Cryptic pathogen-sugar interactions revealed by universal saturation transfer analysis

Abstract

Host-expressed proteins on both host-cell and pathogen surfaces are widely exploited by pathogens, mediating cell entry (and exit) and influencing disease progression and transmission. This is highlighted by the diverse modes of coronavirus entry into cells and their consequent differing pathogenicity that is of direct relevance to the current SARS-CoV-2 pandemic. Host-expressed viral surface proteins bear post-translational modifications such as glycosylation that are essential for function but can confound or limit certain current biophysical methods used for dissecting key interactions. Several human coronaviruses attach to host cell-surface N-linked glycans that include forms of sialic acid. There remains, however, conflicting evidence as to if or how SARS-associated coronaviruses might use such a mechanism. Here, we show that novel protein NMR methods allow a complete and comprehensive analysis of the magnetization transfer caused by interactions between even heavily modified proteins and relevant ligands to generate quantitative binding data in a general manner. Our method couples direct, objective resonance-identification via a deconvolution algorithm with quantitative analysis using Bloch-McConnell equations to obtain interaction parameters (e.g. KD, kEx), which together enable structural modelling. By using an automated and openly available workflow, this method can be readily applied in a range of systems. This complete treatment of so-called 'saturation transfer' between protein and ligand now enables a general analysis of solution-phase ligand-protein binding beyond previously perceived limits of exchange rates, concentration or system - this allows 'universal' saturation transfer analysis (uSTA). uSTA proves critical in mapping direct interaction between natural sialoside sugar ligands and SARS-CoV-2-spike glycoprotein by quantitating ligand signal in spectral regions otherwise occluded by resonances from mobile spike-protein glycans (that also include sialosides). Using uSTA, 'end on'-binding by SARS-CoV-2-spike protein to sialoside glycan is revealed, which contrasts with an observed 'extended surface'-binding for previously validated heparin sugar ligands. Quantitative use of uSTA-derived restraints pinpoints likely binding modes to an intrinsically disordered region of the N-terminal domain of SARS-CoV-2-spike trimer. Consistent with this, glycan binding is minimally perturbed by antibodies that neutralize via binding the ACE2-binding domain (RBD) but strongly disrupted in the B1.1.7 and B1.351 variants-of-concern that possess hotspot mutations around the identified site. An analysis of beneficial genetic variances in cohorts of patients from early 2020 suggests a possible model in which A-lineage-SARS-CoV-2 may have exploited a specific sialylated-polylactosamine motif found on tetraantennary human N-linked-glycoproteins in deeper lung. Since cell-surface glycans are widely relevant to biology and pathology, uSTA can now provide a ready, quantitative method for widespread analysis of complex, host-derived and post-translationally modified proteins with putative ligands relevant to disease even in previously confounding complex systems.

Emergence and spread of SARS-CoV-2 lineage B.1.620 with variant of concern-like mutations and deletions

“…The individual V126A and H245Y substitutions are still largely uncharacterised to the best of our knowledge, but might be counterparts to the R246I substitution in B.1.351, and the latter may interfere with a putative glycan binding pocket in the NTD 16 . All other mutations of B.1.620 in the NTD result in partial loss of neutralisation of convalescent serum and NTD-directed monoclonal antibodies 17 .…”

Glycan-Based Flow-Through Device for the Detection of SARS-COV-2

“…Davis and co-workers have reported that the B1.1.7 and B1.351 spike mutants have reduced NMR STD signal (i.e., weaker contacts) to the NAc protons of α2,3-α sialyllactoside compared to the wild-type, and hence there is potential that the glycan-binding affinity may be decreased. 52 To test the impact of this, 3 mutant truncated spike proteins, B1.1.7, B.1.351, and P1 (variants first detected in Kent (U.K.), South Africa, and Brazil), were expressed in E. coli and tested in our devices. In all cases, a positive test line was seen (Figure 5C, no silver staining), showing that detection capability is retained.…”

The Glycan-Binding Trait of the Sarbecovirus Spike N-Terminal Domain Reveals an Evolutionary Footprint

“…The sialic acid-binding function in transmissible gastroenteritis virus (TGEV) has been suggested to influence viral pathogenicity ( 14 – 16 ), and computational modeling suggests sialic acid binding in SARS-CoV-2 spike may be an immune evasion mechanism ( 17 ). Recent studies also propose that sialic acid or 9- O -Ac sialic acid of glycoproteins and glycolipids might be served as the host factor for SARS-CoV-2 infection ( 18 – 22 ). However, the functional analysis of this phenotype was not fully assessed in cell culture or animal models, leaving the biological impact unknown.…”

“…(B) Alignment of amino acid sequences of S-NTDs (corresponding to aa14 to 292 of SARS-CoV-2). The red rectangle shows the proposed sialic acid-binding sites in the SARS-CoV-2 spike ( 19 ). (C and D) Classical hemagglutination assay.…”