Cryptic pathogen-sugar interactions revealed by universal saturation transfer analysis

  1. Charles J. Buchanan
  2. Ben Gaunt
  3. Peter J. Harrison
  4. Yun Yang
  5. Jiwei Liu
  6. Aziz Khan
  7. Andrew M. Giltrap
  8. Audrey Le Bas
  9. Philip N. Ward
  10. Kapil Gupta
  11. Maud Dumoux
  12. Sergio Daga
  13. Nicola Picchiotti
  14. Margherita Baldassarri
  15. Elisa Benetti
  16. Chiara Fallerini
  17. Francesca Fava
  18. Annarita Giliberti
  19. Panagiotis I. Koukos
  20. Abirami Lakshminarayanan
  21. Xiaochao Xue
  22. Georgios Papadakis
  23. Lachlan P. Deimel
  24. Virgínia Casablancas-Antràs
  25. Timothy D.W. Claridge
  26. Alexandre M.J.J. Bonvin
  27. Quentin J. Sattentau
  28. Simone Furini
  29. Marco Gori
  30. Jiandong Huo
  31. Raymond J. Owens
  32. Christiane Schaffitzel
  33. Imre Berger
  34. Alessandra Renieri
  35. GEN-COVID Multicenter Study
  36. James H. Naismith
  37. Andrew Baldwin
  38. Benjamin G. Davis
1 evaluations Published on
Read the full article Related papers
This article on Sciety

Abstract

Many host pathogen interactions such as human viruses (including non-SARS-coronaviruses) rely on attachment to host cell-surface glycans. There are conflicting reports about whether the Spike protein of SARS-CoV-2 binds to sialic acid commonly found on host cell-surface N-linked glycans. In the absence of a biochemical assay, the ability to analyze the binding of glycans to heavily- modified proteins and resolve this issue is limited. Classical Saturation Transfer Difference (STD) NMR can be confounded by overlapping sugar resonances that compound with known experimental constraints. Here we present ‘universal saturation transfer analysis’ (uSTA), an NMR method that builds on existing approaches to provide a general and automated workflow for studying protein-ligand interactions. uSTA reveals that B-origin-lineage-SARS-CoV-2 spike trimer binds sialoside sugars in an ‘end on’ manner and modelling guided by uSTA localises binding to the spike N-terminal domain (NTD). The sialylated-polylactosamine motif is found on tetraantennary human N-linked-glycoproteins in deeper lung and may have played a role in zoonosis. Provocatively, sialic acid binding is abolished by mutations in some subsequent SARS- CoV-2 variants-of-concern. A very high resolution cryo-EM structure confirms the NTD location and ‘end on’ mode; it rationalises the effect of NTD mutations and the structure-activity relationship of sialic acid analogues. uSTA is demonstrated to be a robust, rapid and quantitative tool for analysis of binding, even in the most demanding systems.

Extended Abstract

The surface proteins found on both pathogens and host cells mediate entry (and exit) and influence disease progression and transmission. Both types can bear host-generated post- translational modifications such as glycosylation that are essential for function but can confound biophysical methods used for dissecting key interactions. Several human viruses (including non- SARS-coronaviruses) attach to host cell-surfaceN-linked glycans that include forms of sialic acid (sialosides). There remains, however, conflicting evidence as to if or how SARS-associated coronaviruses might use such a mechanism. Here, we demonstrate quantitative extension of ‘saturation transfer’ protein NMR methods to a complete mathematical model of the magnetization transfer caused by interactions between protein and ligand. The method couples objective resonance-identification via a deconvolution algorithm with Bloch-McConnell analysis to enable a structural, kinetic and thermodynamic analysis of ligand binding beyond previously-perceived limits of exchange rates, concentration or system. Using an automated and openly available workflow this ‘universal saturation transfer’ analysis (uSTA) can be readily-applied in a range of even heavily-modified systems in a general manner to now obtain quantitative binding interaction parameters (KD, kEx). uSTA proved critical in mapping direct interactions between natural sialoside sugar ligands and relevant virus-surface attachment glycoproteins – SARS-CoV-2-spike and influenza-H1N1-haemagglutinin variants – by quantitating ligand signal in spectral regions otherwise occluded by resonances from mobile protein glycans (that also include sialosides). In B- origin-lineage-SARS-CoV-2 spike trimer ‘end on’-binding to sialoside sugars was revealed contrasting with ‘extended surface’-binding for heparin sugar ligands; uSTA-derived constraints used in structural modelling suggested sialoside-glycan binding sites in a beta-sheet-rich region of spike N-terminal domain (NTD). Consistent with this, uSTA-glycan binding was minimally- perturbed by antibodies that neutralize the ACE2-binding domain (RBD) but strongly disrupted in spike from the B1.1.7/alpha and B1.351/beta variants-of-concern, which possess hotspot mutations in the NTD. Sialoside binding in B-origin-lineage-NTD was unequivocally pinpointed by cryo-EM to a site that is created from residues that are notably deleted in variants (e.g. H69,V70,Y145 in alpha). An analysis of beneficial genetic variances in cohorts of patients from early 2020 suggests a model in which this site in the NTD of B-origin-lineage-SARS-CoV-2 (but not in alpha/beta-variants) may have exploited a specific sialylated-polylactosamine motif found on tetraantennary humanN-linked-glycoproteins in deeper lung. Together these confirm a novel binding mode mediated by the unusual NTD of SARS-CoV-2 and suggest how it may drive virulence and/or zoonosis via modulation of glycan attachment. Since cell-surface glycans are widely relevant to biology and pathology, uSTA can now provide ready, quantitative, widespread analysis of complex, host-derived and post-translationally modified proteins with putative ligands relevant to disease even in previously confounding complex systems.

Related articles

Related articles are currently not available for this article.