Wheat germ agglutinin dimers bind sialyloligosaccharides at four sites in solution: proton nuclear magnetic resonance temperature studies at 360 MHz

Abstract

Equilibrium binding studies have been performed over a range of temperatures from 25.4 to 47.3.degree. C between wheat germ agglutinin isolectin I (WGA I) and the .alpha.2-3 isomer of (N-acetylneuraminyl)lactose (NeuNAc.alpha.2-3Gal.beta.1-4Glc). Proton NMR spectroscopy at 360 MHz has been used to monitor titrations in this system under conditions where the fraction of total ligand which is bound is small, yet the fractional occupation of sites covers a wide range. Several of the ligand resonances, including the N-acetyl methyl and the axial and equatorial hydrogens at carbon 3 of the NeuNAc residue, are shifted and broadened in the presence of WGA due to chemical exchange between the free and bound environments. The lifetime broadening of the N-acetyl resonance at room temperature of a series of related sialyloligosaccharides has been previously used to measure binding affinities to 2 WGA isolectins. The temperature dependence of the apparent bond shifts and the apparent bound line widths of the N-acetyl, H3a, and H3e peaks are reported. The true bound shifts for the 3 resonances have been obtained from these data by using the equations derived by Swift and Connick (1962). The total bound shifts, per monomer, were -1.98, -4.0, and -0.8 ppm for the N-acetyl, the H3a, and the H3e resonances, respectively. The N-acetyl data are consistent with there being 4 NeuNac binding sites on the WGA I dimer, each with a bound shift for the N-acetyl resonance of -0.99 .+-. 0.03 ppm. This in contrast to the X-ray diffraction studies that revealed only 2 NeuNAc sites per dimer. The sources of the ring current shifts for the N-acetyl resonance have been assigned to 2 equivalent sets of tyrosine aromatic side chains per monomer (Tyr-73 and Tyr-159). When the 1H NMR and The X-ray diffraction data are compared, it is apparent that the bound NeuNAc residue occupies a position in each binding site that is analogous to that occupied by the terminal GlcNAc of WGA-bound oligomers of GlcNAc. The N-acteyl of the bound NeuNAc or GlcNAc residue is oriented in the same way over the face of a Tyr ring in each binding site.