open access publication

Article, 2024

Two Receptor Binding Strategy of SARS-CoV‑2 Is Mediated by Both the N‑Terminal and Receptor-Binding Spike Domain

The Journal of Physical Chemistry B, ISSN 1520-6106, 1520-5207, Volume 128, 2, Pages 451-464, 10.1021/acs.jpcb.3c06258


Monti, Michele (Corresponding author) [1] Milanetti, Edoardo 0000-0002-3046-5170 [1] [2] Frans, Myrthe T 0000-0002-6672-0946 [3] Miotto, Mattia 0000-0002-0043-8921 [1] Di Rienzo, Lorenzo 0000-0003-4114-5049 [1] Baranov, Maksim V [3] Gosti, Giorgio 0000-0002-8571-1404 [1] [4] Somavarapu, Arun Kumar 0000-0001-6123-6875 [5] Nagaraj, Madhu [5] Golbek, Thaddeus Wayne 0000-0002-3632-2701 [5] Rossing, Emiel 0000-0002-6381-6475 [6] Moons, Sam J 0000-0001-5507-3811 [6] Boltje, T J Boltje D T J [6] Van Den Bogaart, Geert 0000-0003-2180-6735 [3] Weidner, Tobias 0000-0002-7083-7004 [5] Otzen, Daniel Erik 0000-0002-2918-8989 [5] Tartaglia, Gian Gaetano [1] Ruocco, Giancarlo 0000-0002-2762-9533 [1] [2] Roeters, Steven Joop 0000-0003-3238-2181 (Corresponding author) [5] [7]


  1. [1] Italian Institute of Technology
  2. [NORA names: Italy; Europe, EU; OECD];
  3. [2] Sapienza University of Rome
  4. [NORA names: Italy; Europe, EU; OECD];
  5. [3] Molecular ImmunologyGroningen Biomolecular Sciences and Biotechnology, Nijenborgh 7, 9747 AG, Groningen, The Netherlands
  6. [4] National Research Council
  7. [NORA names: Italy; Europe, EU; OECD];
  8. [5] Aarhus University
  9. [NORA names: AU Aarhus University; University; Denmark; Europe, EU; Nordic; OECD];


It is not well understood why severe acute respiratory syndrome (SARS)-CoV-2 spreads much faster than other β-coronaviruses such as SARS-CoV and Middle East respiratory syndrome (MERS)-CoV. In a previous publication, we predicted the binding of the N-terminal domain (NTD) of SARS-CoV-2 spike to sialic acids (SAs). Here, we experimentally validate this interaction and present simulations that reveal a second possible interaction between SAs and the spike protein via a binding site located in the receptor-binding domain (RBD). The predictions from molecular-dynamics simulations and the previously-published 2D-Zernike binding-site recognition approach were validated through flow-induced dispersion analysis (FIDA)─which reveals the capability of the SARS-CoV-2 spike to bind to SA-containing (glyco)lipid vesicles, and flow-cytometry measurements─which show that spike binding is strongly decreased upon inhibition of SA expression on the membranes of angiotensin converting enzyme-2 (ACE2)-expressing HEK cells. Our analyses reveal that the SA binding of the NTD and RBD strongly enhances the infection-inducing ACE2 binding. Altogether, our work provides in silico, in vitro, and cellular evidence that the SARS-CoV-2 virus utilizes a two-receptor (SA and ACE2) strategy. This allows the SARS-CoV-2 spike to use SA moieties on the cell membrane as a binding anchor, which increases the residence time of the virus on the cell surface and aids in the binding of the main receptor, ACE2, via 2D diffusion.


ACE2, ACE2 binding, AIDS, HEK, HEK cells, Middle East respiratory syndrome, N-terminal, N-terminal domain, SA binding, SA expression, SA moiety, SARS-CoV, SARS-CoV-2, SARS-CoV-2 spike, SARS-CoV-2 virus, acid, analysis, anchor, angiotensin-converting enzyme-2, approach, binding, binding anchors, binding sites, capability, cell membrane, cell surface, cells, cellular evidence, diffusion, dispersion analysis, domain, enzyme-2, evidence, expression, flow cytometry measurements, flow-induced dispersion analysis, inhibition, interaction, measurements, membrane, middle, moiety, molecular dynamics simulations, prediction, previously-published, protein, publications, receptor-binding domain, receptors, recognition, recognition approach, residence time, residents, respiratory syndrome, sialic acid, simulation, sites, spike binding, spike domains, spike protein, spikes, surface, syndrome, time, vesicles, virus


  • European Research Council
  • Lundbeck Foundation
  • Innovation Fund Denmark
  • Novo Nordisk Foundation
  • Novo Nordisk (Denmark)
  • European Commission

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