynamics Simulations of Galectin-1-oligosaccharide Complexes Reveal the Molecular Basis for Ligand Diversity

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.

Variable Oligomerization Modes in Coronavirus Non-Structural Protein 9.

Abstract 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.

A Crystallin Fold in the Interleukin-4-inducing Principle of Schistosoma mansoni Eggs (IPSE/α-1) Mediates IgE Binding for Antigen-independent Basophil Activation

Background: The interleukin-4-inducing principle from Schistosoma mansoni eggs (IPSE/α-1) triggers basophils to release interleukin-4 and interleukin-13 in an IgE-dependent but antigen-independent way. Results: Structural analysis identified IPSE/α-1 as a new member of the βγ-crystallin superfamily with a unique IgE-binding loop. Conclusion: IPSE/α-1 activates basophils via IgE-binding crystallin folds. Significance: Schistosomes use unique mechanisms to manipulate the hosts immune response. 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.

The RNA-Bound Conformation of Neamine as Determined by Transferred NOE Experiments

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.

Low Affinity Carbohydrate Lectin Interactions Examined with Surface Plasmon Resonance

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.

Assaying sialyltransferase activity with surface plasmon resonance

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.

Combined NMR, grid search/MM3 and Metropolis Monte Carlo/GEGOP studies of two l-fucose containing disaccharides: α-l-Fuc-(1,4)-β-d-GlcNAc-OMe and α-l-Fuc-(1,6)-β-d-GlcNAc-OMe

Abstract Complete proton NMR data provided a firm experimental basis to infer the conformational properties of the Lewis type disaccharide α- l -Fuc-(1,4)-β- d -GlcNAc-OMe 1 and the N -glycoprotein type disaccharide α- l -Fuc-(1,6)-β- d -GlcNAc-OMe 2 in aqueous solution. Relaxed potential energy maps from systematic grid searches (GS) using the MM3 force field and Metropolis Monte Carlo (MMC) simulations employing the GEGOP force field were used to calculate corresponding ensemble average NMR data such as 1D transient NOE curves and vicinal coupling constants J ( H , H ). For the disaccharide with a flexible (1,6) linkage ( 2 ), the relaxed potential energy surface based on the MM3 force field was calculated with three variable dihedral angles, φ, ψ and ω. R -factors derived from a comparison of experimental and theoretical NOE data allowed an evaluation of the quality of the conformational models derived from the calculations. Seven inter glycosidic 1D transient NOE curves were measured for each of both disaccharides, 1 and 2 . Overall R -factors of approximately 17% for the 1–4 linked disaccharide and 20% for the 1–6 linked disaccharide indicate a very satisfying agreement between theoretical calculations, both GS/MM3 and MMC/GEGOP, and experimental NOE data, indicating that the gross conformational picture developed is realistic. On the other hand, a close inspection of individual NOE curves and vicinal coupling constants 3 J ( H 5, H 6-pro-R) and 3 J ( H 5, H 6-pro-S) with corresponding theoretical values revealed shortcomings of the computational methods applied. Firstly, the conformational equilibrium around the C5C6 bond of the GlcNAc unit in disaccharide 1 is not correctly described by the GS/MM3 method. This can lead to a false interpretation of NOE data involving protons attached to C6 of GlcNAc. Secondly, relaxation of ring geometry (GS/MM3) was found to have a measurable improvement of intra glycosidic NOEs in both disaccharides, 1 and 2 . In summary, the conformational models derived for disaccharides 1 and 2 represent a starting point for further analysis of potential conformational changes that may occur upon binding of these compounds to specific receptor proteins such as lectins or antibodies.