Many pathogens exploit host cell-surface glycans. However, precise analyses of glycan ligands binding with heavily-modified pathogen proteins can be confounded by overlapping sugar signals and/or compound with known experimental constraints. ‘Universal saturation transfer analysis’ (uSTA) builds on existing nuclear magnetic resonance spectroscopy to provide an automated workflow for quantitating protein-ligand interactions. uSTA reveals that early-pandemic, B-origin lineage SARS-CoV-2 spike trimer binds sialoside sugars in an ‘end-on’ manner. uSTA-guided modelling and a high-resolution cryo-electron microscopy structure implicate the spike N-terminal domain (NTD) and confirm end-on binding. This finding rationalizes the effect of NTD mutations that abolish sugar-binding in SARS CoV 2 variants of concern. Together with genetic variance analyses in early pandemic patient cohorts, this binding implicates a sialylated polylactosamine motif found on tetraantennary N-linked glycoproteins in deeper human lung as potentially relevant to virulence and/or zoonosis.

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.

The fluorination of amino acid residues represents a near-isosteric alteration with the potential to report on biological pathways, yet the site-directed editing of carbon-hydrogen (C-H) bonds in complex biomolecules to carbon-fluorine (C-F) ones is challenging, resulting in its limited exploitation. Here, we describe a protocol for the post-translational and site-directed alteration of native γCH2 to γCF2 in protein sidechains. This alteration allows the installation of difluorinated sidechain analogues of proteinogenic amino acids, in both native and modified states. This chemical editing is robust, mild, fast, and highly efficient, exploiting photochemical and radicalmediated C-C-bonds grafted onto easy-to-access cysteine-derived dehydroalanine-containing proteins as starting materials. The heteroaryl-sulfonyl 'pySOOF' reagent required for generating the key carbon-centred C• radicals that install the sidechain can be synthesized in two-to-six steps from commercially available precursors. This workflow allows the non-expert to create fluorinated proteins within 24 hours, starting from a corresponding purified cysteine-containing protein precursor, without the need for bespoke biological systems. As an example, we readily introduce three γCF2-containing methionines in all three progressive oxidation states (sulfide, sulfoxide and sulfone) as D-/L-forms into Histone eH3.1 at site 4 (a relevant lysine to methionine oncomutation site) and each can be detected by 19 F-nuclear magnetic resonance of the γCF2 group, as well as the two diastereomers of the sulfoxide, even when found in a complex protein mixture of all three.The site-directed editing of C-HC-F enables the use of γCF2 as a highly-sensitive, 'zero-sizezero-background' label in protein sidechains, which may be used to probe biological phenomena, protein structures and/or protein-ligand interactions by 19 F-based detection methods.