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. 2020 Sep:181:104873.
doi: 10.1016/j.antiviral.2020.104873. Epub 2020 Jul 10.

Characterization of heparin and severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) spike glycoprotein binding interactions

So Young Kim  1 , Weihua Jin  2 , Amika Sood  3 , David W Montgomery  4 , Oliver C Grant  4 , Mark M Fuster  5 , Li Fu  2 , Jonathan S Dordick  6 , Robert J Woods  4 , Fuming Zhang  7 , Robert J Linhardt  8
Affiliations

Characterization of heparin and severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) spike glycoprotein binding interactions

So Young Kim et al. Antiviral Res. 2020 Sep.

Abstract

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has resulted in a pandemic and continues to spread around the globe at an unprecedented rate. To date, no effective therapeutic is available to fight its associated disease, COVID-19. Our discovery of a novel insertion of glycosaminoglycan (GAG)-binding motif at S1/S2 proteolytic cleavage site (681-686 (PRRARS)) and two other GAG-binding-like motifs within SARS-CoV-2 spike glycoprotein (SGP) led us to hypothesize that host cell surface GAGs may interact SARS-CoV-2 SGPs to facilitate host cell entry. Using a surface plasmon resonance direct binding assay, we found that both monomeric and trimeric SARS-CoV-2 SGP bind more tightly to immobilized heparin (KD = 40 pM and 73 pM, respectively) than the SARS-CoV and MERS-CoV SGPs (500 nM and 1 nM, respectively). In competitive binding studies, the IC50 of heparin, tri-sulfated non-anticoagulant heparan sulfate, and non-anticoagulant low molecular weight heparin against SARS-CoV-2 SGP binding to immobilized heparin were 0.056 μM, 0.12 μM, and 26.4 μM, respectively. Finally, unbiased computational ligand docking indicates that heparan sulfate interacts with the GAG-binding motif at the S1/S2 site on each monomer interface in the trimeric SARS-CoV-2 SGP, and at another site (453-459 (YRLFRKS)) when the receptor-binding domain is in an open conformation. The current study serves a foundation to further investigate biological roles of GAGs in SARS-CoV-2 pathogenesis. Furthermore, our findings may provide additional basis for further heparin-based interventions for COVID-19 patients exhibiting thrombotic complications.

Keywords: Binding interactions; COVID-19; Glycosaminoglycans; Heparin; SARS-CoV-2; Spike glycoprotein.

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Figures

Fig. 1
Fig. 1
Identification of GAG-binding motif within SARS-CoV-2, SARS-CoV, and MERS-CoV SGPs. Domains in SGP include signal peptide (SP), N-terminal domain (NTD), receptor-binding domain (RBD), fusion peptide (FP), heptad repeat 1/2 (HR 1/2).
Fig. 2
Fig. 2
SPR sensorgrams for binding kinetics/affinity measurements for SGP-HP interactions. (A) SARS-CoV-2 SGP (monomer), concentration of SGP (from top to bottom): 100, 50, 25, 12.5 and 6.25 nM. (B) SARS-CoV SGP, concentrations of SARS-CoV SGP (from top to bottom): 100, 50, 25, 12.5 and 6.25 nM. (C) MERS CoV SGP, concentrations of MERS CoV SGP (from top to bottom): 100, 50, 25, 12.5 and 6.25 nM. (D) SARS-CoV-2 SGP (trimer), concentration of SGP (from top to bottom): 800, 400, 200, 100 and 50 nM. The black curves are the fits using a 1:1 Langmuir model from BIAevaluate 4.0.1.
Fig. 3
Fig. 3
Bar graphs of normalized SARS-CoV-2 SGP binding preference to surface HP by competing with different chemical modified HP in solution. Concentration was 50 nM for SARS-CoV-2 SGP and 1000 nM for different chemical modified HP. All bar graphs based on triplicate experiments.
Fig. 4
Fig. 4
Inhibition analysis of glycans on the interactions between SARS-CoV-2 SGP and HP using SPR; SARS-CoV-2 SGP concentration was 50 nM. (A) Competition SPR sensorgrams of SARS-CoV-2 SGP-HP interaction inhibiting by different concentration of heparin. (B) Dose response curves for IC50 calculation of heparin using SARS-CoV-2 SGP inhibition data from surface competition SPR. (C) Competition SPR sensorgrams of SARS-CoV-2 SGP-HP interaction inhibiting by different concentration of TriS HS. (D) Dose response curves for IC50 calculation of TriS HS using SARS-CoV-2 SGP inhibition data from surface competition SPR. (E) Competition SPR sensorgrams of SARS-CoV-2 SGP-HP interaction inhibiting by different concentration of NACH. (F) Dose response curves for IC50 calculation of NACH using SARS-CoV-2 SGP inhibition data from surface competition SPR.
Fig. 5
Fig. 5
Structure of trimeric SARS-CoV-2 SGP and proposed GAG-binding motifs. (A) Electrostatic potential surface (-ve charge (red) to + ve charge (blue)) computed with Chimera. (B) Electrostatic potential surface showing a top view of the SGP trimer. (C) Solvent accessible surface of the SARS-CoV-2 SGP trimer (pink (Chain A), grey (Chain B), blue (Chain C)) showing the predicted poses of HS hexasaccharides (orange) obtained from unbiased docking, and the three GAG-binding motifs (yellow (Chain A), white (Chain B), red (Chain C)), image generated with VMD (Humphrey et al., 1996). (D) Solvent accessible surface showing a top view of the SGP trimer. Amino acid sequences for GAG-binding motifs site 1, 2, and 3 are YRLFRKS, PRRARS, and SKPSKRS.
Fig. 6
Fig. 6
Proposed model of SARS-CoV-2 host cell entry. SARS-CoV-2 surface is decorated with envelop (E), membrane (M), and SGP (Chan et al., 2020a). (A) Virion lands on host cell surface by binding to heparan sulfate proteoglycan (HSPG). (B) SGP goes through proteolytic digestion by host cell surface protease, which initiates viral-host cell membrane fusion by conformational change caused by host cell receptor binding (HSPG and ACE2). ACE2 is an established host cell surface receptor in SARS-CoV-2 host cell entry (Hoffmann et al., 2020; Wrapp et al., 2020). (C) Virion enters the host cell and may further experience proteolytic processing by endosomal host cell protease. In the case of receptor-dependent endocytic viral entry (Wang et al., 2008), virions may further utilize endocytosed and recycled HSPGs for their advantage.
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