01+/2,345/+)’ $%)’/+1)$,%(glycobiology/NMRPaper021511.pdf · 1 * * * * * * 9! 3,[ n

14
Eur. J. Biochem. 267, 3965±3978 (2000) q FEBS 2000 NMR investigations of protein±carbohydrate interactions Binding studies and refined three-dimensional solution structure of the complex between the B domain of wheat germ agglutinin and N, N 0 , N 00 -triacetylchitotriose Juan Felix Espinosa 1, *, Juan Luis Asensio 1, *, Jose Luis Garcõ Âa 2 , Jose  Laynez 3 , Marta Bruix 4 , Christine Wright 5 , Hans-Christian Siebert 6 , Hans-Joachim Gabius 6 , Francisco Javier Can Ä ada 1 and Jesus Jime  nez-Barbero 1 1 Instituto de Quõ Âmica Orga Ânica General, 2 Centro de Investigaciones Biolo Âgicas, 3 Instituto de Quõ Âmica Fõ Âsica Rocasolano, and 4 Instituto de Estructura de la Materia, CSIC, Madrid, Spain; 5 Department of Pharmacology, University of Virginia, Charlottesville, VA, USA; 6 Institut fu Èr Physiologische Chemie, Tiera Èrztliche Fakulta Èt, Ludwig-Maximilians-Universita Èt, Mu Ènchen, Germany The specific interaction of the isolated B domain of wheat germ agglutinin (WGA-B) with N, N 0 , N 00 - 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. Keywords: hevein; protein/carbohydrate interactions; lectin; microcalorimetry; NMR. 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 (UDA) and wheat germ agglutinin (WGA)] and enzymes (such as class I chitinases) as well as its small size (43 residues) makes this domain an excellent model system for the study of carbohydrate recogni- tion by proteins. Its name stems from hevein, a small protein that is present in laticifers of the rubber tree ( Hevea brasiliensis). It has been shown that hevein inhibits the growth of several chitin-containing fungi protecting in this way the plants from attack by a wide range of potential pathogens [21±23]. From a structural point of view, hevein is a small, single chain protein of 43 amino acids especially rich in glycines and cysteins [24], whose structure has independently been solved in the solid state by X-ray at 2.8 A Ê resolution [25], and in solution by NMR methods both in water [26,27] and in dioxane/water [28], in the last few years. This work has become the basis for further studies on proteins displaying this domain. Wheat germ agglutinin was the first well-characterized member of the chitin-binding class of lectins from the gramineae Correspondence to J. Jime Ânez-Barbero, Department of de Quõ Âmica Orga Ânica Biolo Âgica, Instituto de Quõ Âmica Orga Ânica General, CSIC, Juan de la Cierva 3, 28006 Madrid, Spain. Fax: 1 34 91 5644853, Tel.: 1 34 91 5622900, E-mail: [email protected] Abbreviations: UDA, Urtica dioica agglutinin; WGA, wheat germ agglutinin; CIDNP, chemically-induced dynamic nuclear polarixation; REM, restrained energy minimization; RMD, restrained molecular dynamics.; TPPI, time proportional phase incrementation; ITC, isothermal titration calorimetry. *Note: these two authors contributed equally to this work. (Received 24 February 2000, revised 17 April 2000, accepted 18 April 2000)

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