Showing papers by "Anne Imberty published in 2001"

Binding interactions between barley thaumatin‐like proteins and (1,3)‐β‐D‐glucans

Abstract: The specificity and kinetics of the interaction between the pathogenesis-related group of thaumatin-like proteins (PR5) in higher plants and (1,3)-beta-D-glucans have been investigated. Two thaumatin-like proteins with 60% amino-acid sequence identity were purified from extracts of germinated barley grain, and were designated HvPR5b and HvPR5c. Purified HvPR5c interacted with insoluble (1,3)-beta-D-glucans, but not with cellulose, pustulan, xylan, chitin or a yeast mannoprotein. Tight binding was observed with unbranched and unsubstituted (1,3)-beta-D-glucans, and weaker binding was seen if (1,6)-beta-linked branch points or beta-glucosyl substituents were present in the substrate. The HvPR5b protein interacted weakly with insoluble (1,3)-beta-D-glucans and did not bind to any of the other polysaccharides tested. This indicated that only specific barley PR5 isoforms interact tightly with (1,3)-beta-D-glucans. The complete primary structures of HvPR5b and HvPR5c were determined and used to construct molecular models of HvPR5b and HvPR5c, based on known three-dimensional structures of related thaumatin-like proteins. The models were examined for features that may be associated with (1,3)-beta-D-glucan binding, and a potential (1,3)-beta-D-glucan-binding region was located on the surface of HvPR5c. No obvious structural features that would prevent binding of (1,3)-beta-D-glucan to HvPR5b were identified, but several of the amino acids in HvPR5c that are likely to interact with (1,3)-beta-D-glucans are not present in HvPR5b.

Experimental Proof for the Structure of a Thrombin‐Inhibiting Heparin Molecule

Abstract: Kinetic studies of thrombin inhibition by antithrombin in the presence of heparin have shown that thrombin binds to heparin in a preformed heparin-antithrombin complex. To study the relative position of the thrombin binding domain and the antithrombin binding domain on a heparin molecule we have designed and synthesized heparin mimetics, which structurally are very similar to the genuine polysaccharide. Their inhibitory properties with respect to factor Xa and thrombin provide experimental evidence that in heparin the thrombin binding domain must be located at the nonreducing end of the antithrombin binding domain to observe thrombin inhibition. As expected, factor Xa inhibition is not affected by elongation of the antithrombin binding pentasaccharide sequence, regardless of the position in which this elongation takes place.

Molecular dynamics simulations of solvated UDP-glucose in interaction with Mg2+ cations.

Abstract: Glycosyltransferases are key enzymes involved in biosynthesis of oligosaccharides. Nucleotide-sugars, the glycosyltransferase substrates, serve as activated donors of sugar residues during the enzymatic reaction Although very little is known about the catalytic mechanism of these enzymes, it appears that the catalytic activity in most glycosyltransferases is dependent upon the presence of a divalent cation, for example Mn2+ or Mg2+. It is not known whether the ion is bound to the enzyme before its interaction with the substrate, or if it binds the substrate before the enzymatic reaction to modify its conformation to fit better the active site of the enzyme. We have inspected the latter possibility by running four 2-ns molecular dynamics trajectories on fully solvated UDP-glucose in the presence of Mg2+ ions. Our results indicate that the divalent cation interacts strongly with the nucleotide-sugar in solution, and that it can alter its conformational behavior. It is also shown that a conformation of the pyrophosphate moiety that results in an eclipsed or almost eclipsed orientation of two of the oxygen atoms, and which is found in protein interacting with a nucleotide di- or tri-phosphate X-ray data, is energetically favored. The results are also discussed in light of existing NMR data, and are found to be in a good agreement with them.

Conformational behavior of nucleotide-sugar in solution: molecular dynamics and NMR study of solvated uridine diphosphate-glucose in the presence of monovalent cations.

Abstract: The nucleotide-sugars are metabolites of primary importance in the biosynthesis of polysaccharides and glycoconjugates since they serve as sugar donors in the reactions of glycosyltransferases, enzymes that displays a high specificity for both donors and acceptors. In order to determine the conformational behavior of uridinediphosphoglucose in dilute aqueous solution that includes a physiologically relevant concentration of salt, parallel NMR and molecular modeling investigations have been conducted. Nine molecular dynamics trajectories of 3 ns each were calculated in presence of explicit water and monovalent cations with the use of the AMBER force field with recently developed energy parameters for nucleotide-sugars (P. Petrova, J. Koca, and A. Imberty, Journal of American Chemical Society, 1999, vol. 121, pp. 5535-5547). Theoretical nuclear Overhauser effect data were calculated from these simulations using a model-free approach that takes into account internal motions. Comparison of theoretical and experimental data gives excellent agreement for the region surrounding the glucose-phosphate linkage including the pyrophosphate linkage itself. Less satisfactory agreement is obtained for the ribose ring and the base orientations. On the whole, both NMR and molecular dynamics simulations predict the molecule to be flexible, and to visit a large number of conformations while maintaining an extended overall shape.