Characterization of heparin–protein interaction by saturation transfer difference (STD) NMR

Abstract

The binding affinity and specificity of heparin to proteins is widely recognized to be sulfation-pattern dependent. However, for the majority of heparin-binding proteins (HBPs), it still remains unclear what moieties are involved in the specific binding interaction. Here, we report our study using saturation transfer difference (STD) nuclear magnetic resonance (NMR) to map out the interactions of synthetic heparin oligosaccharides with HBPs, such as basic fibroblast growth factor (FGF2) and fibroblast growth factor 10 (FGF10), to provide insight into the critical epitopes of heparin ligands involved. The irradiation frequency of STD NMR was carefully chosen to excite the methylene protons so that enhanced sensitivity was obtained for the heparin-protein complex. We believe this approach opens up additional application avenues to further investigate heparin-protein interactions.

Current structural biology of the heparin interactome

“…NMR saturation transfer difference of the heparin/ FGF-2 interaction confirms that the binding epitope of an octasaccharide is in the central residues, not at either end [44]. A long-standing study of a set of synthetic hexasaccharides with systematically varying sulfate substitutions [45 ] and papers cited therein, has identified two remarkable structures: one of which binds FGF-1 and Current heparin interactome Pomin and Mulloy 21 …”

Glycosaminoglycan-Protein Interactions by Nuclear Magnetic Resonance (NMR) Spectroscopy

“…STD has been used to investigate the GAG specificity of numerous GAG-binding proteins, including basic fibroblast growth factor (bFGF or FGF2). Crystal structure of FGF2 with heparin showed IdoA2S-GlcNS disaccharide units are essential for strong FGF2-GAG interactions and this was later confirmed by Yu et al through STD ( Figure 5 ) [ 25 ]. In Figure 5 A, the 1D 1 H reference spectrum of 30 μM of FGF2 and 370 μM of a synthetic heparin octasaccharide composed of only IdoA2S-GlcNS disaccharide units ( Figure 5 B) is shown.…”

“…Saturation transfer difference (STD) is a NMR experiment designed to map ligand protons that are involved in the interaction with protein [ 22 , 23 , 24 , 25 ]. In this method a group of protein protons, whose resonance frequencies do not overlap with any resonance frequency of the ligand, is selectively saturated.…”

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Synthetic Oligosaccharide Libraries and Microarray Technology: A Powerful Combination for the Success of Current Glycosaminoglycan Interactomics

“…In glycosaminoglycanomics, for example, GAG‐like molecules (mostly oligosaccharides of well‐defined structures and sizes) have been successfully synthesized . GAG synthesis can be accomplished either by chemoenzymatic routes– or based on modular assembly of pre‐synthesized building blocks –––. Module‐based synthesis is sometimes used in conjunction with additional chemical and/or enzymatic steps to increase yield and diversity of sulfation patterns .…”