NMR investigations of protein-carbohydrate interactions: refined three-dimensional structure of the complex between hevein and methyl β-chitobioside

Juan Luis Asensio 2 Francisco Javier Caada 2 Marta Bruix 1 2 Carlos Gonzlez 1 2 Noureddine Khiar 2 Adela Rodrguez-Romero 0 2 Jess Jimnez-Barbero 2 0 Instituto de Qumica , UNAM, Ciudad Universitaria , Mxico D.F. 04510, Mxico 1 Instituto de Estructura de la Materia , CSIC, Madrid, Spain 2 Instituto de Qumica Orgnica General , CSIC, Madrid, Spain - Received on September 29, 1997; revised on October 29, 1997; accepted on October 30, 1997 3To whom correspondence should be addressed at: Departamento de Qumica Orgnica Biolgica, Instituto de Qumica Orgnica General, CSIC, Juan de la Cierva 3, 28006 Madrid, Spain The specific interaction of hevein with GlcNAc-containing oligosaccharides has been analyzed by 1H-NMR spectroscopy. The association constants for the binding of hevein to a variety of ligands have been estimated from 1H-NMR titration experiments. The association constants increase in the order GlcNAc-(a1 6)-Man < GlcNAc < benzyl-b -GlcNAc < p-nitrophenyl-b -GlcNAc < chitobiose < p-nitrophenyl-b -chitobioside < methyl-b -chitobioside < chitotriose. Entropy and enthalpy of binding for different complexes have been obtained from vant Hoff analysis. The driving force for the binding process is provided by a negative D H0 which is partially compensated by negative D S0. 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 475 accurate protein protonproton distance constraints after employing the MARDIGRAS program. In addition, 15 unambiguous protein/carbohydrate NOEs were detected. All the experimental constraints were used in a refinement protocol including restrained molecular dynamics in order to determine the highly refined solution conformation of this proteincarbohydrate 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 20 refined structures was 0.055 nm, while the heavy atom rmsd was 0.116 nm. It can be deduced that both hydrogen bonds and van der Waals contacts confer stability to the complex. A comparison of the three-dimensional structure of hevein in solution to those reported for wheat germ agglutinin (WGA) and hevein itself in the solid state has also been performed. The polypeptide conformation has also been compared to the NMR-derived structure of a smaller antifungical peptide, Ac-AMP2. Introduction The understanding of how oligosaccharides are recognized (Lasky, 1992; Dwek, 1996; Gabius and Gabius, 1996) by the binding sites of lectins, antibodies, and enzymes is currently a topic of major interest. Detailed information on the three-dimensional structure of proteincarbohydrate complexes has usually been obtained from x-ray crystallography data (Vyas, 1991; Weis and Drickamer, 1996) and modeling (Imberty et al., 1993), since the usually high molecular weight of lectins has prevented their direct studies by means of NMR spectroscopy. However, NMR may also provide information about the driving forces behind proteincarbohydrate interactions in solution (Peters and Pinto, 1996; Richardson et al., 1997). Hevein is a chitin-binding 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. Therefore, it has been suggested that hevein plays a major role in the protection of plants from attack by a wide range of potential pathogens, including fungi (Beintema, 1994; Gidrol et al., 1994). From a structural point of view, hevein is a small, single chain protein of 43 amino acids especially rich in glycines and cysteins (Rodriguez et al., 1986), whose structure has independently been solved in the solid state by x-ray at 0.28 nm resolution (Rodriguez-Romero et al., 1991), and in solution by NMR methods both in water (Asensio et al., 1995a) and in dioxane/water (Andersen et al., 1993), in the last few years. The topology of hevein in solution (Andersen et al., 1993; Asensio et al., 1995a) differs significantly from that observed in the crystal (Rodriguez-Romero et al., 1991). However, it closely resembles the solid state structures of the different domains of wheat germ agglutinin (WGA) (Wright, 1990, 1992) and the solution structure of a smaller polypeptide chain isolated from Amaranthus caudatus, Ac-AMP2 (Martins et al., 1996). According to the x-ray studies (Wright, 1990, 1992), the four domains of WGA have similar three-dimensional structures and, indeed of primary structure, hevein shows a 56% sequence identity to the C domain of WGA. In addition, it has been recently proposed that hevein is involved in the coagulation of latex (Gidrol et al., 1994) by interacting with a 22 kDa glycoprotein through binding to a N-acetyl-glucosamine (GlcNAc). A previous report of our group (Asensio et al., 1995a) demonstrated that hevein binds chitobiose and chitotriose with millimolar affinity and that the binding process is enthalpy driven (Asensio et al., 1995a). This recognition process is not calcium-dependent, in contrast with the proposal made by Gidrol et al. (1994). We also described the interaction between hevein and chitobiose in structural terms, using a NMR-derived three dimensional structure of the protein (Asensio et al., 1995a). Following our studies on the interaction of hevein with chitin-derived oligosaccharides, within a global program directed to the study of proteincarbohydrate interactions in solution (Rivera-Sagredo et al., 1991, 1992; Solis et al., 1993, 1994; Asensio et al., 1995a,b; Espinosa et al., 1996a,b), we now report on the determination of the association constants Fig. 1. Comparison of part of the 1H NMR spectrum of 0.5 mM hevein obtained in the presence of p-nitrophenyl-N-acetyl-b -glucosaminide in 1H2O (A) with that obtained for the free protein (B) (3.8 mM) at 303 K and pH 5.6. between hevein and a variety of N-acetyl glucosamine containing oligosaccharides by using NMR spectroscopy. In addition, the thermodynamic parameters for the methyl b -chitobioside-hevein interactions have been obtained. We also present a highly refined NMR structure (0.055 nm backbone rmsd over residues 341) of the molecular complex between hevein and methyl b -chitobioside in water, based on the accurate analysis of 475 protein protein and 15 protein/carbohydrate NOEs. It has to be mentioned that, although a few examples of solution structures of glycoproteins have been recently solved (Wyss et al., 1995; de Beer et al., 1996; Dwek, 1996; Mer et al., 1996; Weller et al., 1996; Gervais et al., 1997) to the best of our knowledge, this example represents the first case of a highly refined NMR three-dimensional structure of a noncovalent protein/carbohydrate complex in solution (Xu et al., 1995; Johnson et al., 1996; Richardson et al., 1997). Finally, the differences in (...truncated)