Towards understanding the glycan code: next generation structural glycobiology for accurate description of protein-glycan complexes

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The extraordinary properties of living systems emerge as a result of dynamic interactions between their biomolecular components (proteins, nucleic acid, carbohydrates or glycans, ...). Despite the complexity of a system of millions of molecular interactions, the whole network of interactions is highly regulated for healthy organisms, and alterations in the regulation processes underlie all diseases. In this network, different molecules interact with different strengths (affinities), where strong interacting partners produce stable biomolecular complexes and weak binding molecules produce transient assemblies. A well "orchestrated" system of protein-ligand interactions of very different affinities is indeed responsible for most of the regulation processes in living organisms. Strong complexes are formed when sustained biological signals are needed, whereas weak interactions are recalled when quick cellular responses are required after temporary stimuli (signal transduction, reversible cell-cell contacts, transient interactions in host/pathogen recognition, etc.). For a complete understanding of life processes, it is necessary to investigate both strong and weak protein-ligand interactions, which encourages the development of novel approaches to characterize the 3D molecular structures of weak protein complexes. Strong protein-ligand interactions have been extensively investigated and many biologically relevant 3D complexes have been determined, as their intrinsic stability makes them amenable to a number of analytical techniques. However, for weak protein interactions many conventional approaches fail or become unreliable. For example, X-ray crystallography, a very powerful structural technique, show limitations for weak interactions as: (i) obtaining crystals of the complexes including the ligand is difficult, and (ii) the typically poorly defined electronic density that describe the ligand in the binding pocket. NMR spectroscopy, one of the most powerful techniques to study intermolecular interactions, has demonstrated its extraordinary capability for the detection of weak protein-ligand interactions in solution, through the use of ligand-based experiments, like Saturation Transfer Difference (STD) NMR spectroscopy. However, the translation from these experiments to 3D structures is currently not straightforward. We have published some improvements in the set up of STD NMR experiments to determine protein-ligand affinities and study multiple binding modes of ligands in a protein binding pocket. The present proposal stems from recent results in our research group that allow us to propose that STD NMR spectroscopy can provide more structural information for weak interactions than the map of ligand contacts, or group epitope mapping (GEM). Here, we propose to obtain the orientation of the ligand in the binding pocket, a much needed piece of information. We plan to generate new experimental restraints to drive the structural calculations of the complexes, to get accurate 3D structures. Until now, these experimental restraints have remained unexplored. Among the most biologically relevant weak interactions, those of proteins with glycans are essential steps in many cell-cell communication processes, and key in the infectivity of microbial pathogens. In particular, influenza virus exploits this for initial cell recognition, attachment, and release of new virions. In a different strategy, HIV covers its surface with host glycans to evade the immunological response. Interestingly, new broad neutralizing antibodies (bNAb) are being discovered which are able to stop infection and are elicited against those carbohydrates in that glycan shield. In the context of our collaborations with Prof. Rob Field (Norwich) and Dr. Katie Doores (London), we will apply the novel STD NMR approaches to investigate the molecular recognition processes in human and avian influenza virus, and in immunologically active bNAbs against HIV.