Infection of host cells by SARS‐CoV‐2 begins with recognition by the virus S (spike) protein of cell surface heparan sulfate (HS), tethering the virus to the extracellular matrix environment, and causing the subunit S1‐RBD to undergo a conformational change into the ‘open’ conformation. These two events promote the binding of S1‐RBD to the angiotensin converting enzyme 2 (ACE2) receptor, a preliminary step toward viral‐cell membrane fusion. Combining ligand‐based NMR spectroscopy with molecular dynamics, oligosaccharide analogues were used to explore the interactions between S1‐RBD of SARS CoV‐2 and HS, revealing several low‐specificity binding modes and previously unidentified potential sites for the binding of extended HS polysaccharide chains. The evidence for multiple binding modes also suggest that highly specific inhibitors will not be optimal against protein S but, rather, diverse HS‐based structures, characterized by high affinity and including multi‐valent compounds, may be required.

Among the structural proteins of SARS-CoV-2, the nucleocapsid (N) protein stands out for its pronounced structural heterogeneity and multifunctionality throughout the viral life cycle. Recent studies have demonstrated that the N protein localizes to the surface of infected and neighboring non-infected cells, by interacting with heparan sulfate in the extracellular matrix. The N protein (419 residues) comprises two folded domains (⁴⁴NTD¹⁸⁰ and ²⁴⁹CTD³⁶¹) interspersed with three intrinsically disordered regions (¹IDR1⁴³, ¹⁸¹IDR2²⁴⁸, ³⁶²IDR3⁴¹⁹). The coexistence of ordered and disordered elements raises a key question: how does this structural heterogeneity influence N's interactions with biological partners? Here we employ high-resolution NMR spectroscopy as the primary technique to characterize the interaction of three N protein constructs (⁴⁴NTD¹⁸⁰, ¹NTR²⁴⁸, and ¹N⁴¹⁹) with heparin-based ligands of increasing complexity. NMR provides atomic level information on the structured NTD domain and on the otherwise difficult to investigate flexible regions. Molecular dynamics simulations further probe the interaction between NTD and short heparin oligosaccharides. Our data reveal a clear correlation between ligand size and binding affinity: longer saccharide chains promote stronger binding. Additionally, the inclusion of intrinsically disordered regions in the NTR construct significantly enhances the interaction compared to NTD, highlighting the functional relevance of structural disorder. Finally, the full-length protein exhibits distinct spectral behavior with the investigated heparin-based ligand, potentially reflecting additional binding contributions and altered dynamics arising from its complex structure. These findings underscore the utility of NMR spectroscopy in elucidating the dynamic, multivalent nature of protein-polyanion interactions, particularly in highly flexible proteins with a modular domain organization.

The interaction of heparin with antithrombin (AT) involves a specific sequence corresponding to the pentasaccharide GlcNAc/NS6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS6S (AGA*IA). Recent studies have revealed that two AGA*IA-containing hexasaccharides, which differ in the sulfation degree of the iduronic acid unit, exhibit similar binding to AT, albeit with different affinities. However, the lack of experimental data concerning the molecular contacts between these ligands and the amino acids within the protein-binding site prevents a detailed description of the complexes. Differential epitope mapping (DEEP)-STD NMR, in combination with MD simulations, enables the experimental observation and comparison of two heparin pentasaccharides interacting with AT, revealing slightly different bound orientations and distinct affinities of both glycans for AT. We demonstrate the effectiveness of the differential solvent DEEP-STD NMR approach in determining the presence of polar residues in the recognition sites of glycosaminoglycan-binding proteins.

Heparan sulfate proteoglycans serve as initial attachment sites for several viruses and bacteria. Recent studies suggest that SARS-CoV-2 similarly exploits these glycosaminoglycans, facilitating conformational changes in the spike protein that promote the interaction between the receptor-binding domain (S1-RBD) and the cellular angiotensin-converting enzyme 2 receptor (ACE2), thereby triggering the virus internalization process. The molecular details that drive this process, particularly the co-receptor role of heparan sulfate (HS), remain incompletely understood. The interaction between an HS hexasaccharide (hexa) and the N343 glycosylated S1-RBD of the wild-type (WT) and Omicron variant of SARS-CoV-2 was investigated. The conformational properties of hexa with these S1-RBDs in unbound and bound states are explored using multiple independent MD simulations; the protein binding epitope of hexa, as well as the details of its interaction with S1-RBD of the Omicron variant, are characterized by comparing experimental and theoretical ¹H STD NMR signals. This investigation identifies the role played by the glycosyl moiety at N343 in potentially affecting this interaction in both WT and Omicron S1-RBD, explaining the observed low specificity and multi-modal nature of the interaction between HS oligosaccharides and these S1-RBDs.

Heparin, the naturally occurring, highly sulfated glycosaminoglycan drug, acts as a regulator of diverse biological processes by interacting with diverse specific proteins through a variety of sulfated structural motifs with varying degrees of affinity. Despite its rarity, 3-O-sulfation of glucosamine plays a key role in several biological activities, especially anticoagulant activity mediated by interaction with antithrombin (AT), a mechanism that has been extensively studied. The present work primarily focuses on sequences containing 3-O-sulfated glucosamine that are not involved in anticoagulant activity, analysing their relative abundance and structural environments in heparins derived from distinct animal origins. Three heparin samples derived from bovine, ovine and porcine intestinal mucosa (BMH, OMH and PMH) were fractionated by affinity chromatography on AT-Sepharose into no affinity (NA) and high affinity (HA) fractions. Parent heparins and derived NA and HA fractions were enzymatically depolymerised using either a cocktail of heparinases I, II and III or heparinase II alone, and the resulting mixtures of di- and oligosaccharides were analysed by liquid chromatography coupled with mass spectrometry. Digestion with heparinase II, which preserves heparin sequences containing 3-O-sulfated glucosamine, produced a series of 3-O-sulfated trisaccharides containing two glucosamine residues either side of a uronic acid, located at the non-reducing end (NRE) of heparin chains. Treatment with a heparinase cocktail, which cleaves these trisaccharides, released NRE glucosamine monosaccharides, including 3-O-sulfated species. Notably, both the number of NRE trisaccharide species and the overall proportion of 3-O-sulfated monosaccharides were markedly higher in OMH compared to BMH or PMH. Using ¹H/¹³C bi-dimensional nuclear magnetic resonance spectroscopy, the higher proportion of NRE 3-O-sulfated glucosamine in ovine heparin relative to bovine and porcine heparins was found to be preferentially located in chains lacking affinity for AT. These results offer new insights into the localisation of most 3-O-sulfated heparin sequences that are not associated with anticoagulant activity and, importantly, also reveal a possible structural marker that indicates the ovine origin of heparin. Additionally, a tetrasaccharide species was detected that may be indicative of a highly sulfated AT-binding site in BMH.

Heparan sulfate proteoglycans are known to serve as initial attachment site for several viruses and bacteria. Recent studies suggest that SARS-CoV-2 coronavirus similarly exploits these glycosaminoglycans, facilitating conformational changes in the spike protein, that promotes the interaction between the receptor binding domain (S1-RBD) and the cellular angiotensin-converting enzyme 2 receptor (ACE2), thereby triggering the virus internalization process. Until now, the molecular details that drive this process, particularly, the co-receptor role of the heparan sulfate (HS), remain not completely understood. Our research seeks to characterize the interaction between an HS hexasaccharide (hexa) and the N343 glycosylated S1-RBD of the Omicron and wild-type (WT) variants of SARS-CoV-2. The conformational properties of hexa in unbound and bound state with these S1-RBDs are investigated using multiple independent MD simulations; the proton binding epitope of hexa, as well as the details of the interaction between this glycan and S1-RBD of the Omicron variant, are characterized by comparing experimental and theoretical saturation transfer difference NMR signals. This investigation reinforces previous evidence about the low specificity and multi-modal nature of the interaction between HS oligosaccharides and these S1-RBDs, and underlines the role of the glycosyl moiety at N343 in potentially affecting this interaction in both selected variants.