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

Lead Research Organisation: University of East Anglia
Department Name: Pharmacy


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.

Technical Summary

Weak specific protein-glycan interactions are biologically relevant as they constitute the translational elements of the so-called "glycan code" of high importance in the context of glycomics. However, their low affinity poses significant difficulties for the determination of 3D structures: X-ray crystallography can produce incorrect glycan structures, and NMR suffers from a lack of enough experimental restraints.

Many technical issues make difficult the structural calculations of 3D models of weak protein-ligand complexes:
(i) Intermolecular NOEs are extremely difficult to observe (the large excess of ligand "dilutes" the intensities of these NOEs among the signals of the large fraction of free ligand). Thus, the orientation of the ligand is uncertain, even if the structure of the protein (x-ray or NMR) and the ligand (trNOESY) are known.
(ii) Even if very favourable conditions allow determination of a few intermolecular NOEs, full NOE calculation of the structure would involve the full resonance assignment of the protein(iii) For very large receptor size, extremely fast transverse relaxation makes NMR detection not possible, precluding any complete NOE study

We propose to develop a novel "pharmacophoric STD NMR" approach for 3D structure calculation of weak protein-ligand complexes. We will use gradual modification of saturation power, frequencies, saturation times, and solvent composition, to get "functional" protein-ligand information to restrict conformational search of the ligand in the binding pocket. Finally, we propose to develop a protocol to obtain long sought protein-ligand distances restraints from STD NMR, based on a novel protocol for distance calibration. Calculations of 3D structures using the intermolecular semiambiguous restraints will follow using the very recently published "NMR2 molecular replacement" methodology.

Finally, the novel NMR approach will be used to study glycans interactions in flu and HIV infection.

Planned Impact

Pathways to Impact

Overall impacts and beneficiaries:
Developments in the ability to determine accurate 3D molecular structures of weak protein-ligand complexes impact on all current research endeavours focused on deepening our understanding of transient or low affinity biomolecular interactions. Specifically, biologists interested in protein-glycan and protein-metabolite interactions will be significantly benefited from the outcomes from this project. The technical developments will be sought closely by the NMR spectroscopic community, including main companies manufacturing equipment, and the findings in the virus studies will impact pharma companies involved in vaccine design, as well as companies developing selective sensors for flu virus detection. Finally, the PDRA will gain an attractive skill set for future employment in academy or industry.

Impacting a BBSRC strategic priority area:
The development of novel NMR approaches to determine accurate 3D structures of weak biomolecular complexes constitutes a solution to a specific technological gap identified in world-class bioscience. The proposed project is focused on underpinning the "bioscience for health" research priority area. Along the course of the project, the PI will highlight the importance of specific low affinity protein-ligand interactions in key regulatory processes. After successful outcomes from the 3 objectives, the PI will communicate the findings with maximum dissemination to the public with the help of the UEA media relations office.

Novel scientific findings:
A critical deliverable of the project focus on transferring the new knowledge to scientists. This will be carried out through the usual channels of conference presentations and publications in high impact scientific journals in both chemistry and biology. The PI and PDRA will engage with UEA media relations office to announce the work to media through the corresponding press release, and on websites and social media like UEA Facebook page, or Twitter. In 2017 the PI will establish a dedicated webpage to document our studies on weak protein-ligand complexes for the general public. This will help to enhance public knowledge of the biological significance of carbohydrates as regulators of cell-cell and cell-pathogen interactions, and its link to health and disease.
The PI and PDRA will be collaborating with national and international experts and the development and strengthening of such collaboration will increase the global impact of our findings.

Impact on society:
There is a continuous demand from society to the research community for investigating alternative ways to eradicate infectious agents, or to neutralise their pathological effects on humans. Our findings will inform the design of novel compounds for improved molecular sensors for selective detection of human or avian influenza virus, and the design of potential novel immunogenic molecular structures that could lead to the development of broadly acting HIV vaccines.

Impact on industry:
The goal of the project is to provide novel NMR spectroscopic approaches for structure characterization of weak complexes, as well as to unveil structural details of the molecular recognition of glycans in very relevant infection processes, both goals of interest for companies. The PI has identified some of the relevant stakeholders that will be impacted by the project, and they have expressed their interest.

Training and development:
This project will employ a PDRA and contribute to their professional training and development in a variety of technical, academic, and engagement skills. The hired PDRA will be fully engaged in the impact agenda, and will spend a short time in an industrial setting which will provide the PDRA with a very attractive and unique skill set for future employment in academy and industry.

Full details are provided in the "Pathways to Impact" section of the full proposal.


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Dhuna K (2019) Ginsenosides Act As Positive Modulators of P2X4 Receptors. in Molecular pharmacology

Title DEEP-STD NMR spectroscopy to elucidate 3D structures of weak protein-ligand complexes 
Description We have developed a novel spectroscopic approach, based on NMR spectroscopy to assay protein-ligand interactions, in particular to be able to gain structural information at atomic resolution about the interactions of ligands (e.g. drugs) with target receptor proteins, for those cases in which the interaction is weak. This type of protein-ligand interactions are difficult to study at atomic level by other biophysical techniques (e.g. X-ray crystallography) due to the transient nature of the process. With the novel methodology, termed Differential Epitope Mapping STD NMR (DEEP-STD NMR, Angew. Chem. Int. Ed. 2017), it is not only possible to detect the interaction and characterise the main parts of the ligand in contact with the protein, but it allows to provide the "orientation" of the small molecule (i.e. the drug) in the binding pocket of the target protein receptor. This information was not possible to obtain with former approaches (classical STD NMR experiments), and it is fundamental for the generation of 3D models of the structures of protein-ligand complexes of low affinity, which are typically found in the first stages of drug discovery processes. The method has two different implementations that can be run in parallel: i) differential frequency DEEP-STD NMR and ii) differential solvent (D2O / H2O) DEEP-STD NMR. The method is just an extension of the robust STD NMR experiment, which has been heavily used in the last decades by both academic and industrial laboratories. After publication of the novel methodology, we are expanding its applicability to be able to generate NMR-based 3D structures of weak protein-ligand complexes, and have started collaboration with drug-discovery and NMR manufacturer companies. 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? Yes  
Impact The novel method developed in this project has been successfully applied, in the context with a collaboration with biologist at the School of Biology (UEA; Dr Andrew Chantry) to the elucidation of a structural 3D molecular model, for the first time, of an inhibitor of the WWP2 ubiquitin ligase, an E3 ubiquitin ligase associated with tumour outgrowth and spread (Chem. Eur. J. 2018). After publication of the method (Angew. Chem. Int. Ed., 2017), we have initiated a collaboration with a drug discovery company, to expand further the applicability of the method to produce 3D molecular models of receptor-drug interactions, in processes of Fragment Based Drug Discovery. Besides research being in our group, in the context of this collaboration, hey have started trying the novel methodology on-site. After publication of the method, and dissemination of the results in international conferences, we have initiated a collaboration with an NMR manufacturer company to determine the instrumental limits of the method, in terms of the different spectrometers available in the market (different strengths of magnetic field). 
Description DEEP-STD NMR as orientational restraints in docking and MD calculations 
Organisation Vernalis
Country United Kingdom 
Sector Private 
PI Contribution We are trying to develop further the novel DEEP-STD NMR methodology developed in the context of this grant, to be able to produce 3D NMR-validated molecular models of protein-drug complexes, particularly in the context of Fragment Based Drug Discovery
Collaborator Contribution The company is providing biologically relevant target proteins and ligands to develop further our methodology. 3D structures generated with the novel approach in this project are to be compared with existing crystallographic structures from the company.
Impact Preliminary data produced. Currently analysis and further development in progress.
Start Year 2018
Description Novel STD NMR methods for protein-carbohydrate binding in gut microbe-host interactions 
Organisation Quadram Institute Bioscience
Country United Kingdom 
Sector Academic/University 
PI Contribution Expertise in high-resolution NMR spectroscopy and molecular modelling (docking and molecular dynamics) for the study of protein-glycan complexes, as well as knowledge in structural glycobiology to discuss results, ideas, and future projects.
Collaborator Contribution The research group of Nathalie Juge has made fundamental contributions to the good progress of the project. The project is focused on the development of novel structural glycobiology tools based on NMR spectroscopy to deliver relevant structural information of protein-glycan complexes. For the demonstration of the feasibility of the proposed novel approaches, the Juge lab has provided key protein materials and carbohydrate ligands for which high-resolution data were available from X-ray crystallography. This has been fundamental to demonstrate that the first of the novel approaches developed in the context of the present project, DEEP-STD NMR spectroscopy, is working and constitute a novel powerful tool for the glycobiology community, as well as for researchers and companies in the field of drug discovery. Our NMR approaches have also been applied to highly relevant protein-ligand systems in the field of gut microbe-host interactions, with all the biological material provided by the Juge lab.
Impact - Publication in Angewandte Chemie International Edition (2017): "Differential Epitope Mapping by STD NMR Spectroscopy To Reveal the Nature of Protein-Ligand Contacts" / - Publication in Nature Communications (2017): "Unravelling the specificity and mechanism of sialic acid recognition by the gut symbiont Ruminococcus gnavus" / - Publication in Proceedings of the National Academy of Sciences of the United States of America (PNAS) (2018): "Structural basis for the role of serine-rich repeat proteins from Lactobacillus reuteri in gut microbe-host interactions"
Start Year 2017
Description Structural basis of the molecular recognition of substrates by bacterial effectors and glycosyltransferases 
Organisation University of Zaragoza
Country Spain 
Sector Academic/University 
PI Contribution We contribute to this collaboration with our expertise in ligand-based NMR techniques for the study of the molecular recognition of substrates by enzymes. We determine conformations of free and bound species, and provide pharmacophore information on the substrates (which are the structural requirements for ligands to be able to efficiently interact with a given enzyme). We also contribute with our large expertise in molecular dynamics simulations, to predict structural transitions upon interaction of the specific molecular partners. We have also started applying the new STD NMR methods developed in our research group (e.g. DEEP-STD NMR).
Collaborator Contribution Our partner from the University of Zaragoza, led by Prof Ramon Hurtado-Guerrero, a world-leading expert in structural glycobiology of glycosyltransferases, provides us with all the biological material needed for: (i) testing our novel development on biologically relevant systems, and (ii) deepening our structural understanding of the specific interactions of substrates with glycosyltransferases of high relevance in bacterial infection processes, and cholesterol homeostasis.
Start Year 2017