The specific interaction of the isolated B domain of wheat germ agglutinin (WGA-B) with N, N H , N HH-triacetylchitotriose has been analyzed by 1 H-NMR spectroscopy. The association constants for the binding of WGA-B to this trisaccharide have been determined from both 1 H-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 DH which is partially compensated by negative DS. 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 Ê , while the heavy atom rmsd was 2.10 A Ê. 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. Carbohydrates are one of the most extended families of biomolecules in nature. They play a role in energy storage and as constituents of the structural framework of cells and tissues. In addition, due to their extraordinary capacity to encode information stereochemically these molecules take part in a wide variety of recognition processes of biological significance. Thus, carbohydrate recognition by proteins has been shown to be involved in viral and microbial infection, inflammatory responses, innate immunity, fertilization, tumor spread and growth regulation [1±6]. The elucidation of the biochemical and cell biological processes in the cascades from the initial molecular rendezvous to the triggered response has established a burgeoning research field in glycoscience with obvious perspectives for medical application [7±9]. Detailed information on the three-dimensional structure of protein-carbohydrate complexes has frequently been obtained from X-ray crystallography data [10±14] and modeling [15], as the commonly high molecular mass of lectins has prevented their direct studies by means of NMR spectroscopy. However, in favorable cases, NMR may also provide information about the driving forces behind protein±carbohydrate interactions in solution [16±20]. The hevein domain is one of the most common chitin-binding motifs. Its presence in several lectins [such as hevein, pseudohevein, Urtica dioica agglutinin (UD...

The three-dimensional structure of hevein, a small protein isolated from the latex of Heveu brusiliensis (rubber tree), in water solution has been obtained by using 'H-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 'H-NMR. The association constants, Ka, for the binding of hevein to GlcNAc, chitobiose [GlcNAc-, and GlcNAc-a( 1+6)-Man have been estimated from 'H-NMR titration experiments.Since the measured K, 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-a(1+6)-Man GlcNAc < chitobiose < chitotriose. The equilibrium thermodynamic parameters entropy and enthalpy of binding, AS' and AHU, for the hevein-chitobiose and hevein-chitotriose associations have been obtained from van't Hoff analysis of the temperature dependence of the K, values between 25-40°C. The driving force for the binding process is provided by a negative AH0 which is partially compensated by a negative AS'. These negative signs seem to indicate that hydrogen bonding and van der Waals forces are the major interactions stabilizing the complex. Protein-carbohydrate nuclear Overhauser enhancements have allowed a three-dimensional model of the hevein-chitobiose complex to be built. From inspection of this model, a hydrogen bond between Serl9 and the non-reducing N-acetyl carbonyl group is suggested, as well as between Tyr3O and HO-3 of the same sugar residue. The N-acetyl methyl group of the non-reducing GlcNAc displays non-polar contacts to the aromatic Tyr30 and Trp21 residues. In addition, the higher affinities deduced for the P-linked oligosaccharides with respect to GlcNAc and GlcNAc-a( 1+6)-Man can be explained by favourable stacking of the second Blinked GlcNAc moiety and TrpZl.

The first high-resolution solution-state structure of a member of the toxin-agglutinin folding motif with the WGA disulfide linkage is presented. The 1H NMR spectrum of hevein has been 100% assigned from residue 2 through residue 43, the C-terminus, using two-dimensional correlation and NOE spectroscopy. During the course of the NOESY analysis, the three-dimensional structural features of hevein were derived, using nonstereospecific distance constraints (with tight bounds) for XPLOR simulated annealing followed by unconstrained relaxation in the CHARMm force field, at two levels of long-range constraint density. In addition, a large number of low-bound-only constraints, corresponding to unobserved NOE's, were used in both refinements. The first structure elucidation employed a total of 180 distance constraints (60 of which were medium or long range, i/i+n with n < or = 2). The second refinement employed 244 (101 medium or long range) constraints: some conformation-insensitive intraresidue constraints were deleted, two misassigned long-range constraints were corrected, and 41 new i/i+n (n > or = 2) constraints were added. The average bounds precisions of the two refinements were comparable (+/- 0.44 A) and significantly tighter than those that result when a universal low bound corresponding to the sum of the van der Waals radii was used. (The more conservative treatment of NOE's gave the same final structure but required a higher constraint density before assignment errors would stand out during the refinement.) Constraint density also has a significant influence on convergence and accuracy using tight constraints. The study demonstrates that convergence within an ensemble of solution structures is not a dependable criterion for either the accuracy or precision of the derived structure. The best fitting conformers from the refinement at the higher constraint density bear a greater similarity to the solid-state structure of the domains of wheat germ agglutinin (0.95 A rmsd over residues 2-32) than to the recently reported 2.8-A X-ray structure of hevein (1.25 A rmsd over residues 2-32, 2.83 A rmsd over residues 2-42). The consensus conformer from the solution data is defined to a backbone rmsd of < 0.6 A over the full sequence for which NMR data could be collected.(ABSTRACT TRUNCATED AT 400 WORDS)

Giardiasis, the most prevalent intestinal parasitosis in humans, is caused by Giardia lamblia. Current drug therapies have adverse effects on the host, and resistant strains against these drugs have been reported, demonstrating an urgent need to design more specific antigiardiasic drugs. ATP production in G. lamblia depends mainly on glycolysis; therefore, all enzymes of this pathway have been proposed as potential drug targets. We previously demonstrated that the glycolytic enzyme triosephosphate isomerase from G. lamblia (GlTIM), could be completely inactivated by low micromolar concentrations of thiol-reactive compounds, whereas, in the same conditions, the activity of human TIM (HuTIM) was almost unaltered. We found that the chemical modification (derivatization) of at least one Cys, of the five Cys residues per monomer in GlTIM, causes this inactivation. In this study, structural and functional studies were performed to describe the molecular mechanism of GlTIM inactivation by thiol-reactive compounds. We found that the Cys222 derivatization is responsible for GlTIM inactivation; this information is relevant because HuTIM has a Cys residue in an equivalent position (Cys217). GlTIM inactivation is associated with a decrease in ligand affinity, which affects the entropic component of ligand binding. In summary, this work describes a mechanism of inactivation that has not been previously reported for TIMs from other parasites and furthermore, we show that the difference in reactivity between the Cys222 in GlTIM and the Cys217 in HuTIM, indicates that the surrounding environment of each Cys residue has unique structural differences that can be exploited to design specific antigiardiasic drugs.

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