Andrew J. Benie

NMR Analysis of Carbohydrate-Protein Interactions

Carbohydrate-protein interactions are frequently characterized by dissociation constants in the microM to mM range. This is normally associated with fast dissociation rates of the corresponding complexes, in turn leading to fast exchange on the nuclear magnetic resonance (NMR) chemical shift time scale and on the NMR relaxation time scale. Therefore, NMR experiments that take advantage of fast exchange are well suited to study carbohydrate-protein interactions. In general, it is possible to analyze ligand binding by observing either protein signals or ligand resonances. Because most receptor proteins to which carbohydrates bind are rather large with molecular weights significantly exceeding 30 kDa, the analysis of the corresponding protein spectra is not trivial, and only very few studies have been addressing this issue so far. We, therefore, focus on NMR experiments that employ observation of free ligand, that is, carbohydrate signals to analyze the bound state. Two types of NMR experiments have been extremely valuable to analyze carbohydrate-protein interactions at atomic resolution. Whereas transferred nuclear Overhauser effect (NOE) experiments deliver bioactive conformations of carbohydrates binding to proteins, saturation transfer difference (STD) NMR spectra provide binding epitopes and valuable information about the binding thermodynamics and kinetics. We demonstrate the power of a combined transfer NOE/STD NMR approach for the analysis of carbohydrate-protein complexes using selected examples.

Fragment-based screening of the donor substrate specificity of human blood group B galactosyltransferase using saturation transfer difference NMR.

Saturation transfer difference NMR experiments on human blood group B α-(1,3)-galactosyltransferase (GTB) for the first time provide a comprehensive set of binding epitopes of donor substrate analogs in relation to the natural donor UDP-Gal. This study revealed that the enzyme binds several UDP-activated sugars, including UDP-Glc, UDP-GlcNAc, and UDP-GalNAc. In all cases, UDP is the dominant binding epitope. To identify the minimum requirements for specific binding, a detailed analysis utilizing a fragment-based approach was employed. The binding of donor substrate to GTB is essentially controlled by the base as a “molecular anchor.” Uracil represents the smallest fragment that is recognized, whereas CDP, AMP, and GDP do not exhibit any significant binding affinity for the enzyme. The ribose and β-phosphate moieties increase the affinity of the ligands, whereas the pyranose sugar apparently weakens the binding, although this part of the molecule controls the specificity of the enzyme. Accordingly, UDP represents the best binder. The binding affinities of UDP-Gal, UDP-Glc, and UMP are about the same, but lower than that of UDP. Furthermore, we observed that β-d-galactose and α-d-galactose bind weakly to GTB. Whereas β-d-galactose binds to the acceptor and donor sites, it is suggested that α-d-galactose occupies a third hitherto unknown binding pocket. Finally, our experiments revealed that modulation of enzymatic activity by metal ions critically depends on the total enzyme concentration, raising the question as to which of the bivalent metal cations Mg2+ and Mn2+ is more relevant under physiological conditions.

Consistent Bioactive Conformation of the Neu5Acα(2→3)Gal Epitope Upon Lectin Binding

The injured adult mammalian central nervous system has no capacity for axon regeneration, predominantly due to specific inhibitors expressed on residual myelin and on astrocytes recruited to the injury site. Several of these inhibitory proteins have been identified, including the myelin-associated glycoprotein (MAG). 8] MAG is a transmembrane glycoprotein that belongs to a family of sialic acid-binding immunoglobulin-like lectins, the so-called siglecs. There are two classes of welldefined axonal targets of MAG on the surface of neurons: sialylated glycans, specifically the gangliosides GD1a and GT1b, 13] and proteins of the NgR family. 15] Although the relative roles of gangliosides and NgRs as MAG ligands have yet to be resolved, in some systems MAG inhibition is completely reversed by sialidase treatment, suggesting that MAG uses sialylated glycans as its major axonal ligands. Therefore, potent glycan inhibitors of MAG may be a valuable therapeutic approach to enhance axon regeneration. The native carbohydrate ligand with the highest affinity to MAG is the ganglioside GQ1ba. As a starting point for our search for MAG antagonists, data about the minimal binding epitope of 1 and its bioactive conformation are required, since the most abundant solution conformation does not necessarily represent the bound conformation. Recently, the MAG-affinity of partial structures of GQ1ba (1), namely derivatives of tetrasaccharide 2 and trisaccharide 5, was clearly correlated with their ability to reverse MAG-mediated inhibition of axon outgrowth (Scheme 1). 19] Both saccharides 2 and 5 contain a flexible aACHTUNGTRENNUNG(2!3)-glycosidic linkage between the sialic acid (Neu5Ac) and the central galactose (Gal) residue, and it is unknown which of the solution conformations is recognized by the receptor protein. The objective of this study is to analyze the conformation of these aACHTUNGTRENNUNG(2!3)-glycosidic linkages when bound to MAG. It is known that conformational preorganization of ligands may significantly improve binding affinities, and this information is crucial for the design of potent antagonists. A prominent example is conformationally preorganized E-selectin antagonists based on the bioactive conformation of sialyl Lewis. 21] In general, carbohydrate–protein interactions are characterized by exchange reactions that are fast on the NMR chemical shift and relaxation timescales. Therefore, transferred NOE (trNOE) experiments are ideally suited for the analysis of bioactive conformations of protein-bound carbohydrates. Flexible glycosidic linkages have drawn special attention since different bound conformations are possible. Here, we analyze the bioactive conformations of Neu5AcaACHTUNGTRENNUNG(2!3)GalbACHTUNGTRENNUNG(1!3) ACHTUNGTRENNUNG[Neu5Ac a ACHTUNGTRENNUNG(2! 6)]GalNAc (2), Neu5Aca ACHTUNGTRENNUNG(2!3)Galb ACHTUNGTRENNUNG(1!3) ACHTUNGTRENNUNG[Neu5AcaACHTUNGTRENNUNG(2!6)]Gal (3), Neu5AcaACHTUNGTRENNUNG(2!3)GalbACHTUNGTRENNUNG(1!3)GalNAc (5) and GalbACHTUNGTRENNUNG(1!3) [Neu5Aca ACHTUNGTRENNUNG(2!6)]Gal (6), as well as the tetrasaccharide mimic 4 when bound to MAG (Scheme 1; for the syntheses of 2, 3 and 4 see the Supporting Information). This study complements an accompanying paper that describes the analysis of the binding epitopes of trisaccharide 5 and tetrasaccharide 2 when bound to MAG using saturation transfer difference (STD) NMR. From the change in the pattern of specific interglycosidic NOEs between tetrasaccharide 2 free in solution (Figures 1 A and C) and in the bound form (Figures 1 B and D, namely the disappearance of the NOEs H3’’–H3ax’’’ and H3’’–H3eq’’’), it is concluded that the terminal aACHTUNGTRENNUNG(2!3)-linked Neu5Ac residue is bound in a minus-gauche orientation, as it has been observed before for sialyl Lewis binding to E-selectin. The thorough quantitative analysis of the bioactive conformations of the structurally related ganglioside derivatives 2 to 5 (see the Supporting Information) reveals that the “sialyl Lewis-type binding mode” is a common theme among the carbohydrate-protein interactions studied (Table 1, see also Table S7). Interestingly, binding of trisaccharide 6 was too weak to give sizeable trNOEs (Supporting Information), supporting the assertion that an aACHTUNGTRENNUNG(2!3)-linked Neu5Ac residue is absolutely required for MAG binding. 29] Based on a full relaxation and exchange matrix analysis of the trNOE data employing the program CORCEMA, 31] we deduced a docking model (Supporting Information) for the interaction of the ganglioside derivatives with MAG. A complete set of NOESY spectra of oligosaccharides 2 to 5 in the absence and presence of MAG is found in the Supporting Information (Figures S1 and S2). As an example, Figure 1 [a] Dr. O. Schwardt, Dr. G.-P. Gao, Prof. Dr. B. Ernst Institute of Molecular Pharmacy, University of Basel Klingelbergstrasse 50, 4056 Basel (Switzerland) Fax: (+ 41) 61-267-15-52 E-mail : beat.ernst@unibas.ch [b] Dr. A. Bhunia, Dr. A. J. Benie, Prof. Dr. T. Peters Institute of Chemistry, University of Luebeck Ratzeburger Allee 160, 23538 Luebeck (Germany) [c] Dr. M. Hricovini Institute of Chemistry, Slovak Academy of Science 84238 Bratislava (Slovakia) [d] H. G thje, Prof. Dr. S. Kelm Department of Physiological Biochemistry, University of Bremen 28334 Bremen (Germany) Supporting information for this article is available on the WWW under http ://www.chembiochem.org or from the author: inter alia : full relaxation matrix calculations and docking procedures.

Characterization of ligand binding to N-acetylglucosamine kinase studied by STD NMR.

Saturation transfer difference (STD) NMR experiments on human N-acetylglucosamine kinase (GlcNAc kinase) have been used to determine binding epitopes for the GlcNAc and ATP substrates and their analogues. The study reveals that during the enzyme reaction the binding mode of both substrates is conserved, although the binding affinity of the sugar is reduced. This suggests that the protein does not undergo any significant structural changes during catalysis. Our experiments also demonstrate that GlcNAc kinase has residual activity in the absence of Mg(2+). Furthermore, our experiments clearly show that the GlcNAc kinase predominately, if not exclusively, produces the beta anomer of phosphorylated sugars. To identify the minimum requirements for substrate binding, a detailed analysis of different natural occurring as well as synthetic sugars was employed. Modifications at the 1, 2, 3, 4 and 6 position as well as the N-acetyl group greatly reduce the binding affinity. In addition, the binding mode of these substrate analogues is often also changed. The high beta anomeric preference of GlcNAc kinase along with the drastically reduced binding affinity for sugars other than GlcNAc, suggests that GlcNAc kinase phosphorylates beta-GlcNAc in cells.

Functional binding of hexanucleotides to 3C protease of hepatitis A virus

Oligonucleotides as short as 6 nt in length have been shown to bind specifically and tightly to proteins and affect their biological function. Yet, sparse structural data are available for corresponding complexes. Employing a recently developed hexanucleotide array, we identified hexadeoxyribonucleotides that bind specifically to the 3C protease of hepatitis A virus (HAV 3Cpro). Inhibition assays in vitro identified the hexanucleotide 5′-GGGGGT-3′ (G5T) as a 3Cpro protease inhibitor. Using 1H NMR spectroscopy, G5T was found to form a G-quadruplex, which might be considered as a minimal aptamer. With the help of 1H, 15N-HSQC experiments the binding site for G5T was located to the C-terminal β-barrel of HAV 3Cpro. Importantly, the highly conserved KFRDI motif, which has previously been identified as putative viral RNA binding site, is not part of the G5T-binding site, nor does G5T interfere with the binding of viral RNA. Our findings demonstrate that sequence-specific nucleic acid–protein interactions occur with oligonucleotides as small as hexanucleotides and suggest that these compounds may be of pharmaceutical relevance.