Characterizing carbohydrate-protein interactions by nuclear magnetic resonance spectroscopy

Abstract

Interactions between proteins and soluble carbohydrates and/or surface displayed glycans are central to countless recognition, attachment and signaling events in biology. The physical chemical features associated with these binding events vary considerably, depending on the biological system of interest. For example, carbohydrate-protein interactions can be stoichiometric or multivalent, the protein receptors can be monomeric or oligomeric, and the specificity of recognition can be highly stringent or rather promiscuous. Equilibrium dissociation constants for carbohydrate binding are known to vary from micromolar to millimolar, with weak interactions being far more prevalent; and individual carbohydrate-binding sites can be truly symmetrical or merely homologous, and hence, the affinities of individual sites within a single protein can vary, as can the order of binding. Several factors, including the weak affinities with which glycans bind their protein receptors, the dynamic nature of the glycans themselves, and the nonequivalent interactions among oligomeric carbohydrate receptors, have made nuclear magnetic resonance (NMR) an especially powerful tool for studying and defining carbohydrate-protein interactions. Here, we describe those NMR approaches that have proven to be the most robust in characterizing these systems, and explain what type of information can (or cannot) be obtained from each. Our goal is to provide the reader the information necessary for selecting the correct experiment or sets of experiments to characterize their carbohydrate-protein interaction of interest.

Keywords: complex-type glycan; glycan binding; multivalent; oligomannose.

Figure 1

Schematic representation of different types of carbohydrate-protein interactions.

Figure 2

NMR characterization of Manα1-2Man binding to CV-N. a) Chemical structures of Man₉GlcNAc₂ (Man-9) and Manα1-2Man; b) location of two symmetrically located binding sites on CV-N, identified by chemical shift mapping; c, d) chemical shift perturbations occurring with addition of 1 and 2 eq Manα1-2Man.

Figure 3

Chemical shift mapping and stoichiometry of α1,2-mannobiose binding to microvirin (MVN). a) Chemical shift perturbations as a function of residue number. No perturbations were observed for the second domain; b) location of the carbohydrate binding site on MVN, colored as in panel (a).

Figure 4

Schematic of and STD NMR experiment. Depiction of a) on-resonance (saturated) and b) off-resonance (control) experiments where saturation diffuses to the ligand in the on-resonance experiment. c) Subtraction of the spectra give a difference spectrum where the peak intensities are proportional to the distance of the ¹Hs from the receptor.

Figure 5

STD NMR spectrum of a complex-type glycan binding to the HIV-1 neutralizing antibody PG16. The difference spectrum and chemical shift assignments of the glycan demonstrate recognition of complex-type glycan through the Sial-Gal termini.

Figure 6

Single ligand titration STD NMR of Man₅GlcNAc₂ binding to the HIV-1 neutralizing antibody PG9. a) Difference spectrum indicates recognition of oligomannose; b) integration and c) fitting of the N-acetyl methyl signals allows determination of the K_d of binding.

Figure 7

STD NMR spectrum of α/β L-fucose bound to norovirus P domain protein. Binding of the unnatural β-anomer dominates binding to the protein.