CHARACTERIZATION OF THE INTERACTION BETWEEN HEPARAN SULFATE AND THE SPIKE (S) PROTEIN OF THE LATEST SARS-COV-2 VARIANTS

Heparan sulfate is a linear, highly sulfated polysaccharide that belongs to the glycosaminoglycan (GAG) family. The disaccharide repeating unit includes a uronic acid residue (either D-glucuronic or L-iduronic) 1→4 linked to a glucosamine (GlcN). The glucosamine residues can be mono- or polysulfated at the N-, 6-O, and rarely at the 3-O positions, while the uronic acids can be sulfated at the 2-O position. Furthermore, HS displays heterogeneity in chain length and block composition, in which acetyl- and sulfate-rich sequences alternate. HS is covalently attached to the membrane protein (via the O- or the N-glycosylation of a Ser or Asn residue, respectively), forming heparan sulfate proteoglycans (HSPGs). Due to the pronounced density of negative charge of HS, HSPGs play a key role in several biological processes, including cell hydration, intercellular communication, storage, protection, and exchange of biomolecules (e.g., cytokines, chemokines), and the regulation of receptors and proteolytic enzymes (e.g., tyrosine kinase-type growth factor). Moreover, HS is also involved in diverse physiological and pathological events, such as embryonic development, inflammatory response, blood coagulation, and bacterial and viral infection. In the latter context, several studies show that a wide range of pathogens can interact with HS during the early stage of the infection. This aspect has recently drawn significant attention following the outbreak of the SARS-CoV-2 pandemic. Indeed, the virus exploits HS in the extracellular matrix to tether viral particles to the host cell surface, thereby promoting the specific interaction between the cell surface human angiotensin-converting enzyme 2 receptor (hACE2) and the receptor binding domain (RBD) that decorates the capsid of SARS-CoV-2. In fact, this event triggers the fusion between the cell and virus membranes. The RBD region (and, more broadly, the S1 subunit) contains several positively charged amino acids that are solvent-exposed and form a positive channel in which HS chains can be hosted without overlapping the hACE2 binding site. The primary goal of this study is to understand how the role of HS in these early molecular recognition events differs in the latest and most widespread variants of SARS-CoV-2, through the application of complementary computational and experimental approaches that combine NMR spectroscopy and different computational tools. We have selected two hexasaccharides as HS mimetics to probe their interactions with different RBDs using docking calculations. We then focused our study on the Omicron variant and one of the hexasaccharide mimetics, performing molecular dynamic simulations and 1H-NMR Saturation Transfer Difference experiments. Structural insights into its binding modes were obtained using RedMat, an NMR-based analysis tool for the analysis of molecular dynamic simulation trajectories, and they were compared with the ligand interactions observed for the wild-type RBD. This knowledge will contribute to the structural biology knowledge of SARS-CoV-2 and to the development of new antiviral strategies and antiviral drugs.