In:
Science, American Association for the Advancement of Science (AAAS), Vol. 377, No. 6604 ( 2022-07-22)
Abstract:
The surface proteins found on both pathogens and host cells mediate cell entry (and exit) and influence disease progression and transmission. Both types of proteins can bear host-generated posttranslational modifications, such as glycosylation, that are essential for function but can confound current 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 whether or how SARS-associated coronaviruses might use such a mechanism. In the absence of an appropriate biochemical assay, the ability to analyze the binding of such glycans to heavily modified proteins and resolve this issue is limited. RATIONALE We developed and demonstrated a quantitative extension of “saturation transfer” protein nuclear magnetic resonance (NMR) methods to a complete mathematical model of the magnetization transfer caused by interactions between protein and ligand. The designed method couples objective resonance identification and intensity measurement in NMR spectra (via a deconvolution algorithm) with Bloch-McConnell analysis of magnetization transfer (as judged by this resonance signal intensity) to enable a structural, kinetic, and thermodynamic analysis of ligand binding. Such quantification is beyond previously perceived limits of exchange rates, concentration, or system and therefore represents a potentially universal saturation transfer analysis (uSTA) method. RESULTS In an automated workflow, uSTA can be applied to a range of even heavily modified protein systems in a general manner to obtain quantitative binding interaction parameters ( K D , k Ex ). uSTA proved critical in mapping direct interactions between sialoside sugar ligands and relevant virus surface attachment glycoproteins, including multiple variants of both severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and influenza H1N1 hemagglutinin protein. It was successful in quantitating ligand NMR signals in spectral regions otherwise occluded by resonances from mobile protein glycans. In early-pandemic (December 2019) B-origin-lineage SARS-CoV-2 spike trimer, a clear “end-on” binding mode of sialoside sugars to spike was revealed by uSTA. This mode contrasted with “extended-surface side”-binding for heparin sugar ligands. uSTA-derived restraints used in structural modeling suggested sialoside-glycan binding sites in a β sheet–rich region of spike N-terminal domain (NTD), distant from the receptor-binding domain (RBD) that binds ACE2 co-receptor and that has been identified as the site for other sugar interactions. Consistent with this NTD site being a previously unknown sialoside sugar-binding pocket, uSTA-sialoside binding was minimally perturbed by antibodies that neutralize the ACE2-binding RBD domain. Strikingly, uSTA also shows that this sialoside binding is disrupted in spike from multiple variants of concern (B1.1.7/alpha, B1.351/beta, B.1.617.2/delta, and B.1.1.529/omicron) that emerged later in the pandemic (September 2020 onward). Notably, these variants possess multiple hotspot mutations in the NTD. End-on sialoside binding in a B-origin-lineage spike-NTD pocket was pinpointed by cryo-EM to a previously unknown site that is created from residues that are notably mutated or are in regions where mutations occur in variants of concern (e.g., His 69 , Val 70 , and Tyr 145 in alpha and omicron). An analysis of beneficial genetic variances correlated with disease severity 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 later variants) may have exploited a specific sialylated polylactosamine motif found on tetraantennary human N-linked glycoproteins, known to be present in deeper human lung. CONCLUSION Together, these results confirm a distinctive sugar-binding mode mediated by the unusual NTD of B-origin-lineage SARS-CoV-2 spike protein that is lost in later variants. This may implicate modulation of binding by SARS-CoV-2 virus to human cell surface sugars as a determinant of virulence and/or zoonosis. More generally, because cell surface glycans are widely relevant to biology and pathology, the uSTA method can now provide ready, quantitative, widespread analysis of complex, host-derived, and posttranslationally modified proteins in their binding to putative ligands, which may be relevant to disease, even in previously confounding complex systems. uSTA identifies a sugar-binding site and pose in the unusual NTD of early-pandemic SARS-CoV-2 spike. ( A ) This site and pose, which were confirmed by cryo–electron microscopy, are heavily mutated in variants of concern. ( B ) Analyses reveal a loss of sialoside-spike binding, rationalized by clustering of mutations around the binding site. *All alpha-variant mutations are in 〉 1 variant.
Type of Medium:
Online Resource
ISSN:
0036-8075
,
1095-9203
DOI:
10.1126/science.abm3125
Language:
English
Publisher:
American Association for the Advancement of Science (AAAS)
Publication Date:
2022
detail.hit.zdb_id:
128410-1
detail.hit.zdb_id:
2066996-3
detail.hit.zdb_id:
2060783-0
SSG:
11
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