Hevein domains

The arsenal of plant defense peptides contains members capable of binding carbohydrate residues, namely /31-4 linked A -acetyl glucosamine residues that form the biopolymer chitin. The actual mode of action remains unclear. Antifungal and antimicrobial activity has been shown in vitro. For example Ac-AMP2 is a small disulfide-rich chitin-binding peptide isolated from the seeds of Amaranthus caudatus with antimicrobial activity. It differs from Ac-AMP 1 by one additional arginine residue at the C-terminus. The structure was determined by NMR and contains a cystine knot motif. Ac-AMP2 displays a so-called hevein domain partly... 

Posch, A., et al. (1999). Class I endochitinase containing a hevein domain is the causative allergen in latex-associated avocado allergy, Clin. Exp. Allergy, 29, 5, 667-672. 

HEVEIN DOMAINS AN ATTRACTIVE MODEL TO STUDY CARBOHYDRATE-PROTEIN INTERACTIONS AT ATOMIC RESOLUTION... 

III. The Hevein Domain Basic Features and Biological Relevance 305... 

The small size of hevein (43 residues), and the ease of its availability by biochemical purification or methods of peptide synthesis make this domain an excellent model system for the study of carbohydrate recognition by proteins. Herein, and taking the hevein domain as a model, we focus on the study of those molecular-recognition features relevant for the interactions between carbohydrates and proteins. We detail all of the techniques that are instrumental for tackling this problem, and how these can strategically be combined in an efficient manner. Particular emphasis is placed on the acquisition and analysis of data at atomic resolution (by NMR and/or X-ray ), and how these structural data relate with thermodynamic and kinetic information in reaching an understanding of the forces and interactions that play decisive roles in the interactions between carbohydrates and proteins. 

Hevein domains included in ibis article and their origin... 

Fig. 1. NMR-titration experiments permit the detection of binding as well as delineating the protein region responsible for interaction with the sugar. The different spectra illustrate the variations in chemical shifts of a hevein domain upon addition of increasing molar amounts of (G1cNAc)3. Distinct key resonances are labeled.

Representative Data on Association Constants of Sugar Binding to Hevein Domains as Measured by Different Methods... 

Fig. 2. Chemical-shift perturbation analysis of a hevein domain. The bars indicate the maximum chemical shift differences for the backbone protons between free hevein and the hevein-(GlcNAc)3 complex.

Fig. 3. Titration data obtained by DOSY experiments for (GlcNAc) when added in portions to a hevein domain-containing solution. The plot is of the change in log D (Alog D) as a function of stoichiometrically added (GlcNAc) to a hevein domain containing solution. The predicted AlogZ) values for free hevein (1 0), as well as 1 1 and 2 1 protein-oligosaccharide complexes, are indicated in the right part of the panel.

Representative studies on the oligomerization state of hevein domains in the ligand-free and ligand-containing states, as determined by different... 

Fig. 4. Section of the NOESY spectrum for a complex between (GlcNAc)3 and a hevein domain. Key intermolecular protein-sugar NOEs are highlighted. These NOE peaks permit location of the position of the binding site and to gauge the orientation of the sugar in the 3D structure of the complex.

Representative Studies on Application of Laser Photo-CIDNP, Fluorescence, and IR to Monitor Ligand Interaction with Hevein Domains... 

Other biophysical methods, such as fluorescence can be used to monitor sugar binding to lectins.The presence of Trp units bound in the hevein domain provides a key point for spectroscopic probing of hevein-sugar interactions, and fluorescence can be used to detect and measure the binding affinity of lectin... 

Analytical ultracentrifugation experiments determine shape parameters and the average molecular weight of large molecules in solution, and can thus be used to deduce the stoichiometry of the complexes between hevein domains and... 

Several complexes of single hevein domains bound to oligosaccharide ligands have been studied at atomic resolution and their binding-energy features analyzed. 

Statistics of the NMR-Based Complexes between Hevein Domains and (GlcNAc)3... 

Comparisons have been made of the 3D structure of hevein in solution with the structures reported for other hevein domains, including WGA and hevein itself in the solid state.Despite differences in the number and nature of several amino acid residues, the polypeptide conformation has also been compared with the NMR-derived structure of a smaller antifungal peptide (30 amino acids) termed Ac-AMP2. The interactions just described have also been observed in the crystal structures of WGA-chitobiose and WGA-sialyllactose (see later). In all cases, the obtained conformations and intermolecular protein-sugar interactions are indeed similar (see later), regardless of the experimental method used to determine the 3D structure. 

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