Francisco Javier Cañada

Carbohydrate-aromatic interactions.

The recognition of saccharides by proteins has far reaching implications in biology, technology, and drug design. Within the past two decades, researchers have directed considerable effort toward a detailed understanding of these processes. Early crystallographic studies revealed, not surprisingly, that hydrogen-bonding interactions are usually involved in carbohydrate recognition. But less expectedly, researchers observed that despite the highly hydrophilic character of most sugars, aromatic rings of the receptor often play an important role in carbohydrate recognition. With further research, scientists now accept that noncovalent interactions mediated by aromatic rings are pivotal to sugar binding. For example, aromatic residues often stack against the faces of sugar pyranose rings in complexes between proteins and carbohydrates. Such contacts typically involve two or three CH groups of the pyranoses and the π electron density of the aromatic ring (called CH/π bonds), and these interactions can exhibit a variety of geometries, with either parallel or nonparallel arrangements of the aromatic and sugar units. In this Account, we provide an overview of the structural and thermodynamic features of protein-carbohydrate interactions, theoretical and experimental efforts to understand stacking in these complexes, and the implications of this understanding for chemical biology. The interaction energy between different aromatic rings and simple monosaccharides based on quantum mechanical calculations in the gas phase ranges from 3 to 6 kcal/mol range. Experimental values measured in water are somewhat smaller, approximately 1.5 kcal/mol for each interaction between a monosaccharide and an aromatic ring. This difference illustrates the dependence of these intermolecular interactions on their context and shows that this stacking can be modulated by entropic and solvent effects. Despite their relatively modest influence on the stability of carbohydrate/protein complexes, the aromatic platforms play a major role in determining the specificity of the molecular recognition process. The recognition of carbohydrate/aromatic interactions has prompted further analysis of the properties that influence them. Using a variety of experimental and theoretical methods, researchers have worked to quantify carbohydrate/aromatic stacking and identify the features that stabilize these complexes. Researchers have used site-directed mutagenesis, organic synthesis, or both to incorporate modifications in the receptor or ligand and then quantitatively analyzed the structural and thermodynamic features of these interactions. Researchers have also synthesized and characterized artificial receptors and simple model systems, employing a reductionistic chemistry-based strategy. Finally, using quantum mechanics calculations, researchers have examined the magnitude of each propertys contribution to the interaction energy.

The cyclopentenone 15-deoxy-delta 12,14-prostaglandin J2 binds to and activates H-Ras

The cyclopentenone 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) induces cell proliferation and mitogen-activated protein kinase activation. Here, we describe that these effects are mediated by 15d-PGJ2-elicited H-Ras activation. We demonstrate that this pathway is specific for H-Ras through the formation of a covalent adduct of 15d-PGJ2 with Cys-184 of H-Ras, but not with N-Ras or K-Ras. Mutation of C184 inhibited H-Ras modification and activation by 15d-PGJ2, whereas serum-elicited stimulation was not affected. These results describe a mechanism for the activation of the Ras signaling pathway, which results from the chemical modification of H-Ras by formation of a covalent adduct with cyclopentenone prostaglandins.

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.

Aromatic–Carbohydrate Interactions: An NMR and Computational Study of Model Systems

The interactions of simple carbohydrates with aromatic moieties have been investigated experimentally by NMR spectroscopy. The analysis of the changes in the chemical shifts of the sugar proton signals induced upon addition of aromatic entities has been interpreted in terms of interaction geometries. Phenol and aromatic amino acids (phenylalanine, tyrosine, tryptophan) have been used. The observed sugar-aromatic interactions depend on the chemical nature of the sugar, and thus on the stereochemistries of the different carbon atoms, and also on the solvent. A preliminary study of the solvation state of a model monosaccharide (methyl beta-galactopyranoside) in aqueous solution, both alone and in the presence of benzene and phenol, has also been carried out by monitoring of intermolecular homonuclear solvent-sugar and aromatic-sugar NOEs. These experimental results have been compared with those obtained by density functional theory methods and molecular mechanics calculations.

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 1H‐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 Å, whereas the heavy atom RMSD was 2.18 Å. Two different orientations of the trisaccharide within the pseudohevein binding site are suggested, furnishing an explanation in structural terms for the lectins 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. Proteins 2000;40:218–236.

The Interaction of Hevein with N-acetylglucosamine-containing Oligosaccharides. Solution Structure of Hevein Complexed to Chitobiose

The three-dimensional structure of hevein, a small protein isolated from the latex of Hevea brasiliensis (rubber tree), in water solution has been obtained by using 1H-NMR spectroscopy and dynamic simulated annealing calculations. The average root-mean-square deviation (rmsd) of the best 20 refined structures generated using DIANA prior to simulated annealing was 0.092 nm for the backbone atoms and 0.163 nm for all heavy atoms (residues 3-41). The specific interaction of hevein with N-acetylglucosamine-containing oligosaccharides has also been analyzed by 1H-NMR. The association constants, Ka, for the binding of hevein to GlcNAc, chitobiose [GlcNAc-beta(1-->4)-GlcNAc], chitotriose [GlcNAc-beta(1-->4)-GlcNAc-beta(1-->4)-GlcNAc], and GlcNAc-alpha(1-->6)-Man have been estimated from 1H-NMR titration experiments. Since the measured Ka values for chitobiose binding are almost identical with and without calcium ions, it is shown that these cations are not required for sugar binding. The association increases in the order GlcNAc-alpha(1-->6)-Man 6)-Man can be explained by favourable stacking of the second beta-linked GlcNAc moiety and Trp21.

NMR and Modeling Studies of Protein–Carbohydrate Interactions: Synthesis, Three-Dimensional Structure, and Recognition Properties of a Minimum Hevein Domain with Binding Affinity for Chitooligosaccharides

HEV32, a 32‐residue, truncated hevein lacking eleven C‐terminal amino acids, was synthesized by solid‐phase methodology and correctly folded with three cysteine bridge pairs. The affinities of HEV32 for small chitin fragments—in the forms of N,N′,N′′‐triacetylchitotriose ((GlcNAc)3) (millimolar) and N,N′,N′′,N′′′,N′′′′,N′′′′′‐hexaacetylchitohexaose ((GlcNAc)6) (micromolar)—as measured by NMR and fluorescence methods, are comparable with those of native hevein. The HEV32 ligand‐binding process is enthalpy driven, while entropy opposes binding. The NMR structure of ligand‐bound HEV32 in aqueous solution was determined to be highly similar to the NMR structure of ligand‐bound hevein. Solvated molecular‐dynamics simulations were performed in order to monitor the changes in side‐chain conformation of the binding site of HEV32 and hevein upon interaction with ligands. The calculations suggest that the Trp21 side‐chain orientation of HEV32 in the free form differs from that in the bound state; this agrees with fluorescence and thermodynamic data. HEV32 provides a simple molecular model for studying protein–carbohydrate interactions and for understanding the physiological relevance of small native hevein domains lacking C‐terminal residues.

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 β‐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 α‐ and/or β‐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′α1‐3Galβ1‐R in DMSO as solvent disclosed that such an additional crosspeak, that is, Gal′ OH2–Gal OH4, 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.

α‐O‐Linked Glycopeptide Mimetics: Synthesis, Conformation Analysis, and Interactions with Viscumin, a Galactoside‐Binding Model Lectin

Efficient cycloaddition of a silylidene-protected galactal with a suitable heterodiene yielded the basis for a facile diastereoselective route to a glycopeptide-mimetic scaffold. Its carbohydrate part was further extended by beta1-3-linked galactosylation. The pyranose rings retain their (4)C(1) chair conformation, as shown by molecular modeling and NMR spectroscopy, and the typical exo-anomeric geometry was observed for the disaccharide. The expected bioactivity was ascertained by saturation-transfer-difference NMR spectroscopy by using the galactoside-specific plant toxin viscumin as a model lectin. The experimental part was complemented by molecular docking. The described synthetic route and the strategic combination of computational and experimental techniques to reveal conformational properties and bioactivity establish the prepared alpha-O-linked glycopeptide mimetics as promising candidates for further exploitation of this scaffold to give O-glycans for lectin blocking and vaccination.

Hevein Domains: An Attractive Model to Study Carbohydrate–Protein Interactions at Atomic Resolution

Publisher Summary This chapter focuses on hevein domains and presents an attractive model to study carbohydrate–protein interactions at atomic resolution. Among the various biological processes in which carbohydrates are involved as biochemical signals, it is noteworthy that many plants harbor defense proteins (lectins) against pathogenic attack. These proteins are able to bind to chitin. This natural biopolymer is a key structural component of the cell wall of fungi and of the exoskeleton of invertebrates such as insects, nematodes, and arthropods. Direct binding to the saccharide can occur for the respective lectin, while a particular domain can also be instrumental for chitin-degrading enzymes. The antifungal activity of plant chitinases is largely restricted to those chitinases that contain a noncatalytic, plant-specific, chitin-binding domain (ChBD), also termed as “hevein domain.” This domain displays a common structural motif of 30–43 residues, rich in glycine and cysteine residues in highly conserved positions and organized around a four-disulfide core. The chapter explains the concepts related to protein–carbohydrate interactions and elaborates the basic techniques for analyzing sugar–hevein interactions. It also discusses the structure of the Hevein–Saccharide complexes.