- Email: [email protected]
Free and protein-bound carbohydrate structures
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Free and protein-bound carbohydrate structures Jesús Jiménez-Barbero*†, Juan Luis Asensio*‡, Francisco Javier Cañada*§ and Ana Poveda# 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 13C-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. Addresses *Dept Química Orgánica Biológica, Instituto Química Orgánica, CSIC, Juan de la Cierva 3, 28006 Madrid, Spain † e-mail: [email protected] ‡ e-mail: [email protected] § e-mail: [email protected] # Servicio Interdepartamental de Investigación, Universidad Autónoma Madrid, 28047 Madrid, Spain; e-mail: [email protected] Current Opinion in Structural Biology 1999, 9:549–555 0959-440X/99/$ — see front matter © 1999 Elsevier Science Ltd. All rights reserved. Abbreviations Con A concanavalin A DGL Dioclea grandiflora lectin DMSO dimethyl sulfoxide Fuc fucose Gal galactose GalNAc N-acetylgalactosamine GlcNAc N-acetylglucosamine ITC isothermal titration calorimetry NOE nuclear Overhauser enhancement TRNOE transferred NOE
Introduction Carbohydrate–protein interactions are involved in a range of biological mechanisms, starting with fertilization and extending to pathologies such as tumor spread. They also mediate diverse cellular activities, including cell recognition, growth control and apoptosis [1,2]. It is obvious that a detailed knowledge of the structure and dynamics of carbohydrates, both free and bound to proteins, is indeed relevant [3]. This information may be extracted by different means — X-ray crystallography has been widely employed to characterize large free and complexed biomolecules. Thus, examples of the application of X-ray crystallography to the study of sugar–protein intermolecular interactions are of prime interest. Carbohydrates are rather difficult to crystallize, however, even when they form part of glycoproteins, probably as a result of their inherent flexibility. Besides, X-ray crystallography only provides indirect information on the dynamics of biomolecules and, moreover, for flexible structures, only one
conformation may be analyzed. Therefore, NMR spectroscopy has been applied to this field, as it can provide conformational and dynamical information. As a result of the characteristics of sugars, it is recognized that relaxation NMR parameters should be complemented by computational methods (molecular mechanics/dynamics or Monte Carlo calculations) in order to define the structural features of the carbohydrate in an unambiguous way. Fortunately, the limits of NMR spectroscopy as a result of the size and/or complexity of the studied biomolecule are continuously extending [4 •]. Access to fully 13C-labeled oligosaccharides and the availability of new NMR experiments have provided parameters [5•] that can be used to gain structural information on the carbohydrate in both free and bound states. This review addresses issues for which papers that appeared in literature after mid 1998 have been considered. The publications have been arbitrarily arranged in different sections for free oligosaccharides, noncovalently bound protein–carbohydrate complexes, glycosidase–carbohydrate interactions, carbohydrate analogs and glycolipids, glycopeptides and glycoproteins. Several reviews have also appeared that are intimately related to the topic addressed herein. An up-todate view of the application of NMR spectroscopy as a tool to study protein–carbohydrate interactions in solution has been presented [6•]. A detailed analysis of present knowledge on the conformations of free and conjugated carbohydrates has also been published [3].
Free oligosaccharides Theoretical and experimental questions have been addressed; from the theoretical perspective, the calculation of a potential of mean force to describe the conformation of disaccharides in solution has been proposed [7]. This approach could compete with the frequently used molecular mechanics approach. A detailed chemometric analysis of the energy and geometry results provided by the most popular molecular mechanics programs has demonstrated that their output strongly depends on the force field employed [8]. The search for restraints other than NOEs (nuclear Overhauser enhancements) to describe the structures of oligosaccharides in solution has always been a major concern for experimentalists. Thus, an empirical Karplus-type relationship for C-O-C-C spin-coupling constants [9] and a new NMR sequence to obtain C-O-C-H couplings have been proposed [10]. Vicinal glycosylation has been shown to markedly affect the conformational properties of a branched oligosaccharide compared with its parent disaccharides [11]. In fact, rigidification of the glycosidic linkages has been demonstrated by NMR spectroscopy and molecular mechanics calculations.
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The influence of the medium
The characterization of hydrogen-bonded hydroxyl groups using secondary hydrogen/deuterium NMR isotope effects has been achieved in dimethyl sulfoxide (DMSO) solution [12]. The application of this or similar protocols to a water medium poses some experimental problems that can be overcome [13]. It is clear that the nature and polarity of the solvent strongly influences the conformation around the C5–C6 hydroxymethyl group of glucose derivatives [14] and that there is a net effect on the conformational properties of hyaluronan as a result of the competition between OH...water and OH…OH hydrogen bonding [15]. On the other hand, the medium has a minor influence on the conformations of several glycosyl phosphatidyl inositol (GPI)-like saccharides in water, DMSO and within SDS micelles [16]. New NMR experiments
The major advance in the experimental derivation of carbohydrates structures in solution has arrived from NMR experiments that combine isotope-labeled entities with liquid-crystal technology [17]. A new area of NMR spectroscopy is opened that uses the anisotropic characteristics of molecular alignment in strong magnetic fields [5•]. Under particular conditions, residual dipolar C–H couplings can be obtained and used to derive the relative orientation of the corresponding C–H vectors with respect to the magnetic field and, therefore, reveal the average conformation of the oligosaccharide [18•,19•]. The extraction of similar information for H–H vectors has also been demonstrated [20•]. Although the exact protocol for obtaining fine structural information poses some experimental and analysis problems, several examples of the application of this methodology have been already reported [18•–20•]. These types of experiment have also been applied to deduce the conformation of a trisaccharide ligand within the binding site of the Β-subunit pentamer of a toxin [21•].
Noncovalently bound protein–carbohydrate complexes In contrast to the relatively large number of structures of these complexes determined by X-ray crystallography, there are fewer examples of their complete determination in solution by NMR spectroscopy. Both methods may provide deep insights into the origin of the sugar specificity of receptors. Thus, X-ray analysis of Dolichos biflorus lectin has permitted the explanation of its GalNAc versus Gal specificity in structural terms [22]. Moreover, the study of crystal structures of complexes of human galectin-7 with Gal, GalNAc, lactose and N-acetyllactosamine has permitted the identification of some differences that could explain the selectivity of galectins towards their natural ligands [23•]. In solution, the structure of the complex between hevein, an allergenic protein isolated from latex, and a disaccharide has been determined by NMR spectroscopy [24•], thus permitting the explanation of the binding affinities of chito-oligosaccharides in structural
terms. Also, a xylan-binding domain (XBD) has been structurally characterized within family IIb from Cellulomonas fimi Cex [25]. As X-ray crystallography also has severe limitations in the study of membrane proteins and their complexes, new NMR techniques have been developed to tackle these problems. The selectivity of 13C-labeled substrates for FucP, an integral membrane symport protein with weak L-Fuc binding, has been studied using solidstate NMR [26]. No doubt, the extension to bigger proteins enabled by new NMR techniques, such as TROSY [4•], will permit the determination of large-sized complexes. In fact, data for a 46 ns correlation time carbohydrate-binding protein have been already collected [27•]. Application of TRNOE and related NMR experiments
When a complete protein–sugar complex can not be analyzed in solution, information on the conformation of the bound saccharides can at least be derived from transferred NOE (TRNOE) studies, provided that exchange between the complexed and uncomplexed states is sufficiently fast. Notably, the conditions required to monitor TRNOEs appear to be frequently satisfied by sugar receptors [6•]. Following this methodology, several cases have been described, including the conformational analysis of Kdo-containing disaccharides bound to monoclonal antibodies [28,29]. Thus, intermolecular NOEs between both biomolecules were observed that facilitated the location of the binding pocket [28]. QUIET-NOESY-type experiments have been proposed to complement regular TRNOEs in order to detect spin-diffusion effects [29]. The usual TRROE experiment may lead to ambiguous conclusions if cross-peaks caused by direct and three-spin effects have similar intensities and cancel each other. On the other hand, TR-QUIET-NOESY permits the separation of direct effects provided that the third spin resonates out of the inverted window. Using TRNOE, the solution three-dimensional structure of the complex between the B-subunit pentamer of Escherichia coli verotoxin VT-1 and a trisaccharide moiety of globotriaosylceramide has also been determined [30]. Maybe the most elegant use of TRNOE experiments has been achieved by the identification of an E-selectin antagonist in a substance mixture [31••]. A similar approach has been described using saturation transfer-based experiments [32]. As, in complexes, the cross-relaxation rates of the bound entity are opposite in sign to those of the free ligand and produce negative NOEs [6•], the change in sign can be used to distinguish those ligands that are bound in the mixture. Following the controversy concerning the conformation of sialyl LewisX bound to E-selectin, the bioactive conformation of the 13C-enriched tetrasaccharide has been determined through heteronuclear full-relaxation-matrix analysis [33•]. The reported conformation significantly differs from those deduced in previous studies. With regard to oligosaccharide flexibility, it has been demonstrated that an antibody is able to provide the binding energy required to change the glycosidic torsion of an oligosaccharide, so that the less stable anti-α conformer is the only one that is recognized
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[34]. Additional cases of conformational selection by the protein binding sites have been reported [35,36]. Structure and thermodynamics
The binding of pre-organized tethered oligosaccharides to a monoclonal antibody has been studied [37•]. In this case, the introduction of rigidity has minor effects on the complexation parameters. On the other hand, the binding of a rigidified, methylene-bridged trisaccharide to concanavalin A (Con A) displays a more favorable entropy that is offset by the less favorable enthalpy of binding than that for the flexible linear compound [38•]. The role of water in specific binding may be of great importance in these two cases, as also demonstrated by the X-ray and thermodynamic study of the binding of mannose-containing saccharides to Con A [39]. Probably the most in-depth investigation into the structural and thermodynamic characteristics of sugar binding by lectins has been performed by Brewer and co-workers [40•–42•]. In a series of papers, the recognition of a typical core trimannoside by the lectin from Dioclea grandiflora (DGL) has been compared with related data obtained from the highly homologous lectin Con A. First, the importance of each hydroxyl group of the trimannoside in DGL binding was deduced from isothermal titration calorimetry (ITC) using a complete set of monodeoxy and other derivatives of the trisaccharide [40•]. Thus, the differences in selectivity presented by DGL versus Con A for binding the trimannoside or the extended biantennary pentasaccharide (plus two nonreducing GlcNAc rings) have been explained in structural terms by analyzing several sugar–lectin complexes using X-ray crystallography and modeling [41•]. Finally, the differences in solvation energies (from primary solvent hydrogen/deuterium isotope effects) for the binding of the monodeoxy derivatives to both DGL and Con A have been correlated with the differential position of the structured water molecules observed in the crystal structures [42•]; however, this multidisciplinary approach has still not been able to explain in structural terms the fine details of the ligand selectivity observed in the ITC experiments [42•]. New NMR experiments
Access to 13C-labeled carbohydrates has also opened new avenues to identifying key experimental information for sugar–protein complexes in solution. For instance, it has been possible to deduce the hydrogen-bond geometry of a protein-bound monosaccharide from water-exchangemediated cross-relaxation NMR experiments [43]. In perspective, the present possibility of using NMR spectroscopy to obtain direct experimental measurements on angles between bond vectors through cross-correlated relaxation measurements [4•,5•] will have direct consequences for the quality of the determined carbohydrate conformation. Moreover, as the measurement of crosscorelated relaxation depends on the average tumbling time of the molecule [44•,45•], these measurements can be applied to the determination of protein-bound conformations, thus complementing TRNOE measurements.
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Knowledge of the key interacting groups in the bound conformation of a carbohydrate has permitted the elegant design of an artificial receptor for cholera toxin, in order to find hydrolyzable saccharide analogs that may have therapeutic properties [46]. Regarding molecular recognition, it has been shown, using a variety of biophysical techniques, that peanut lectin in the molten-globule form is still able to retain its carbohydrate specificity [47].
Glycosidase–carbohydrate interactions A new view of the protonation step in terms of the direction of the proton’s approach to the glycosidic oxygen of the substrate has been presented for retaining glycosidases. This view is based on inhibition studies using transitionstate analogs and molecular modeling [48•]. The use of a slow substrate, 2,4-dinitrophenyl 2-deoxy-2-fluoro-cellotriose, has allowed the characterization of its Michaelis complex with a cellulase from Bacillus agaradhaerens (Cel5A) [49••]. The substrate is bound in a skew boat conformation. This structural observation supports the mechanism of action of retaining glycosidases, whereby some sort of substrate deformation towards the glycosyloxocarbonium-like transition-state structure is needed. Further ahead in the reaction pathway, the structure of the covalent 2-fluoroglycosyl–enzyme intermediate has been also determined for both Cel5A [49••] and endoglucanase Cel2B from Streptomyces lividans [50]. The α configuration at the anomeric carbon that is bonded to the enzyme has been demonstrated. Alternatively, using a mutant of the β(1-4)-glycosidase Cex from C. fimi, the X-ray structure determination of the covalent intermediate has permitted the analysis of the interactions between the sugar 2-hydroxyl group and the enzyme [51]. In addition, for both Cel5A and Cel2B, the structures of the enzyme–product complexes have been obtained to complete the full picture of the reaction [49••,50]. Related enzyme–product structures have also been determined for a xylanase from Penicillium simplicissimum, whereby xylo-oligosaccharides were used as cryoprotectant for the X-ray analysis [52]. Additional information on the sugar-binding subsites could be extracted from structures of the oligosaccharide–enzyme complexes. Alternatively, the binding subsites of the thermophilic xylanase from Thermomyces lanuginosus have been investigated by molecular modeling [53]. Interestingly, some enzyme conformational changes necessary for catalysis to occur were predicted at the binding cleft. In the case of human lysozyme, an unexpected new feature has been described by affinity labeling the two essential carboxylates at the active site [54]. Two ligand molecules were step-wise bonded to the enzyme and, from the crystal structure of the complex, the authors proposed that the first ligand assisted the recognition of the second one. It has also been possible to explain the preferred recognition of a Thermomonospora fusca β-mannanase for mannans, as opposed to β(1-4) cellulose oligomers. According to the structure, both hydrogen-bond stabilizing interactions and steric conflicts are responsible for the observed manno specificity [55].
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S- and C-glycoside analogs In contrast with the known conformational behavior of S-glycosides [56], there is ongoing controversy concerning the conformational similarities between C- and O-glycosides. The components of a C-mannobiose–O-mannobiose pair with an α(1→2) linkage show rather different conformational distributions in water [57•]. In particular, the C-analog shows a 20% population of conformers in the nonexoanomeric Φ region, in contrast with the natural compound (the O-glycoside), for which no population is found within this area. On the other hand, Kishi and co-workers [58•] have proposed that C- and O-lactoses share the same conformational characteristics in the free state. Using X-ray crystallography, they also demonstrated that peanut agglutinin recognizes the same syn-Φ conformation of both sugars [58•]. This result contrasts with the previously reported conformational equilibrium for C-lactose in water [6•]. Moreover, it has been proven using TRNOE that the high-energy gauche-gauche (or anti-Φ) conformer of C-lactose (not detected for natural lactose and 5% populated for C-lactose) is selected by E. coli β-galactosidase [59••]. The topological features of the binding sites that restrict the ligand conformation, thus altering the equilibrium of the flexible C-glycoside, can contribute to the observed results. When a protein establishes interactions with several sugar units, only certain topologically favored conformers will fit into the binding site. Along this reasoning, the 2.7 Å resolution X-ray data on C-lactose–peanut lectin [58•] can readily be reconciled with this interpretation. In this particular case, two hydrogen bonds between glucose O-3 and two amino acids can only exist if C-lactose adopts the syn conformation. Identical interactions have been identified for the binding of C-lactose to the nonhomologous bovine heart galectin-1 [60•]. The distortion is limited to accommodating just one conformation in the binding site, which is also populated in solution, and no drastic rotation of the glycosidic angle is required.
Glycolipids, glycopeptides and glycoproteins Knowledge of the conformation and phase characteristics of a self-assembling monoglucosyl diacylglycerol analog has been obtained by NMR spectroscopy and modeling [61]. Several reports have dealt both with the study of the threedimensional structures of the glycans of glycoproteins and glycopeptides, and with the conformational and dynamical implications of glycosylation. In fact, it seems that there are cases for which the glycan induces local changes in the polypeptide chain [62,63,64•], although it has been shown that the flexibility of an oligomannose does not alter the overall topology of the complete glycoprotein [65]. In the case of the antimicrobial glycopeptide drosocin, it has been shown that, although the Gal(β1-3)GalNAc(α1-O)-Thr linkage did not modify the random coil preference, it did produce subtle population shifts of folded conformers towards those presenting more extended conformations [66]. A much greater stabilizing conformational effect of O-glycosidic chains has been determined for a synthetic pentapeptide with up to three sugar chains that is used as model of polyglycosylated mucins [64•]. The multiple
placement of oligosaccharides initiated by α-GalNAc linkages to hydroxy amino acids is required for an elongated mucin motif to emerge. In this context, both molecular [67•] and theoretical [68] bases for glycosylation-induced conformational switching have been proposed.
Conclusions Multidisciplinary approaches to the study of protein–carbohydrate complexes are probably the best way to gain an understanding of the structure/activity relationships of these systems. Therefore, the combination of organic (chemical or chemoenzymatical) synthesis to prepare labeled and/or modified glycans with the conformational analysis of the free ligand using both experimental (X-ray crystallography and/or NMR spectroscopy) and theoretical data, as well as using molecular biology and/or protein purification techniques to get decent amounts of pure material, followed by the structural analysis of the complex either in the solid state using X-ray crystallography or in solution using the newest NMR techniques, together with thermodynamic measurements of the association process, should provide answers to both general and specific questions.
Acknowledgements We thank the Dirección General de Enseñanza Superior (DGES) (PB960837) for funding. JLA thanks Communidad Autónoma De Madrid (CAM) and Ministerio de Educación y Cultura (MEC) for fellowships.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
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interactions between the two GlcNAc chairs and tryptophan and tyrosine rings, as well as two protein–sugar hydrogen bonds, are responsible for the specific association.
11. Kozar T, Nifant’ev EN, Grosskurth H, Dabrowski U, Dabrowski J: Conformational changes due to vicinal glycosylation: the β-D-Galp(1-3)]β β-D-Glc1-OMe branched α-L-Rhap(1-2)[β trisaccharide compared with its parent disaccharides. Biopolymers 1998, 46:417-432.
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Ottiger M, Bax A: Characterization of magnetically oriented phospholipid micelles for measurement of dipolar couplings in macromolecules. J Biomol NMR 1998, 12:361-372.
18. Rundlof T, Landersjo C, Lycknert K, Maliniak A, Widmalm G: NMR • investigation of oligosaccharide conformation using dipolar couplings in an aqueous dilute liquid crystalline medium. Magn Reson Chem 1998, 36:773-776. The authors show how it is possible to deduce the average conformation of a tetrasaccharide in solution using residual dipolar C–H couplings, without using nuclear Overhauser enhancement information. 19. Kiddle GR, Homans SW: Residual dipolar couplings as new • conformational restraints in isotopically 13C-enriched oligosaccharides. FEBS Lett 1998, 436:128-130. C–H residual dipolar couplings within a simulated annealing protocol were used to deduce the solution conformation of sialyl lactose. No nuclear Overhauser enhancement information is necessary to deduce the major conformation in solution. 20. Bolon PJ, Prestegard JH: COSY cross peaks from 1H-1H dipolar • couplings in NMR spectra of field oriented oligosaccharides. J Am Chem Soc 1998, 120:9366-9367. H–H residual dipolar couplings, which depend on the distance according to 3 1/r , were estimated using easy experimental NMR methods. The potential use of this method to relatively position remote rings is described. 21. Shimizu H, Donohue-Rolfe A, Homans SW: Derivation of the bound • state conformation of a ligand in a weakly aligned ligand-protein complex. J Am Chem Soc 1999, 121:5815-5816. As the alignment of a protein–ligand complex is substantially larger than that of the free ligand, residual dipolar couplings were used to deduce the toxinbound conformation of a trisaccharide, without using TR-NOE experiments. 22. Hamelryck TA, Loris R, Bouckaert J, Dao-Thi M, Strecker G, Imberty A, Fernandez E, Wyns L, Etzler ME: Carbohydrate binding, quaternary structure, and a novel hydrophobic binding site in two legume lectin oligomers from Dolichos biflorus. J Mol Biol 1999, 286:1161-1177. 23. Leonidas DD, Vatzaki EH, Vorum H, Celis JE, Madsen P, Acharya KR: • Structural basis for the recognition of carbohydrates by human galectin 7. Biochemistry 1998, 37:13930-13940. A comparison of the structural features of galectin-7 with other galectins explains their different selectivity for natural ligands. 24. Asensio JL, Cañada FJ, Bruix M, Gonzalez C, Khiar N, Rodriguez • Romero A, Jimenez-Barbero J: NMR investigations of carbohydrate-protein interactions: refined three dimensional structure of the complex between hevein and methyl chitobioside. Glycobiology 1998, 8:569-577. One of the few examples of a detailed three-dimensional structural analysis of a protein–carbohydrate complex in solution is described. Van der Waals
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30. Shimizu H, Field RA, Homans SW, Donohue-Rolfe A: Solution structure of the complex between the B-subunit homopentamer of verotoxin VT-1 from Escherichia coli and the trisaccharide moiety of globotriaosylceramide. Biochemistry 1998, 37:11078-11082. 31. Henrichsen D, Ernst B, Magnani JL, Wang WT, Meyer B, Peters T: •• Bioaffinity NMR spectroscopy: identification of an E-selectin antagonist in a substance mixture by transfer NOE. Angew Chem Int Ed 1999, 38:98-102. Transferred nuclear Overhauser enhancement methods can discriminate an E-selectin antagonist from a complex mixture of similar compounds. Perspectives on the application and drawbacks of the method are discussed. 32. Mayer M, Meyer B: Characterization of ligand binding by saturation transfer difference NMR spectroscopy. Angew Chem Int Ed 1999, 38:1784-1788 33. Harris R, Kiddle GR, Field RA, Milton MJ, Ernst B, Magnani JL, • Homans SW: Stable isotope assisted NMR studies on 13C-enriched sialyl LewisX in solution and bound to E-selectin. J Am Chem Soc 1999, 121:2546-2551. 13C-labeled sialyl LewisX was used to explore its conformation while bound to E-selectin. Quantitative nuclear Overhauser enhancement (NOE)-derived distances and vicinal scalar C–H couplings were used as input for time-averaged molecular dynamics simulations to derive the free and bound conformations of the tetrasaccharide. The reported conformations significantly differs from those deduced in previous studies. 34. Milton MJ, Bundle DR: Observation of the anti-conformation of a glycosidic linkage in an antibody bound oligosaccharide. J Am Chem Soc 1998, 120:10547-10548. 35. Gilleron M, Siebert HC, Kaltner H, von der Lieth CW, Kozár T, Halkes KM, Korchagina EY, Bovin NV, Gabius HJ, Vliegenthart JFG: Conformer selection and differential restriction of ligand mobility by a plant lectin. Conformational behaviour of β1-3GlcNAcβ β1-R, Galβ β1-3GalNAcβ β1-R and Galβ β1-2Galβ β1-R' in Galβ the free state and complexed with galactoside-specific mistletoe lectin as revealed by random walk and conformational clustering molecular mechanics calculations, molecular dynamics simulations and nuclear Overhauser experiments. Eur J Biochem 1998, 252:416-427. 36. Hricovini M, Guerrini M, Bisio A: Structure of heparin-derived tetrasaccharide complexed to the plasma protein antithrombin derived from NOEs, J-couplings and chemical shifts. Eur J Biochem 1998, 261:789-801. 37. •
Bundle DR, Alibes R, Nilar S, Otter A, Warwas M, Zhang P: Thermodynamic and conformational implications of glycosidic rotamers preorganized for binding. J Am Chem Soc 1998, 120:5317-5318. Significant reduction of torsional flexibility failed to produce important entropic gains with the interaction of tethered ligands with a monoclonal antibody. Changes in the water structure around the tethers may contribute to the observed thermodynamic parameters.
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38. Navarre N, Amiot N, van Oijen A, Imberty A, Poveda A, Jimenez • Barbero J, Cooper A, Nutley MA, Boons G-J: Synthesis and conformational analysis of a conformationally constrained trisaccharide and complexation properties with concanavalin A. Chem Eur J 1999, 5:2281-2294. In contrast with the previous paper [37•], an important entropy gain is observed with the binding of a synthetically rigidified trisaccharide to the lectin concanavalin A. The entropy gain is offset by a significant lost of enthalpy. 39. Swaminathan CP, Surolia N, Surolia A: Role of water in the specific binding of mannose and mannooligosaccharides to concanavalin A. J Am Chem Soc 1998, 120:5153-5159. 40. Dam TK, Oscarson S, Brewer CF: Thermodynamics of binding of • the core trimannoside of asparagine-linked carbohydrates and deoxy analogs to Dioclea grandiflora lectin. J Biol Chem 1998, 273:32812-32817. The binding of monodeoxy derivatives of the typical trimannoside core of glycoproteins to Dioclea grandiflora lectin was studied by microcalorimetry. The role of every hydroxyl group was deduced from the thermodynamic binding parameters. 41. Rozwarski DA, Swami BM, Brewer CF, Sacchettini JC: Crystal • structure of the lectin from Dioclea grandiflora complexed with core trimannoside of asparagine-linked carbohydrates. J Biol Chem 1998, 273:32818-32825. The analysis of the crystal structure of the Dioclea grandiflora lectin–trimannoside complex permits the interpretation of the thermodynamic data in structural terms. The role of water molecules is also explained. 42. Dam TK, Oscarson S, Sacchettini JC, Brewer CF: Differential • solvation of “core” trimannoside complexes of the Dioclea grandiflora lectin and concanavalin A detected by primary solvent isotope effects in isothermal titration microcalorimetry. J Biol Chem 1998, 273:32826-32832. The complete picture of the binding process between Dioclea grandiflora lectin and the core trimannoside is described and compared with that for concanavalin A. Although most of the binding features are explained, the fine details of the differential binding selectivity remain unclear. 43. Sayers EW, Weaver JL, Prestegard JH: Hydrogen bonding geometry of a protein-bound carbohydrate from water exchange-mediated cross relaxation. J Biomol NMR 1998, 12:209-222. 44. Carlomagno T, Felli IC, Czech M, Fischer R, Sprinzl M, Griesinger C: • Transferred cross correlated relaxation: application to the determination of sugar pucker in an aminoacylated tRNA-mimetic weakly bound to EF-Tu. J Am Chem Soc 1999, 121:1945-1948. The puckering of a protein-bound sugar within a RNA mimetic was determined using new NMR experiments. The information gained depends on the correlation time of the molecule and, therefore, the collected data are dominated by the complexed state. 45. Blommers MJJ, Stark W, Jones CE, Head D, Owen CE, Jahnke W: • Transferred cross correlated relaxation complements TR-NOE. Structure of an IL-4R derived peptide bound to STAT-6. J Am Chem Soc 1999, 121:1949-1953. An identical approach to that described in [44•] was used to examine the conformational changes of a peptide when bound by a protein. Although isotope labeling is necessary, this methodology permits the expansion of knowledge concerning bound conformations of biomolecules. 46. Bernardi A, Checchia A, Brocca P, Sonnino S, Zuccotto F: Sugar mimics: an artificial receptor for cholera toxin. J Am Chem Soc 1999, 121:2032-2036. 47.
Reddy GB, Srinivas VR, Ahmad N, Surolia A: Molten globule-like state of peanut lectin monomer retains its carbohydrate specificity. J Biol Chem 1999, 274:4500-4503.
48. Heightman TD, Vasella AT: Recent insights into inhibition, structure, • and mechanism of configuration-retaining glycosidases. Angew Chem Int Ed 1999, 38:750-770. A detailed revision of the current view of the structure/mechanism relationship for retaining glycosidase enzymes. 49. Davies GJ, Mackenzie L, Varrot A, Dauter M, Brzozowski AM, •• Schulein M, Withers SG: Snapshots along an enzymatic reaction coordinate: analysis of a retaining beta-glycoside hydrolase. Biochemistry 1998, 37:11707-11713. A complete structural view of the pathway of the hydrolysis of a fluorosugar by a retaining glycosidase is provided by X-ray crystallography. 50. Sulzenbacher G, Mackenzie LF, Wilson KS, Withers SG, Dupont C, Davies GJ: The crystal structure of a 2-fluorocellotriosyl complex of the Streptomyces lividans endoglucanase CelB2 at 1.2 Å resolution. Biochemistry 1999, 38:4826-4833.
51. Notenboom V, Birsan C, Nitz M, Rose DR, Warren RA, Withers SG: Insights into transition state stabilization of the β-1,4-glycosidase Cex by covalent intermediate accumulation in active site mutants. Nat Struct Biol 1998, 5:812-818. 52. Schmidt A, Gbitz GM, Kratky C: Xylan binding subsite mapping in the xylanase from Penicillium simplicissimum using xylooligosaccharides as cryo-protectant. Biochemistry 1999, 38:2403-2412. 53. Gruber K, Klintschar G, Hayn M, Schlacher A, Steiner W, Kratky C: Thermophilic xylanase from Thermomyces lanuginosus: high-resolution X- ray structure and modeling studies. Biochemistry 1998, 37:13475-13485. 54. Muraki M, Harata K, Sugita N, Sato K: Dual affinity labeling of the active site of human lysozyme with an N-acetyllactosamine derivative: first ligand assisted recognition of the second ligand. Biochemistry 1999, 38:540-548. 55. Hilge M, Gloor SM, Rypniewski W, Sauer O, Heightman TD, Zimmermann W, Winterhalter K, Piontek K: High-resolution native and complex structures of thermostable beta-mannanase from Thermonospora fusca-substrate specificity in glycosyl hydrolase family 5. Structure 1998, 6:1433-1444. 56. Aguilera B, Jimenez-Barbero J, Fernandez-Mayoralas A: Conformational differences between Fuc(1-3)GlcNAc and its thioglycoside analogue. Carbohydr Res 1998, 308:19-27. 57. •
Espinosa JF, Bruix M, Jarreton O, Skrydstrup T, Beau JM, Jiménez Barbero J: Conformational differences between C- and O-glycosides: the C-mannobiose/O-mannobiose case. Chem Eur J 1999, 4:442-448. C-mannobiose presents a high population of conformers with a nonexoanomeric orientation of the α angle, demonstrating that, in general, C-and O-glycoside conformations are different. 58. Ravishankar R, Surolia A, Vijayan M, Lim S, Kishi Y: Preferred • conformation of C-lactose at the free and peanut lectin bound states. J Am Chem Soc 1998, 120:11297-11303. C-lactose is bound by peanut lectin in the same conformation as O-lactose. Key hydrogen bonds between the protein and a glucose residue in the syn conformation provide the required interactions for the exclusive recognition of this conformer. 59. Espinosa JF, Montero E, Vian A, Garcia JL, Dietrich H, Schmidt RR, •• Martin-Lomas M, Imberty A, Cañada J, Jimenez-Barbero J: E. coli galactoside recognizes a high energy conformation of C-lactose, a non hydrolyzable substrate analogue. NMR investigations of the molecular complex. J Am Chem Soc 1998, 120:1309-1316. NMR spectroscopy and molecular modeling were used to demonstrate the exclusive recognition of a local minimum conformation of C-lactose by a glycosidase enzyme. This fact indirectly reveals the intrinsic flexibility of C-lactose and provides experimental proof for conformational distortion at an enzyme-binding and/or catalytic site. 60. Asensio JL, Espinosa JF, Dietrich H, Cañada FJ, Schmidt RR, • Martín Lomas M, André S, Gabius HJ, Jiménez-Barbero J: Bovine heart galectin-1 selects an unique (syn) conformation of C-lactose, a flexible lactose analogue. J Am Chem Soc 1999, in press. Three different proteins, namely galectin-1, E. coli β-galactosidase and ricinB, select three different conformations of the same synthetic analog, C-lactose. Despite its lack of homology to peanut agglutinin, identical interactions to those described in [58•] provide the required interactions for the exclusive recognition of the syn-Φψ conformer by bovine heart galectin-1. 61. Song J, Hollingsworth IR: Synthesis, conformational analysis and phase characterization of a versatile self-assembling monoglucosyl diacylglycerol analogue. J Am Chem Soc 1999, 121:1851-1861. 62. Wu W, Pasternack L, Hwanh DH, Koeller KM, Lin CC, Seitz O, Wong C-H: Structural study on O-glycopeptides: glycosylation induced conformational changes of O-GlcNac, O-LacNAc, O-sialyl-LacNAc, and O-sialyl-Lewis X peptides of the mucin domain of MAdCAM-1. J Am Chem Soc 1999, 121:2409-2417. 63. Yamaguchi Y, Kato K, Shindo M, Aoki S, Furusho K, Koga K, Takahashi N, Arata Y, Shimada I: Dynamics of the carbohydrate chains attached to the Fc portion of immunoglobulin G as studied by NMR spectroscopy assisted by selective 13C labeling of the glycans. J Biomol NMR 1998, 12:385-394. 64. Live DH, Williams LJ, Kuduk SD, Schwarz JB, Glunz PW, Chen XT, • Sames D, Kumar RA, Danishefsky SJ: Probing cell-surface architecture through synthesis: an NMR-determined structural
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motif for tumor-associated mucins. Proc Natl Acad Sci USA 1999, 96:3489-3493. Glycopeptide synthesis, coupled with detailed NMR analysis, provides insights into the design of a basic sequence that is able to mimic an elongated mucin motif.
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non-glycosylated derivative: effects of glycosylation on solution conformation. Biochemistry 1999, 38:705-714.
65. Woods RJ, Pathiaseril A, Wormald MR, Edge CJ, Dwek EA: The high degree of internal flexibility observed for an oligomannose oligosaccharide does not alter the overall topology of the molecule. Eur J Biochem 1998, 258:372-386.
67. O’Connor SE, Imperiali B: A molecular basis for glycosylation • induced conformational switching. Chem Biol 1998, 5:427-437. The conformation and dynamics of different glycopeptides with natural and modified chitobioses was analyzed by NMR spectroscopy. The authors state that the N-acetyl groups on the sugars have a key influence on the glycopeptide conformation.
66. McManus AM, Otvos L Jr, Hoffmann R, Craik DJ: Conformational studies by NMR of the antimicrobial peptide, drosocin, and its
68. Hoffmann D, Florcke H: A structural role for glycosylation: lessons from the hp model. Fold Des 1998, 3:337-343.
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Free and protein-bound carbohydrate structures Jesús Jiménez-Barbero*†, Juan Luis Asensio*‡, Francisco Javier Cañada*§ and Ana Poveda# 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 13C-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. Addresses *Dept Química Orgánica Biológica, Instituto Química Orgánica, CSIC, Juan de la Cierva 3, 28006 Madrid, Spain † e-mail: [email protected] ‡ e-mail: [email protected] § e-mail: [email protected] # Servicio Interdepartamental de Investigación, Universidad Autónoma Madrid, 28047 Madrid, Spain; e-mail: [email protected] Current Opinion in Structural Biology 1999, 9:549–555 0959-440X/99/$ — see front matter © 1999 Elsevier Science Ltd. All rights reserved. Abbreviations Con A concanavalin A DGL Dioclea grandiflora lectin DMSO dimethyl sulfoxide Fuc fucose Gal galactose GalNAc N-acetylgalactosamine GlcNAc N-acetylglucosamine ITC isothermal titration calorimetry NOE nuclear Overhauser enhancement TRNOE transferred NOE
Introduction Carbohydrate–protein interactions are involved in a range of biological mechanisms, starting with fertilization and extending to pathologies such as tumor spread. They also mediate diverse cellular activities, including cell recognition, growth control and apoptosis [1,2]. It is obvious that a detailed knowledge of the structure and dynamics of carbohydrates, both free and bound to proteins, is indeed relevant [3]. This information may be extracted by different means — X-ray crystallography has been widely employed to characterize large free and complexed biomolecules. Thus, examples of the application of X-ray crystallography to the study of sugar–protein intermolecular interactions are of prime interest. Carbohydrates are rather difficult to crystallize, however, even when they form part of glycoproteins, probably as a result of their inherent flexibility. Besides, X-ray crystallography only provides indirect information on the dynamics of biomolecules and, moreover, for flexible structures, only one
conformation may be analyzed. Therefore, NMR spectroscopy has been applied to this field, as it can provide conformational and dynamical information. As a result of the characteristics of sugars, it is recognized that relaxation NMR parameters should be complemented by computational methods (molecular mechanics/dynamics or Monte Carlo calculations) in order to define the structural features of the carbohydrate in an unambiguous way. Fortunately, the limits of NMR spectroscopy as a result of the size and/or complexity of the studied biomolecule are continuously extending [4 •]. Access to fully 13C-labeled oligosaccharides and the availability of new NMR experiments have provided parameters [5•] that can be used to gain structural information on the carbohydrate in both free and bound states. This review addresses issues for which papers that appeared in literature after mid 1998 have been considered. The publications have been arbitrarily arranged in different sections for free oligosaccharides, noncovalently bound protein–carbohydrate complexes, glycosidase–carbohydrate interactions, carbohydrate analogs and glycolipids, glycopeptides and glycoproteins. Several reviews have also appeared that are intimately related to the topic addressed herein. An up-todate view of the application of NMR spectroscopy as a tool to study protein–carbohydrate interactions in solution has been presented [6•]. A detailed analysis of present knowledge on the conformations of free and conjugated carbohydrates has also been published [3].
Free oligosaccharides Theoretical and experimental questions have been addressed; from the theoretical perspective, the calculation of a potential of mean force to describe the conformation of disaccharides in solution has been proposed [7]. This approach could compete with the frequently used molecular mechanics approach. A detailed chemometric analysis of the energy and geometry results provided by the most popular molecular mechanics programs has demonstrated that their output strongly depends on the force field employed [8]. The search for restraints other than NOEs (nuclear Overhauser enhancements) to describe the structures of oligosaccharides in solution has always been a major concern for experimentalists. Thus, an empirical Karplus-type relationship for C-O-C-C spin-coupling constants [9] and a new NMR sequence to obtain C-O-C-H couplings have been proposed [10]. Vicinal glycosylation has been shown to markedly affect the conformational properties of a branched oligosaccharide compared with its parent disaccharides [11]. In fact, rigidification of the glycosidic linkages has been demonstrated by NMR spectroscopy and molecular mechanics calculations.
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The influence of the medium
The characterization of hydrogen-bonded hydroxyl groups using secondary hydrogen/deuterium NMR isotope effects has been achieved in dimethyl sulfoxide (DMSO) solution [12]. The application of this or similar protocols to a water medium poses some experimental problems that can be overcome [13]. It is clear that the nature and polarity of the solvent strongly influences the conformation around the C5–C6 hydroxymethyl group of glucose derivatives [14] and that there is a net effect on the conformational properties of hyaluronan as a result of the competition between OH...water and OH…OH hydrogen bonding [15]. On the other hand, the medium has a minor influence on the conformations of several glycosyl phosphatidyl inositol (GPI)-like saccharides in water, DMSO and within SDS micelles [16]. New NMR experiments
The major advance in the experimental derivation of carbohydrates structures in solution has arrived from NMR experiments that combine isotope-labeled entities with liquid-crystal technology [17]. A new area of NMR spectroscopy is opened that uses the anisotropic characteristics of molecular alignment in strong magnetic fields [5•]. Under particular conditions, residual dipolar C–H couplings can be obtained and used to derive the relative orientation of the corresponding C–H vectors with respect to the magnetic field and, therefore, reveal the average conformation of the oligosaccharide [18•,19•]. The extraction of similar information for H–H vectors has also been demonstrated [20•]. Although the exact protocol for obtaining fine structural information poses some experimental and analysis problems, several examples of the application of this methodology have been already reported [18•–20•]. These types of experiment have also been applied to deduce the conformation of a trisaccharide ligand within the binding site of the Β-subunit pentamer of a toxin [21•].
Noncovalently bound protein–carbohydrate complexes In contrast to the relatively large number of structures of these complexes determined by X-ray crystallography, there are fewer examples of their complete determination in solution by NMR spectroscopy. Both methods may provide deep insights into the origin of the sugar specificity of receptors. Thus, X-ray analysis of Dolichos biflorus lectin has permitted the explanation of its GalNAc versus Gal specificity in structural terms [22]. Moreover, the study of crystal structures of complexes of human galectin-7 with Gal, GalNAc, lactose and N-acetyllactosamine has permitted the identification of some differences that could explain the selectivity of galectins towards their natural ligands [23•]. In solution, the structure of the complex between hevein, an allergenic protein isolated from latex, and a disaccharide has been determined by NMR spectroscopy [24•], thus permitting the explanation of the binding affinities of chito-oligosaccharides in structural
terms. Also, a xylan-binding domain (XBD) has been structurally characterized within family IIb from Cellulomonas fimi Cex [25]. As X-ray crystallography also has severe limitations in the study of membrane proteins and their complexes, new NMR techniques have been developed to tackle these problems. The selectivity of 13C-labeled substrates for FucP, an integral membrane symport protein with weak L-Fuc binding, has been studied using solidstate NMR [26]. No doubt, the extension to bigger proteins enabled by new NMR techniques, such as TROSY [4•], will permit the determination of large-sized complexes. In fact, data for a 46 ns correlation time carbohydrate-binding protein have been already collected [27•]. Application of TRNOE and related NMR experiments
When a complete protein–sugar complex can not be analyzed in solution, information on the conformation of the bound saccharides can at least be derived from transferred NOE (TRNOE) studies, provided that exchange between the complexed and uncomplexed states is sufficiently fast. Notably, the conditions required to monitor TRNOEs appear to be frequently satisfied by sugar receptors [6•]. Following this methodology, several cases have been described, including the conformational analysis of Kdo-containing disaccharides bound to monoclonal antibodies [28,29]. Thus, intermolecular NOEs between both biomolecules were observed that facilitated the location of the binding pocket [28]. QUIET-NOESY-type experiments have been proposed to complement regular TRNOEs in order to detect spin-diffusion effects [29]. The usual TRROE experiment may lead to ambiguous conclusions if cross-peaks caused by direct and three-spin effects have similar intensities and cancel each other. On the other hand, TR-QUIET-NOESY permits the separation of direct effects provided that the third spin resonates out of the inverted window. Using TRNOE, the solution three-dimensional structure of the complex between the B-subunit pentamer of Escherichia coli verotoxin VT-1 and a trisaccharide moiety of globotriaosylceramide has also been determined [30]. Maybe the most elegant use of TRNOE experiments has been achieved by the identification of an E-selectin antagonist in a substance mixture [31••]. A similar approach has been described using saturation transfer-based experiments [32]. As, in complexes, the cross-relaxation rates of the bound entity are opposite in sign to those of the free ligand and produce negative NOEs [6•], the change in sign can be used to distinguish those ligands that are bound in the mixture. Following the controversy concerning the conformation of sialyl LewisX bound to E-selectin, the bioactive conformation of the 13C-enriched tetrasaccharide has been determined through heteronuclear full-relaxation-matrix analysis [33•]. The reported conformation significantly differs from those deduced in previous studies. With regard to oligosaccharide flexibility, it has been demonstrated that an antibody is able to provide the binding energy required to change the glycosidic torsion of an oligosaccharide, so that the less stable anti-α conformer is the only one that is recognized
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[34]. Additional cases of conformational selection by the protein binding sites have been reported [35,36]. Structure and thermodynamics
The binding of pre-organized tethered oligosaccharides to a monoclonal antibody has been studied [37•]. In this case, the introduction of rigidity has minor effects on the complexation parameters. On the other hand, the binding of a rigidified, methylene-bridged trisaccharide to concanavalin A (Con A) displays a more favorable entropy that is offset by the less favorable enthalpy of binding than that for the flexible linear compound [38•]. The role of water in specific binding may be of great importance in these two cases, as also demonstrated by the X-ray and thermodynamic study of the binding of mannose-containing saccharides to Con A [39]. Probably the most in-depth investigation into the structural and thermodynamic characteristics of sugar binding by lectins has been performed by Brewer and co-workers [40•–42•]. In a series of papers, the recognition of a typical core trimannoside by the lectin from Dioclea grandiflora (DGL) has been compared with related data obtained from the highly homologous lectin Con A. First, the importance of each hydroxyl group of the trimannoside in DGL binding was deduced from isothermal titration calorimetry (ITC) using a complete set of monodeoxy and other derivatives of the trisaccharide [40•]. Thus, the differences in selectivity presented by DGL versus Con A for binding the trimannoside or the extended biantennary pentasaccharide (plus two nonreducing GlcNAc rings) have been explained in structural terms by analyzing several sugar–lectin complexes using X-ray crystallography and modeling [41•]. Finally, the differences in solvation energies (from primary solvent hydrogen/deuterium isotope effects) for the binding of the monodeoxy derivatives to both DGL and Con A have been correlated with the differential position of the structured water molecules observed in the crystal structures [42•]; however, this multidisciplinary approach has still not been able to explain in structural terms the fine details of the ligand selectivity observed in the ITC experiments [42•]. New NMR experiments
Access to 13C-labeled carbohydrates has also opened new avenues to identifying key experimental information for sugar–protein complexes in solution. For instance, it has been possible to deduce the hydrogen-bond geometry of a protein-bound monosaccharide from water-exchangemediated cross-relaxation NMR experiments [43]. In perspective, the present possibility of using NMR spectroscopy to obtain direct experimental measurements on angles between bond vectors through cross-correlated relaxation measurements [4•,5•] will have direct consequences for the quality of the determined carbohydrate conformation. Moreover, as the measurement of crosscorelated relaxation depends on the average tumbling time of the molecule [44•,45•], these measurements can be applied to the determination of protein-bound conformations, thus complementing TRNOE measurements.
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Knowledge of the key interacting groups in the bound conformation of a carbohydrate has permitted the elegant design of an artificial receptor for cholera toxin, in order to find hydrolyzable saccharide analogs that may have therapeutic properties [46]. Regarding molecular recognition, it has been shown, using a variety of biophysical techniques, that peanut lectin in the molten-globule form is still able to retain its carbohydrate specificity [47].
Glycosidase–carbohydrate interactions A new view of the protonation step in terms of the direction of the proton’s approach to the glycosidic oxygen of the substrate has been presented for retaining glycosidases. This view is based on inhibition studies using transitionstate analogs and molecular modeling [48•]. The use of a slow substrate, 2,4-dinitrophenyl 2-deoxy-2-fluoro-cellotriose, has allowed the characterization of its Michaelis complex with a cellulase from Bacillus agaradhaerens (Cel5A) [49••]. The substrate is bound in a skew boat conformation. This structural observation supports the mechanism of action of retaining glycosidases, whereby some sort of substrate deformation towards the glycosyloxocarbonium-like transition-state structure is needed. Further ahead in the reaction pathway, the structure of the covalent 2-fluoroglycosyl–enzyme intermediate has been also determined for both Cel5A [49••] and endoglucanase Cel2B from Streptomyces lividans [50]. The α configuration at the anomeric carbon that is bonded to the enzyme has been demonstrated. Alternatively, using a mutant of the β(1-4)-glycosidase Cex from C. fimi, the X-ray structure determination of the covalent intermediate has permitted the analysis of the interactions between the sugar 2-hydroxyl group and the enzyme [51]. In addition, for both Cel5A and Cel2B, the structures of the enzyme–product complexes have been obtained to complete the full picture of the reaction [49••,50]. Related enzyme–product structures have also been determined for a xylanase from Penicillium simplicissimum, whereby xylo-oligosaccharides were used as cryoprotectant for the X-ray analysis [52]. Additional information on the sugar-binding subsites could be extracted from structures of the oligosaccharide–enzyme complexes. Alternatively, the binding subsites of the thermophilic xylanase from Thermomyces lanuginosus have been investigated by molecular modeling [53]. Interestingly, some enzyme conformational changes necessary for catalysis to occur were predicted at the binding cleft. In the case of human lysozyme, an unexpected new feature has been described by affinity labeling the two essential carboxylates at the active site [54]. Two ligand molecules were step-wise bonded to the enzyme and, from the crystal structure of the complex, the authors proposed that the first ligand assisted the recognition of the second one. It has also been possible to explain the preferred recognition of a Thermomonospora fusca β-mannanase for mannans, as opposed to β(1-4) cellulose oligomers. According to the structure, both hydrogen-bond stabilizing interactions and steric conflicts are responsible for the observed manno specificity [55].
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S- and C-glycoside analogs In contrast with the known conformational behavior of S-glycosides [56], there is ongoing controversy concerning the conformational similarities between C- and O-glycosides. The components of a C-mannobiose–O-mannobiose pair with an α(1→2) linkage show rather different conformational distributions in water [57•]. In particular, the C-analog shows a 20% population of conformers in the nonexoanomeric Φ region, in contrast with the natural compound (the O-glycoside), for which no population is found within this area. On the other hand, Kishi and co-workers [58•] have proposed that C- and O-lactoses share the same conformational characteristics in the free state. Using X-ray crystallography, they also demonstrated that peanut agglutinin recognizes the same syn-Φ conformation of both sugars [58•]. This result contrasts with the previously reported conformational equilibrium for C-lactose in water [6•]. Moreover, it has been proven using TRNOE that the high-energy gauche-gauche (or anti-Φ) conformer of C-lactose (not detected for natural lactose and 5% populated for C-lactose) is selected by E. coli β-galactosidase [59••]. The topological features of the binding sites that restrict the ligand conformation, thus altering the equilibrium of the flexible C-glycoside, can contribute to the observed results. When a protein establishes interactions with several sugar units, only certain topologically favored conformers will fit into the binding site. Along this reasoning, the 2.7 Å resolution X-ray data on C-lactose–peanut lectin [58•] can readily be reconciled with this interpretation. In this particular case, two hydrogen bonds between glucose O-3 and two amino acids can only exist if C-lactose adopts the syn conformation. Identical interactions have been identified for the binding of C-lactose to the nonhomologous bovine heart galectin-1 [60•]. The distortion is limited to accommodating just one conformation in the binding site, which is also populated in solution, and no drastic rotation of the glycosidic angle is required.
Glycolipids, glycopeptides and glycoproteins Knowledge of the conformation and phase characteristics of a self-assembling monoglucosyl diacylglycerol analog has been obtained by NMR spectroscopy and modeling [61]. Several reports have dealt both with the study of the threedimensional structures of the glycans of glycoproteins and glycopeptides, and with the conformational and dynamical implications of glycosylation. In fact, it seems that there are cases for which the glycan induces local changes in the polypeptide chain [62,63,64•], although it has been shown that the flexibility of an oligomannose does not alter the overall topology of the complete glycoprotein [65]. In the case of the antimicrobial glycopeptide drosocin, it has been shown that, although the Gal(β1-3)GalNAc(α1-O)-Thr linkage did not modify the random coil preference, it did produce subtle population shifts of folded conformers towards those presenting more extended conformations [66]. A much greater stabilizing conformational effect of O-glycosidic chains has been determined for a synthetic pentapeptide with up to three sugar chains that is used as model of polyglycosylated mucins [64•]. The multiple
placement of oligosaccharides initiated by α-GalNAc linkages to hydroxy amino acids is required for an elongated mucin motif to emerge. In this context, both molecular [67•] and theoretical [68] bases for glycosylation-induced conformational switching have been proposed.
Conclusions Multidisciplinary approaches to the study of protein–carbohydrate complexes are probably the best way to gain an understanding of the structure/activity relationships of these systems. Therefore, the combination of organic (chemical or chemoenzymatical) synthesis to prepare labeled and/or modified glycans with the conformational analysis of the free ligand using both experimental (X-ray crystallography and/or NMR spectroscopy) and theoretical data, as well as using molecular biology and/or protein purification techniques to get decent amounts of pure material, followed by the structural analysis of the complex either in the solid state using X-ray crystallography or in solution using the newest NMR techniques, together with thermodynamic measurements of the association process, should provide answers to both general and specific questions.
Acknowledgements We thank the Dirección General de Enseñanza Superior (DGES) (PB960837) for funding. JLA thanks Communidad Autónoma De Madrid (CAM) and Ministerio de Educación y Cultura (MEC) for fellowships.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
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Simanek EE, McGarvey GJ, Jablonowski JA, Wong CH: Selectin-carbohydrate interactions: from natural ligands to designed mimics. Chem Rev 1998, 98:833-862.
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Bush CA, Martin-Pastor M, Imberty A: Structure and conformation of complex carbohydrates of glycoproteins, glycolipids and bacterial polysaccharides. Annu Rev Biophys Biomol Struct 1999, 28:269-293.
4. Wüthrich K: The second decade- into the third millennium. • Nat Struct Biol 1998, 5:492-496. New perspectives for NMR spectroscopy are described. 5. Prestegard JH: New techniques in structural NMR-anisotropic • interactions. Nat Struct Biol 1998, 5:517-522. Modern NMR techniques that permit the determination of structures using methods other than nuclear Overhauser enhancements are clearly revealed. 6. Poveda A, Jiménez-Barbero J: NMR investigations of carbohydrate • protein interactions in solution. Chem Soc Rev 1998, 27:133-143. An up-to-date review of the applications of NMR spectroscopy to studying sugar–protein molecular recognition processes. 7.
Naidoo KJ, Brady JW: Calculation of the Ramachandran potential of mean force for a disaccharide in aqueous solution. J Am Chem Soc 1999, 121:2244-2252.
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Perez S, Imberty A, Engelsen SB, Gruza J, Mazeau K, Jimenez-Barbero J, Poveda A, Espinosa JF, van Eick BP, Johnson G et al.: A comparison and chemometric analysis of several molecular mechanics force fields and parameters sets applied to carbohydrates. Carbohydr Res 1999, 314:141-155.
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Bose B, Zhao S, Stenutz R, Cloran F, Bondo PB, Bondo G, Hertz B, Carmichael I, Serianni AS: Three bond C-O-C-C spin coupling constants in carbohydrates: development of a Karplus relationship. J Am Chem Soc 1998, 120:11158-11173.
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interactions between the two GlcNAc chairs and tryptophan and tyrosine rings, as well as two protein–sugar hydrogen bonds, are responsible for the specific association.
11. Kozar T, Nifant’ev EN, Grosskurth H, Dabrowski U, Dabrowski J: Conformational changes due to vicinal glycosylation: the β-D-Galp(1-3)]β β-D-Glc1-OMe branched α-L-Rhap(1-2)[β trisaccharide compared with its parent disaccharides. Biopolymers 1998, 46:417-432.
25. Simpson PJ, Bolam DN, Cooper A, Ciruela A, Hazlewood GP, Gilbert HJ, Williamson MP: A family IIb xylan-binding domain has a similar secondary structure to a homologous family IIa cellulose-binding domain but different ligand specificity. Structure 1999, 7:853-864.
12. Dabrowski J, Grosskurth H, Baust C, Nifant’ev EN: Secondary H/D isotope effect on hydrogen bonded hydroxyl groups as a tool for recognizing distance constraints in conformational analysis of oligosaccharides. J Biomol NMR 1998, 12:161-172.
26. Spooner PJ, O’Reilly WJ, Homans SW, Rutherford NG, Henderson PJ, Watts A: Weak substrate binding to transport proteins studied by NMR. Biophys J 1998, 75:2794-2800.
13. Sandström C, Baumann H, Kenne L: The use of chemical shifts of hydroxy protons of oligosaccharides as conformational probes for NMR studies in aqueous solution. Evidence for persistent hydrogen bond interaction in branched trisaccharides. J Chem Soc Perkin Trans II 1998:2385-2393. 14. Rockwell GC, Grindley TB: Effect of solvation on the rotation of the hydroxymethyl groups in carbohydrates. J Am Chem Soc 1998, 120:10953-10963. 15. Almond A, Brass A, Sheehan JK: Dynamic exchange between stabilized conformations predicted for hyaluronan tetrasaccharides: comparison of molecular dynamic simulations with available NMR data. Glycobiology 1998, 6:1433-1444. 16. Dietrich HJ, Chiara JL, Espinosa JF, Jimenez-Barbero J, Leon Y, Varela-Nieto I, Mato JM, Cano FH, Foces-Foces C, Martin-Lomas M: Glycosyl inositol derivatives related to inositolphosphoglycan mediators: synthesis, structure and biological activity. Chem Eur J 1999, 5:320-336. 17.
Ottiger M, Bax A: Characterization of magnetically oriented phospholipid micelles for measurement of dipolar couplings in macromolecules. J Biomol NMR 1998, 12:361-372.
18. Rundlof T, Landersjo C, Lycknert K, Maliniak A, Widmalm G: NMR • investigation of oligosaccharide conformation using dipolar couplings in an aqueous dilute liquid crystalline medium. Magn Reson Chem 1998, 36:773-776. The authors show how it is possible to deduce the average conformation of a tetrasaccharide in solution using residual dipolar C–H couplings, without using nuclear Overhauser enhancement information. 19. Kiddle GR, Homans SW: Residual dipolar couplings as new • conformational restraints in isotopically 13C-enriched oligosaccharides. FEBS Lett 1998, 436:128-130. C–H residual dipolar couplings within a simulated annealing protocol were used to deduce the solution conformation of sialyl lactose. No nuclear Overhauser enhancement information is necessary to deduce the major conformation in solution. 20. Bolon PJ, Prestegard JH: COSY cross peaks from 1H-1H dipolar • couplings in NMR spectra of field oriented oligosaccharides. J Am Chem Soc 1998, 120:9366-9367. H–H residual dipolar couplings, which depend on the distance according to 3 1/r , were estimated using easy experimental NMR methods. The potential use of this method to relatively position remote rings is described. 21. Shimizu H, Donohue-Rolfe A, Homans SW: Derivation of the bound • state conformation of a ligand in a weakly aligned ligand-protein complex. J Am Chem Soc 1999, 121:5815-5816. As the alignment of a protein–ligand complex is substantially larger than that of the free ligand, residual dipolar couplings were used to deduce the toxinbound conformation of a trisaccharide, without using TR-NOE experiments. 22. Hamelryck TA, Loris R, Bouckaert J, Dao-Thi M, Strecker G, Imberty A, Fernandez E, Wyns L, Etzler ME: Carbohydrate binding, quaternary structure, and a novel hydrophobic binding site in two legume lectin oligomers from Dolichos biflorus. J Mol Biol 1999, 286:1161-1177. 23. Leonidas DD, Vatzaki EH, Vorum H, Celis JE, Madsen P, Acharya KR: • Structural basis for the recognition of carbohydrates by human galectin 7. Biochemistry 1998, 37:13930-13940. A comparison of the structural features of galectin-7 with other galectins explains their different selectivity for natural ligands. 24. Asensio JL, Cañada FJ, Bruix M, Gonzalez C, Khiar N, Rodriguez • Romero A, Jimenez-Barbero J: NMR investigations of carbohydrate-protein interactions: refined three dimensional structure of the complex between hevein and methyl chitobioside. Glycobiology 1998, 8:569-577. One of the few examples of a detailed three-dimensional structural analysis of a protein–carbohydrate complex in solution is described. Van der Waals
27. •
Yang D, Kay LE: TROSY triple resonance four dimensional spectroscopy of a 46 ns tumbling protein. J Am Chem Soc 1999, 121:2571-2575. Modern NMR techniques expand the range of application of NMR spectroscopy to the study large complexes in the near future. 28. Sokolowski T, Haselhorst T, Scheffer K, Weisemann R, Kosma P, Brade H, Brade L, Peters T: Conformational analysis of Chlamydiaspecific disaccharide Kdo-(2->8)-Kdp-(2->O)-allyl in aqueous solution and bound to a monoclonal antibody: observation of intermolecular transfer NOEs. J Biomol NMR 1998, 12:123-133. 29. Haselhorst T, Espinosa JF, Jimenez-Barbero J, Sokolowski T, Kosma P, Brade H, Brade L, Peters T: NMR experiments reveal distinct antibody-bound conformations of a synthetic disaccharide representing a general structural element of bacterial lipopolysaccharide epitopes. Biochemistry 1999, 38:6449-6459.
30. Shimizu H, Field RA, Homans SW, Donohue-Rolfe A: Solution structure of the complex between the B-subunit homopentamer of verotoxin VT-1 from Escherichia coli and the trisaccharide moiety of globotriaosylceramide. Biochemistry 1998, 37:11078-11082. 31. Henrichsen D, Ernst B, Magnani JL, Wang WT, Meyer B, Peters T: •• Bioaffinity NMR spectroscopy: identification of an E-selectin antagonist in a substance mixture by transfer NOE. Angew Chem Int Ed 1999, 38:98-102. Transferred nuclear Overhauser enhancement methods can discriminate an E-selectin antagonist from a complex mixture of similar compounds. Perspectives on the application and drawbacks of the method are discussed. 32. Mayer M, Meyer B: Characterization of ligand binding by saturation transfer difference NMR spectroscopy. Angew Chem Int Ed 1999, 38:1784-1788 33. Harris R, Kiddle GR, Field RA, Milton MJ, Ernst B, Magnani JL, • Homans SW: Stable isotope assisted NMR studies on 13C-enriched sialyl LewisX in solution and bound to E-selectin. J Am Chem Soc 1999, 121:2546-2551. 13C-labeled sialyl LewisX was used to explore its conformation while bound to E-selectin. Quantitative nuclear Overhauser enhancement (NOE)-derived distances and vicinal scalar C–H couplings were used as input for time-averaged molecular dynamics simulations to derive the free and bound conformations of the tetrasaccharide. The reported conformations significantly differs from those deduced in previous studies. 34. Milton MJ, Bundle DR: Observation of the anti-conformation of a glycosidic linkage in an antibody bound oligosaccharide. J Am Chem Soc 1998, 120:10547-10548. 35. Gilleron M, Siebert HC, Kaltner H, von der Lieth CW, Kozár T, Halkes KM, Korchagina EY, Bovin NV, Gabius HJ, Vliegenthart JFG: Conformer selection and differential restriction of ligand mobility by a plant lectin. Conformational behaviour of β1-3GlcNAcβ β1-R, Galβ β1-3GalNAcβ β1-R and Galβ β1-2Galβ β1-R' in Galβ the free state and complexed with galactoside-specific mistletoe lectin as revealed by random walk and conformational clustering molecular mechanics calculations, molecular dynamics simulations and nuclear Overhauser experiments. Eur J Biochem 1998, 252:416-427. 36. Hricovini M, Guerrini M, Bisio A: Structure of heparin-derived tetrasaccharide complexed to the plasma protein antithrombin derived from NOEs, J-couplings and chemical shifts. Eur J Biochem 1998, 261:789-801. 37. •
Bundle DR, Alibes R, Nilar S, Otter A, Warwas M, Zhang P: Thermodynamic and conformational implications of glycosidic rotamers preorganized for binding. J Am Chem Soc 1998, 120:5317-5318. Significant reduction of torsional flexibility failed to produce important entropic gains with the interaction of tethered ligands with a monoclonal antibody. Changes in the water structure around the tethers may contribute to the observed thermodynamic parameters.
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38. Navarre N, Amiot N, van Oijen A, Imberty A, Poveda A, Jimenez • Barbero J, Cooper A, Nutley MA, Boons G-J: Synthesis and conformational analysis of a conformationally constrained trisaccharide and complexation properties with concanavalin A. Chem Eur J 1999, 5:2281-2294. In contrast with the previous paper [37•], an important entropy gain is observed with the binding of a synthetically rigidified trisaccharide to the lectin concanavalin A. The entropy gain is offset by a significant lost of enthalpy. 39. Swaminathan CP, Surolia N, Surolia A: Role of water in the specific binding of mannose and mannooligosaccharides to concanavalin A. J Am Chem Soc 1998, 120:5153-5159. 40. Dam TK, Oscarson S, Brewer CF: Thermodynamics of binding of • the core trimannoside of asparagine-linked carbohydrates and deoxy analogs to Dioclea grandiflora lectin. J Biol Chem 1998, 273:32812-32817. The binding of monodeoxy derivatives of the typical trimannoside core of glycoproteins to Dioclea grandiflora lectin was studied by microcalorimetry. The role of every hydroxyl group was deduced from the thermodynamic binding parameters. 41. Rozwarski DA, Swami BM, Brewer CF, Sacchettini JC: Crystal • structure of the lectin from Dioclea grandiflora complexed with core trimannoside of asparagine-linked carbohydrates. J Biol Chem 1998, 273:32818-32825. The analysis of the crystal structure of the Dioclea grandiflora lectin–trimannoside complex permits the interpretation of the thermodynamic data in structural terms. The role of water molecules is also explained. 42. Dam TK, Oscarson S, Sacchettini JC, Brewer CF: Differential • solvation of “core” trimannoside complexes of the Dioclea grandiflora lectin and concanavalin A detected by primary solvent isotope effects in isothermal titration microcalorimetry. J Biol Chem 1998, 273:32826-32832. The complete picture of the binding process between Dioclea grandiflora lectin and the core trimannoside is described and compared with that for concanavalin A. Although most of the binding features are explained, the fine details of the differential binding selectivity remain unclear. 43. Sayers EW, Weaver JL, Prestegard JH: Hydrogen bonding geometry of a protein-bound carbohydrate from water exchange-mediated cross relaxation. J Biomol NMR 1998, 12:209-222. 44. Carlomagno T, Felli IC, Czech M, Fischer R, Sprinzl M, Griesinger C: • Transferred cross correlated relaxation: application to the determination of sugar pucker in an aminoacylated tRNA-mimetic weakly bound to EF-Tu. J Am Chem Soc 1999, 121:1945-1948. The puckering of a protein-bound sugar within a RNA mimetic was determined using new NMR experiments. The information gained depends on the correlation time of the molecule and, therefore, the collected data are dominated by the complexed state. 45. Blommers MJJ, Stark W, Jones CE, Head D, Owen CE, Jahnke W: • Transferred cross correlated relaxation complements TR-NOE. Structure of an IL-4R derived peptide bound to STAT-6. J Am Chem Soc 1999, 121:1949-1953. An identical approach to that described in [44•] was used to examine the conformational changes of a peptide when bound by a protein. Although isotope labeling is necessary, this methodology permits the expansion of knowledge concerning bound conformations of biomolecules. 46. Bernardi A, Checchia A, Brocca P, Sonnino S, Zuccotto F: Sugar mimics: an artificial receptor for cholera toxin. J Am Chem Soc 1999, 121:2032-2036. 47.
Reddy GB, Srinivas VR, Ahmad N, Surolia A: Molten globule-like state of peanut lectin monomer retains its carbohydrate specificity. J Biol Chem 1999, 274:4500-4503.
48. Heightman TD, Vasella AT: Recent insights into inhibition, structure, • and mechanism of configuration-retaining glycosidases. Angew Chem Int Ed 1999, 38:750-770. A detailed revision of the current view of the structure/mechanism relationship for retaining glycosidase enzymes. 49. Davies GJ, Mackenzie L, Varrot A, Dauter M, Brzozowski AM, •• Schulein M, Withers SG: Snapshots along an enzymatic reaction coordinate: analysis of a retaining beta-glycoside hydrolase. Biochemistry 1998, 37:11707-11713. A complete structural view of the pathway of the hydrolysis of a fluorosugar by a retaining glycosidase is provided by X-ray crystallography. 50. Sulzenbacher G, Mackenzie LF, Wilson KS, Withers SG, Dupont C, Davies GJ: The crystal structure of a 2-fluorocellotriosyl complex of the Streptomyces lividans endoglucanase CelB2 at 1.2 Å resolution. Biochemistry 1999, 38:4826-4833.
51. Notenboom V, Birsan C, Nitz M, Rose DR, Warren RA, Withers SG: Insights into transition state stabilization of the β-1,4-glycosidase Cex by covalent intermediate accumulation in active site mutants. Nat Struct Biol 1998, 5:812-818. 52. Schmidt A, Gbitz GM, Kratky C: Xylan binding subsite mapping in the xylanase from Penicillium simplicissimum using xylooligosaccharides as cryo-protectant. Biochemistry 1999, 38:2403-2412. 53. Gruber K, Klintschar G, Hayn M, Schlacher A, Steiner W, Kratky C: Thermophilic xylanase from Thermomyces lanuginosus: high-resolution X- ray structure and modeling studies. Biochemistry 1998, 37:13475-13485. 54. Muraki M, Harata K, Sugita N, Sato K: Dual affinity labeling of the active site of human lysozyme with an N-acetyllactosamine derivative: first ligand assisted recognition of the second ligand. Biochemistry 1999, 38:540-548. 55. Hilge M, Gloor SM, Rypniewski W, Sauer O, Heightman TD, Zimmermann W, Winterhalter K, Piontek K: High-resolution native and complex structures of thermostable beta-mannanase from Thermonospora fusca-substrate specificity in glycosyl hydrolase family 5. Structure 1998, 6:1433-1444. 56. Aguilera B, Jimenez-Barbero J, Fernandez-Mayoralas A: Conformational differences between Fuc(1-3)GlcNAc and its thioglycoside analogue. Carbohydr Res 1998, 308:19-27. 57. •
Espinosa JF, Bruix M, Jarreton O, Skrydstrup T, Beau JM, Jiménez Barbero J: Conformational differences between C- and O-glycosides: the C-mannobiose/O-mannobiose case. Chem Eur J 1999, 4:442-448. C-mannobiose presents a high population of conformers with a nonexoanomeric orientation of the α angle, demonstrating that, in general, C-and O-glycoside conformations are different. 58. Ravishankar R, Surolia A, Vijayan M, Lim S, Kishi Y: Preferred • conformation of C-lactose at the free and peanut lectin bound states. J Am Chem Soc 1998, 120:11297-11303. C-lactose is bound by peanut lectin in the same conformation as O-lactose. Key hydrogen bonds between the protein and a glucose residue in the syn conformation provide the required interactions for the exclusive recognition of this conformer. 59. Espinosa JF, Montero E, Vian A, Garcia JL, Dietrich H, Schmidt RR, •• Martin-Lomas M, Imberty A, Cañada J, Jimenez-Barbero J: E. coli galactoside recognizes a high energy conformation of C-lactose, a non hydrolyzable substrate analogue. NMR investigations of the molecular complex. J Am Chem Soc 1998, 120:1309-1316. NMR spectroscopy and molecular modeling were used to demonstrate the exclusive recognition of a local minimum conformation of C-lactose by a glycosidase enzyme. This fact indirectly reveals the intrinsic flexibility of C-lactose and provides experimental proof for conformational distortion at an enzyme-binding and/or catalytic site. 60. Asensio JL, Espinosa JF, Dietrich H, Cañada FJ, Schmidt RR, • Martín Lomas M, André S, Gabius HJ, Jiménez-Barbero J: Bovine heart galectin-1 selects an unique (syn) conformation of C-lactose, a flexible lactose analogue. J Am Chem Soc 1999, in press. Three different proteins, namely galectin-1, E. coli β-galactosidase and ricinB, select three different conformations of the same synthetic analog, C-lactose. Despite its lack of homology to peanut agglutinin, identical interactions to those described in [58•] provide the required interactions for the exclusive recognition of the syn-Φψ conformer by bovine heart galectin-1. 61. Song J, Hollingsworth IR: Synthesis, conformational analysis and phase characterization of a versatile self-assembling monoglucosyl diacylglycerol analogue. J Am Chem Soc 1999, 121:1851-1861. 62. Wu W, Pasternack L, Hwanh DH, Koeller KM, Lin CC, Seitz O, Wong C-H: Structural study on O-glycopeptides: glycosylation induced conformational changes of O-GlcNac, O-LacNAc, O-sialyl-LacNAc, and O-sialyl-Lewis X peptides of the mucin domain of MAdCAM-1. J Am Chem Soc 1999, 121:2409-2417. 63. Yamaguchi Y, Kato K, Shindo M, Aoki S, Furusho K, Koga K, Takahashi N, Arata Y, Shimada I: Dynamics of the carbohydrate chains attached to the Fc portion of immunoglobulin G as studied by NMR spectroscopy assisted by selective 13C labeling of the glycans. J Biomol NMR 1998, 12:385-394. 64. Live DH, Williams LJ, Kuduk SD, Schwarz JB, Glunz PW, Chen XT, • Sames D, Kumar RA, Danishefsky SJ: Probing cell-surface architecture through synthesis: an NMR-determined structural
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motif for tumor-associated mucins. Proc Natl Acad Sci USA 1999, 96:3489-3493. Glycopeptide synthesis, coupled with detailed NMR analysis, provides insights into the design of a basic sequence that is able to mimic an elongated mucin motif.
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non-glycosylated derivative: effects of glycosylation on solution conformation. Biochemistry 1999, 38:705-714.
65. Woods RJ, Pathiaseril A, Wormald MR, Edge CJ, Dwek EA: The high degree of internal flexibility observed for an oligomannose oligosaccharide does not alter the overall topology of the molecule. Eur J Biochem 1998, 258:372-386.
67. O’Connor SE, Imperiali B: A molecular basis for glycosylation • induced conformational switching. Chem Biol 1998, 5:427-437. The conformation and dynamics of different glycopeptides with natural and modified chitobioses was analyzed by NMR spectroscopy. The authors state that the N-acetyl groups on the sugars have a key influence on the glycopeptide conformation.
66. McManus AM, Otvos L Jr, Hoffmann R, Craik DJ: Conformational studies by NMR of the antimicrobial peptide, drosocin, and its
68. Hoffmann D, Florcke H: A structural role for glycosylation: lessons from the hp model. Fold Des 1998, 3:337-343.