The C-terminal domains of two homologous Oleaceae ß-1,3-glucanases recognise carbohydrates differently: Laminarin binding by NMR.

Ole e 9 and Fra e 9 are two allergenic ß-1,3-glucanases from olive and ash tree pollens, respectively. Both proteins present a modular structure with a catalytic N-terminal domain and a carbohydrate-binding module (CBM) at the C-terminus. Despite their significant sequence resemblance, they differ in some functional properties, such as their catalytic activity and the carbohydrate-binding ability. Here, we have studied the different capability of the recombinant C-terminal domain of both allergens to bind laminarin by NMR titrations, binding assays and ultracentrifugation. We show that rCtD-Ole e 9 has a higher affinity for laminarin than rCtD-Fra e 9. The complexes have different exchange regimes on the NMR time scale in agreement with the different affinity for laminarin observed in the biochemical experiments. Utilising NMR chemical shift perturbation data, we show that only one side of the protein surface is affected by the interaction and that the binding site is located in the inter-helical region between α1 and α2, which is buttressed by aromatic side chains. The binding surface is larger in rCtD-Ole e 9 which may account for its higher affinity for laminarin relative to rCtD-Fra e 9.

Ligand-based nuclear magnetic resonance screening techniques.

A critical step in the drug discovery process is the identification of high-affinity ligands for macromolecular targets, and, over the last 10 years, NMR spectroscopy has become a powerful tool in the pharmaceutical industry. Instrumental improvements in recent years have contributed significantly to this development. Digital recording, cryogenic probes, autosamplers, and higher magnetic fields shorten the time for data acquisition and improve the spectral quality. In addition, new experiments and pulse sequences make a vast amount of information available for the drug discovery process. All these techniques take advantage of the fact that upon complex formation between a target molecule and a ligand, significant perturbations can be observed in NMR-sensitive parameters of either the target or the ligand. These perturbations can be used qualitatively to detect ligand binding or quantitatively to assess the strength of the binding interaction. In addition, some of the techniques allow the identification of the ligand-binding site or which part of the ligand is responsible for interacting with the target.In this chapter, we will use examples from our own research to illustrate how NMR experiments to characterize ligand binding may be used to both screen for novel compounds during the process of lead generation, and provide structural information useful for lead optimization during the latter stages of a discovery program.