NMR Investigation of the Bound Conformation of Natural and Synthetic Oligomannosides to Banana Lectin

Clavel, Caroline; Canales, Ángeles; Gupta, Garima; Cañada, F. Javier; Penadés, Soledad; Surolia, Avadhesha; Jiménez‐Barbero, Jesús

doi:10.1002/ejoc.200600897

Cited by 3 publications

(8 citation statements)

References 51 publications

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“…Here, binding of transition-state probes is linked to energy gains, resulting from binding of high-energy conformers on a transition-state itinerary of hydrolytic enzymes, where oxocarbonium ion-like-shaped intermediates have been suggested to play key roles (11). These events have been investigated predominantly by X-ray diffraction (e.g., refs 15 and (5052)), although STD NMR techniques have contributed significantly (27,28,53). Against this background, the goal of our study was to shed light on the structural basis of binding of S-and O-linked glucoand manno-configured aryl-β-D-glycosides to the Os3BGlu7 and HvBII enzymes that preferentially hydrolyze β-D-glucoside and β-D-mannoside substrates, respectively (3)(4)(5)(6)(7).…”

Section: Discussionmentioning

confidence: 99%

“…The trNOESY experiments with HvBII were performed with freshly prepared mixtures containing HvBII and 4NP-S-Glc or 4NP-S-Man inhibitors, at approximately 30:1 molar ratios, in 20 mM phosphate buffer (pH 5.0) (near the pH optimum), with mixing times of 50, 100, 150, and 200 ms (28), resulting in final thio inhibitor concentrations of approximately 2-3 mM. No purging spin-lock period was employed to remove the NMR signals arising from the HvBII background, because typically the background signals are not observed with large proteins such as HvBII.…”

Section: Methodsmentioning

confidence: 99%

“…First, line broadening of the inhibitor protons was monitored after the addition of the enzyme. The theoretical analysis of the trNOEs of 4NP-S-Glc or 4NP-S-Man protons was performed using Complete Relaxation and Conformational Exchange Matrix (CORCEMA), and a relaxation matrix with exchange as described previously (28). Varying exchange rate constants were employed to obtain optimal matches between the experimental and theoretical results of the methylene protons of 4NP-S-Glc or 4NP-S-Man at the C6 atoms, which could be separated by a fixed distance of 1.8 A ˚.…”

Section: Methodsmentioning

confidence: 99%

“…Selective T 1 measurements were performed on anomeric and several other protons to obtain the values mentioned above. Experimental NOEs were fitted to the double-exponential function f ( t ) = p 0 (e − p 1 t )(1 − e − p 2 t ), as described previously , , where p 0 , p 1 , and p 2 are adjustable parameters. The initial slope was determined from the first derivative at time zero [ f (0) = p 0 p 2 ].…”

Section: Methodsmentioning

confidence: 99%

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Binding of β-d-Glucosides and β-d-Mannosides by Rice and Barley β-d-Glycosidases with Distinct Substrate Specificities

et al. 2010

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Predominantly, rice Os3BGlu7 operates as a β-d-glucosidase (EC 3.2.1.21), while barley HvBII acts as a β-d-mannosidase (EC 3.2.1.25). Saturation transfer difference nuclear magnetic resonance (STD NMR) and transferred nuclear Overhauser effect (trNOE) spectroscopy in conjunction with quantum mechanics/molecular mechanics (QM/MM) modeling and docking at the 6-31+G* level were used to investigate binding of S- and O-linked gluco- and manno-configured aryl-β-d-glycosides to Os3BGlu7 and HvBII. Kinetic analyses with 4-nitrophenyl β-d-thioglucoside (4NP-S-Glc) and 4-nitrophenyl β-d-thiomannoside (4NP-S-Man) indicated that the inhibitions were competitive with apparent K(i) constants of 664 and 710 μM for Os3BGlu7 and 95 and 266 μM for HvBII, respectively. The STD NMR and trNOESY experiments revealed that 4NP-S-Glc and 4NP-S-Man bound weakly in (4)C(1) conformations to Os3BGlu7; 4NP-S-Glc adopted (3)S(5) (B(3,O)) or (1)S(3) ((1,4)B) conformations, and 4NP-S-Man preferred (4)C(1) geometry, when bound to HvBII. The QM modeling and docking, based on GLIDE scores, predicted that 4NP-O-Glc, 4NP-O-Man, and 4NP-S-Man bound preferentially in (1)S(3) geometries to both enzymes, contrary to 4NP-S-Glc that could also adopt a (4)C(1) conformation, although in a "flipped-down" ring position. The experimental and computational data suggested that in glycoside recognition and substrate specificity of Os3BGlu7 and HvBII, a combination of the following determinants is likely to play key roles: (i) the inherent conformational and spatial flexibilities of gluco- and manno-configured substrates in the enzymes' active sites, (ii) the subtle differences in the spatial disposition of active site residues and their capacities to form interactions with specific groups of substrates, and (iii) the small variations in the charge distributions and shapes of the catalytic sites.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Binding of β-d-Glucosides and β-d-Mannosides by Rice and Barley β-d-Glycosidases with Distinct Substrate Specificities

et al. 2010

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“…The unusual sugar specificity of Banlec among the JRLs has been explored and the investigation of bound conformations of various ligands to Banlec has been done to characterize the binding sites 15, 16. This study, on the other hand, focuses on the structural stability of Banlec in the presence of chaotropes.…”

Section: Introductionmentioning

confidence: 99%

Unfolding energetics and stability of banana lectin

2008

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The unfolding pathway of banana lectin from Musa paradisiaca was determined by isothermal denaturation induced by the chaotrope GdnCl. The unfolding was found to be a reversible process. The data obtained by isothermal denaturation provided information on conformational stability of banana lectin. The high values of DeltaG of unfolding at various temperatures indicated the strength of intersubunit interactions. It was found that banana lectin is a very stable and denatures at high chaotrope concentrations only. The basis of the stability may be attributed to strong hydrogen bonds of the order 2.5-3.1 A at the dimeric interface along with the presence of water bridges. This is perhaps very unique example in proteins where subunit association is not a consequence of the predominance of hydrophobic interactions.

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Docking, synthesis, and NMR studies of mannosyl trisaccharide ligands for DC-SIGN lectin

et al. 2008

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DC-SIGN, a lectin, which presents at the surface of immature dendritic cells, constitutes nowadays a promising target for the design of new antiviral drugs. This lectin recognizes highly glycosylated proteins present at the surface of several pathogens such as HIV, Ebola virus, Candida albicans, Mycobacterium tuberculosis, etc. Understanding the binding mode of this lectin is a topic of tremendous interest and will permit a rational design of new and more selective ligands. Here, we present computational and experimental tools to study the interaction of di- and trisaccharides with DC-SIGN. Docking analysis of complexes involving mannosyl di- and trisaccharides and the carbohydrate recognition domain (CRD) of DC-SIGN have been performed. Trisaccharides Manalpha1,2[Manalpha1,6]Man 1 and Manalpha1,3[Manalpha1,6]Man 2 were synthesized from an orthogonally protected mannose as a common intermediate. Using these ligands and the soluble extracellular domain (ECD) of DC-SIGN, NMR experiments based on STD and transfer-NOE were performed providing additional information. Conformational analysis of the mannosyl ligands in the free and bound states was done. These studies have demonstrated that terminal mannoses at positions 2 or 3 in the trisaccharides are the most important moiety and present the strongest contact with the binding site of the lectin. Multiple binding modes could be proposed and therefore should be considered in the design of new ligands.

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NMR Investigation of the Bound Conformation of Natural and Synthetic Oligomannosides to Banana Lectin

Cited by 3 publications

References 51 publications

Binding of β-d-Glucosides and β-d-Mannosides by Rice and Barley β-d-Glycosidases with Distinct Substrate Specificities

Binding of β-d-Glucosides and β-d-Mannosides by Rice and Barley β-d-Glycosidases with Distinct Substrate Specificities

Unfolding energetics and stability of banana lectin

Docking, synthesis, and NMR studies of mannosyl trisaccharide ligands for DC-SIGN lectin

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