Weak Carbohydrate–Protein Interactions by NMR


Nuclear magnetic resonance (NMR) provides several complementary tools to characterise and study biomolecular interactions between molecules. In particular, the fast and weak interactions between a small ligand and a large receptor can be analysed in the equilibrium state. In adequate conditions, it can characterise if a small molecule is interacting with a protein, if the conformation of the ligand changes upon binding, the regions of the ligand that are involved in the binding, and even the bound conformation within the complex. The process of association‐dissociation should be fast, and consequently, a single set of averaged signals carrying the information of both states detected. Typically, the protein is in lower proportion than the ligand and practically the signals from the ligand dominate the spectra. Besides, as the magnitudes used depend on the tumbling of the molecule (NOE, transfer of magnetisation or relaxation times) the average becomes very displaced towards the complex due to its larger correlation times.

Key Concepts

  • Fast equilibrium in the NMR time scale between the free and the bound ligand is needed.
  • Magnitudes depending on the molecular correlation time (dipolar coupling, saturation transference, relaxation times) are monitored.
  • Small amounts of complex thank the favourable molecular correlation time dominate the average of the corresponding magnitude.
  • The behaviour of the ligand complexed can be deduced from the observation of the free ligand signals.
  • In Transfer NOESY, dipolar transference within the bound ligand is observed.
  • In STD‐NMR transference between the ligand and the receptor, which is proportional to the closeness between ligand and receptor, is monitored.
  • In relaxation times analysis, differences between bound and free ligand are compared.
  • The WaterLOGSY experiment traces the magnetisation transfer between water – ligand and receptor.

Keywords: NMR ; protein–carbohydrate interaction; STD‐NMR ; transfer NOESY ; WaterLOGSY ; transient interactions

Figure 1. Structure of a heparin trisaccharide with the central iduronate in: (a) 1C4 and (b) 2SO conformation; (c) NOESY 500 MHz and 600 ms t mix; (d) transfer NOESY (800 MHz, 200 ms t mix) – the labels correspond to the assignment of cross‐peaks – notice the peak I2 H2–H5, a NOESY peak exclusive to the 2SO conformation; and (e) STD – affinity factor growing curves for the extraction of the initial rates for quantitative analysis. Reproduced with permission from Muñoz‐García et al. (). © American Chemical Society.
Figure 2. (a) Detail of the binding‐site structure of a mannose pseudodisaccharide bound to DC‐SIGN ECD (ExtraCellular Domain), showing the interactions between the Ca2+ ion and the terminal mannose through hydroxyls 3 and 4, and STD growing rates for selected protons; (b) experimental; and (c) CORCEMA‐calculated using the structure shown in (a). Reproduced with permission from Thepaut et al. (). © American Chemical Society.
Figure 3. Differential Epitope Mapping (0.6/6.55 ppm) of 2,7‐anhydro‐Neu5Ac in complex with RgNanH‐GH33 (left). (a) ΔSTD histogram: positive ΔSTDs (above the limit of +0.75) after aliphatic irradiation (0.6 ppm) are shown in cyan and negative ΔSTDs (below −0.75) after aromatic irradiation (6.55 ppm) in magenta. (b) DEEP‐STD map of the ligand. Cyan surfaces highlight ligand contacts with aliphatic side chains; magenta surfaces show contacts with aromatic side chains. (c) Crystal structure of the complex (PDB ID: 4X4A). Differential epitope mapping (D2O/H2O) of 2,7‐anhydro‐Neu5Ac in complex with RgNanH‐GH33 (right). (d) ΔSTD histogram: protons with an ΔSTD factor <−0.75 are shown in green. (e) DEEP‐STD map of the ligand. Green surfaces indicate ligand contacts with protein side chains carrying slowly exchanging protons. (f) Crystal structure of the complex (PDB ID: 4X4A). The slowly exchangeable protons in the binding pocket are depicted with green surfaces. Source: https://onlinelibrary.wiley.com/doi/full/10.1002/anie.201707682. Licensed under CC by 4.0.


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Further Reading

Arda A and Jimenez‐Barbero J (2018) The recognition of glycans by protein receptors. Insights from NMR spectroscopy. Chemical Communications 54: 4761–4769.

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Nieto, Pedro M(Mar 2019) Weak Carbohydrate–Protein Interactions by NMR . In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0028398]