Carbohydrate–Protein Interactions: A 3D View by NMR

ANA ARDÁ, ANGELES CANALES, F. JAVIER CAÑADA AND JESÚS JIMENEZ-BARBERO

1.1 Introduction

Nowadays it is well established that carbohydrates have exceptional properties for coding information. This also holds true for carbohydrate receptors, lectins, antibodies and enzymes, which translate the sugar-based signals into cellular effects. In fact, one of the important roles of carbohydrates in Nature is to serve as recognition points for molecular receptors (which can be grouped into enzymes, lectins and antibodies), giving rise to a specific molecular recognition process that triggers a given biological response. The knowledge of the structural elements that govern such molecular recognition events is fundamental, and furthermore opens the possibilities to intervene in them with therapeutic purposes.

Oligosaccharides are involved in a plethora of regulatory processes, such as bacterial/viral infection, angiogenesis, inflammation, cell growth and development. Understanding the chemical basis of carbohydrate–receptor interactions not only gives a functional meaning to structures and changes occurring in diseases but also helps devise innovative therapeutic approaches. Therefore, the comprehension of the conformational, dynamics and spatial presentation features of saccharides is of paramount importance. NMR spectroscopy has been demonstrated to be a robust tool for carbohydrate research. In fact, different NMR approaches are widely employed to study the interactions of carbohydrates and chemical analogues (glyco-mimetics) with their receptors up to the level of atomic resolution, both from the perspective of the carbohydrate ligand and from the receptor. The most accessible observables related to the structure are chemical shifts (δ), scalar couplings (J) and nuclear Overhauser effects (NOEs). However, in studies with complex oligosaccharides there are limits to the amount of relevant structural information provided by these observables, due to problems of signal overlapping, strong coupling and/or the scarcity of the key NOE information. In this sense, there is increasing use of additional parameters with structural information, such as residual dipolar couplings (RDCs), paramagnetic relaxation enhancements (PREs) or pseudo contact shifts (PCSs) induced by a paramagnetic ion. We will discuss all these parameters in this chapter.

Carbohydrates are rather flexible molecules. Therefore, NMR observables do not always correlate with a single conformer but with an ensemble of low free-energy conformers that can be accessed by thermal fluctuations. In this regard, NMR parameters should be complemented by computational methods in attempts to unravel the structural and conformational features of the molecular recognition process unambiguously.

Depending on the system under study, different NMR approaches can be followed to characterize protein–carbohydrate interactions; the standard methodologies can usually be classified as "ligand-based" or "receptor- based". The selection of the proper methodology is usually determined by the size of the receptor, the dissociation constant of the complex (KD), the availability of labelled protein (15N, 13C) and the access to soluble receptors at enough concentration for NMR measurements.

1.2 Ligand-Based Approach

This is the most frequently employed methodology in NMR-based screening applied to drug discovery programs, both in academia and in industry. As the "ligand-observed detection" name suggests, detection takes place on the resonances of the free ligand. Ligand recognition can be identified thanks to the different motions of the receptor and ligand molecules (Figure 1.1): upon carbohydrate recognition the motional properties of the ligand are similar to the receptor and this change in mobility can be detected by different NMR experiments.

Ligand-based methods require complexes with relatively fast kinetics. This means dissociation constants in the range KD ≥ 100 μM. If kon is well approximated by a diffusion-limited value (107–109 M-1 s-1), then the slowest exchange rate (kon) values lie in the range 1000<koff 100 000 s-1. Even though, in the biological context, carbohydrates are generally attached to proteins or lipids (glycoproteins and glycolipids), most of the studies of the interactions between carbohydrates and their receptors by NMR in solution are carried out by using free sugars. Thus, in the absence of multivalent effects, their binding strengths to proteins are usually rather weak. Thereby, in neutral carbohydrates the dissociation constants are usually in the mM to low μM range and the ligand-based approach can be successfully used.

In such a situation, we profit from the fact that, in most cases, carbohydrate ligands dissociate relatively fast from the receptor's binding site. If the receptor is much larger than the ligand, its larger correlation time will dominate the...