Distance mapping of protein-binding sites using spin-labeled oligosaccharide ligands

Abstract

The binding of a nitroxide spin-labeled analog of N-acetyllactosamine to galectin-3, a mammalian lectin of 26 kD size, is studied to map the binding sites of this small oligosaccharide on the protein surface. Perturbation of intensities of cross-peaks in the (15)N heteronuclear single quantum coherence (HSQC) spectrum of full-length galectin-3 owing to the bound spin label is used qualitatively to identify protein residues proximate to the binding site for N-acetyllactosamine. A protocol for converting intensity measurements to a more quantitative determination of distances between discrete protein amide protons and the bound spin label is then described. This protocol is discussed as part of a drug design strategy in which subsequent perturbation of chemical shifts of distance mapped amide cross-peaks can be used effectively to screen a library of compounds for other ligands that bind to the target protein at distances suitable for chemical linkage to the primary ligand. This approach is novel in that it bypasses the need for structure determination and resonance assignment of the target protein.

Fig. 1.

Comparison of HSQC plots of (A) galectin-3 with no spin label and (B) galectin-3 with 30 times excess spin label. Crosspeaks in the primary binding site are labeled with corresponding assignments. Crosspeaks in the secondary binding site are labeled with asterisks.

Fig. 2.

X-ray structure of galectin-3 modeled with LacNAc-TEMPO. The region in dark corresponds to residues affected primarily by the spin label. The distance to the most severely broadened resonance corresponding to residue 184 (shown in green) is indicated by the dashed line.

Fig. 3.

Scheme for the synthesis of LacNAc-TEMPO. (*) Refer to text for numbered compounds.