Cryptic pathogen-sugar interactions revealed by universal saturation transfer analysis
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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-surface N -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 (K D , k Ex ). 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 human N -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.
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SciScore for 10.1101/2021.04.14.439284: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
Antibodies Sentences Resources Pseudovirus was detected via CR3022, anti-Human IgG-HRP (Jackson ImmunoResearch Europe) and 1-Step Ultra TMB ELISA substrate (Thermo Fisher). 1×104 ACE-2-expressing MDCK cells in PBS were seeded in a flat-bottom 96-well plate and treated with 0.3mU Arthrobacter ureafaciens neuraminidase (Merck) or PBS (mock) for 30 minutes (37°C, 5% CO2). anti-Human IgG-HRPsuggested: NoneExperimental Models: Cell Lines Sentences Resources Expi293 cells (Thermofisher Scientific) were used to express the Spike-Bap protein. Expi293suggested: RRID:CVCL_D615)Pseudoviral Cell-entry Asssay: A spike-expressing lentivirus in HEK … SciScore for 10.1101/2021.04.14.439284: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
Antibodies Sentences Resources Pseudovirus was detected via CR3022, anti-Human IgG-HRP (Jackson ImmunoResearch Europe) and 1-Step Ultra TMB ELISA substrate (Thermo Fisher). 1×104 ACE-2-expressing MDCK cells in PBS were seeded in a flat-bottom 96-well plate and treated with 0.3mU Arthrobacter ureafaciens neuraminidase (Merck) or PBS (mock) for 30 minutes (37°C, 5% CO2). anti-Human IgG-HRPsuggested: NoneExperimental Models: Cell Lines Sentences Resources Expi293 cells (Thermofisher Scientific) were used to express the Spike-Bap protein. Expi293suggested: RRID:CVCL_D615)Pseudoviral Cell-entry Asssay: A spike-expressing lentivirus in HEK 293T cells was generated using a two-plasmid system. HEK 293Tsuggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)Pseudovirus was detected via CR3022, anti-Human IgG-HRP (Jackson ImmunoResearch Europe) and 1-Step Ultra TMB ELISA substrate (Thermo Fisher). 1×104 ACE-2-expressing MDCK cells in PBS were seeded in a flat-bottom 96-well plate and treated with 0.3mU Arthrobacter ureafaciens neuraminidase (Merck) or PBS (mock) for 30 minutes (37°C, 5% CO2). MDCKsuggested: NoneSoftware and Algorithms Sentences Resources The values we obtain performing this analysis on BSA/Trp closely match those measured by ITC, and the values we measure for ligand 2 and SPIKE are in good agreement with those measured by SPR as described in the text. SPIKEsuggested: (SPIKE, RRID:SCR_010466)The uSTA analysis pipeline then provides a user with a report that shows the results of the various stages of analysis, and uses pymol to render the surfaces. pymolsuggested: (PyMOL, RRID:SCR_000305)Genetic analysis of clinical samples: Variant calling: Reads were mapped to the hg19 reference genome by the Burrow-Wheeler aligner BWA. BWAsuggested: (BWA, RRID:SCR_010910)Variants were annotated by ANNOVAR. ANNOVARsuggested: (ANNOVAR, RRID:SCR_012821)All data pre-processing and the RFE procedure was coded in Python; the LR model was used, as included, in the scikit-learn module with the liblinear coordinate descent optimization algorithm. Pythonsuggested: (IPython, RRID:SCR_001658)Results from OddPub: Thank you for sharing your data.
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: We found the following clinical trial numbers in your paper:
Identifier Status Title NCT04549831 Recruiting Genetic Bases of COVID-19 Clinical Variability Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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