Role of aromatic amino acids in carbohydrate binding of plant lectins: Laser photo chemically induced dynamic nuclear polarization study of hevein domain-containing lectins

Abstract

Carbohydrate recognition by lectins often involves the side chains of tyrosine, tryptophan, and histidine residues. These moieties are able to produce chemically induced dynamic nuclear polarization (CIDNP) signals after laser irradiation in the presence of a suitable radical pair-generating dye. Elicitation of such a response in proteins implies accessibility of the respective groups to the light-absorbing dye. In principle, this technique is suitable to monitor surface properties of a receptor and the effect of ligand binding if CIDNP-reactive amino acids are affected. The application of this method in glycosciences can provide insights into the protein-carbohydrate interaction process, as illustrated in this initial study. It focuses on a series of N-acetylglucosamine-binding plant lectins of increasing structural complexity (hevein, pseudohevein, Urtica dioica agglutinin and wheat germ agglutinin and its domain B), for which structural NMR- or X-ray crystallographic data permit a decision of the validity of the CIDNP method-derived conclusions. On the other hand, the CIDNP data presented in this study can be used for a rating of our molecular models of hevein, pseudohevein, and domain B obtained by various modeling techniques. Experimentally, the shape and intensity of CIDNP signals are determined in the absence and in the presence of specific glycoligands. When the carbohydrate ligand is bound, CIDNP signals of side chain protons of tyrosine, tryptophan, or histidine residues are altered, for example, they are broadened and of reduced intensity or disappear completely. In the case of UDA, the appearance of a new tryptophan signal upon ligand binding was interpreted as an indication for a conformational change of the corresponding indole ring. Therefore, CIDNP represents a suitable tool to study protein-carbohydrate interactions in solution, complementing methods such as X-ray crystallography, high-resolution multidimensional nuclear magnetic resonance, transferred nuclear Overhauser effect experiments, and molecular modeling.

Analysis of Protein−Carbohydrate Interaction at the Lower Size Limit of the Protein Part (15-Mer Peptide) by NMR Spectroscopy, Electrospray Ionization Mass Spectrometry, and Molecular Modeling

“…Further analysis (distance matrix approach) of the probability of pseudoatom residence in relation to any other sequence part (boundary set to distance <10 Å) derived a measure of spatial correlation of the complete set of building blocks. As positive control for these calculations, the lectin-ligand complexes of hevein and PNA were also subjected to these calculations using the parameters of the NMR solution structure of hevein (22)(23)(24)27) and the crystallographic parameters of PNA (47,48,63) to start the MD runs at 300 K in a water box. Any occurrence of spatial vicinity indicative of presence of secondary/tertiary structural elements or contact to the ligand will show up above or beyond the diagonal line.…”

“…The major secondary structural elements are an antiparallel -sheet formed by two tetrapeptide stretches within the sequence Leu16-Ser26 and a short R-helix from Asp28 to Ser32 together with four disulfide bridges (22)(23)(24)(25)(26). For heveinligand interaction, Trp21 and Tyr30 provide favorable stacking and a hydrogen bond (Tyr30 with OH-3 of the nonreducing GlcNAc residue) so that the ligand comes into contact with the aromatic residues (23,24,27). The antimicrobial protein Ac-AMP2 with three disulfide bridges shares the antiparallel -sheet from Met13-Lys23 to an extent that the root-mean-square deviations of backbone atoms (residues 12-32) to those of hevein amount to only 0.1 nm (24,28).…”

NMR investigations of protein–carbohydrate interactions

“…WGA‐B was essentially prepared as previously described [35] with some modifications. In short, a Sca l– Kpn l fragment encoding the WGA‐B domain was obtained from plasmid pCWB [35] and subcloned into plasmid pCUZ1 [45] previously digested with the same enzymes. The resulting plasmid pJFC encoding a fusion protein C‐LYTA‐WGA‐B (where C‐LYTA is the C‐terminal domain of LytA) was transformed into Escherichia coli DH5α.…”

A New Combined Computational and NMR-Spectroscopical Strategy for the Identification of Additional Conformational Constraints of the Bound Ligand in an Aprotic Solvent

“…[51, 52, 71±73] Glycosylated poly-L-lysine was prepared by reductive amination with NaCNBH 3 as described previously. [69] Hevein was purified as described previously, [41] and its ligands were purchased from Sigma. The disaccharides Gal'a1-3Galb1-R, Gal'b1-2Galb1-R, and Gal'b1-3Galb1-R were synthesized as previously described in detail.…”