A tethered ligand assay to probe SARS-CoV-2:ACE2 interactions

Autor: Bauer, Magnus S., Gruber, Sophia, Hausch, Adina, Gomes, Priscila S.F.C., Milles, Lukas F., Nicolaus, Thomas, Schendel, Leonard C., Navajas, Pilarlopez, Procko, Erik, Lietha, Daniel, Melo, Marcelo C.R., Bernardi, Rafael C., Gaub, Hermann E., Lipfert, Jan, Sub Molecular Biophysics, Soft Condensed Matter and Biophysics
Přispěvatelé: Sub Molecular Biophysics, Soft Condensed Matter and Biophysics, German Research Foundation, Human Frontier Science Program, European Molecular Biology Laboratory, Auburn University, Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), European Commission, Bauer, Magnus Sebastian, Gomes, Priscila S. F. C., Milles, Lukas Frederik, Nicolaus, Thomas, Schendel, Leonard C., Procko, Erik, Lietha, Daniel, Melo, Marcelo C. R., Bernardi, Rafael C., Gaub, Hermann Eduard, Lipfert, Jan, Bauer, Magnus Sebastian [0000-0003-1357-2852], Gomes, Priscila S. F. C. [0000-0001-7370-9596], Milles, Lukas Frederik [0000-0001-8417-3205], Nicolaus, Thomas [0000-0001-8417-3205], Schendel, Leonard C. [0000-0002-1986-2693], Procko, Erik [0000-0002-0028-490X], Lietha, Daniel [0000-0002-6133-6486], Melo, Marcelo C. R. [0000-0001-6901-1646], Bernardi, Rafael C. [0000-0003-0758-2026], Gaub, Hermann Eduard [0000-0002-4220-6088], Lipfert, Jan [0000-0003-3613-7896]
Rok vydání: 2022
Předmět:
Zdroj: Digital.CSIC. Repositorio Institucional del CSIC
instname
Proceedings of the National Academy of Sciences of the United States of America, 119(14). National Academy of Sciences
ISSN: 1091-6490
0027-8424
DOI: 10.1073/pnas.2114397119
Popis: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections are initiated by attachment of the receptor-binding domain (RBD) on the viral Spike protein to angiotensin-converting enzyme-2 (ACE2) on human host cells. This critical first step occurs in dynamic environments, where external forces act on the binding partners and avidity effects play an important role, creating an urgent need for assays that can quantitate SARS-CoV-2 interactions with ACE2 under mechanical load. Here, we introduce a tethered ligand assay that comprises the RBD and the ACE2 ectodomain joined by a flexible peptide linker. Using magnetic tweezers and atomic force spectroscopy as highly complementary single-molecule force spectroscopy techniques, we investigate the RBD:ACE2 interaction over the whole physiologically relevant force range. We combine the experimental results with steered molecular dynamics simulations and observe and assign fully consistent unbinding and unfolding events across the three techniques, enabling us to establish ACE2 unfolding as a molecular fingerprint. Measuring at forces of 2 to 5 pN, we quantify the force dependence and kinetics of the RBD:ACE2 bond in equilibrium. We show that the SARS-CoV-2 RBD:ACE2 interaction has higher mechanical stability, larger binding free energy, and a lower dissociation rate compared to SARS-CoV-1, which helps to rationalize the different infection patterns of the two viruses. By studying how free ACE2 outcompetes tethered ACE2, we show that our assay is sensitive to prevention of bond formation by external binders. We expect our results to provide a way to investigate the roles of viral mutations and blocking agents for targeted pharmaceutical intervention.
This study was supported by German Research Foundation Projects 386143268 and 111166240, a Human Frontier Science ProgramCross Disciplinary Fellowship (LT000395/2020C) and European Molecular Biology Organization Non-Stipendiary long-term fellowship (ALTF 1047-2019) to L.F.M., and the Physics Department of LMU Munich. R.C.B. and P.S.F.C.G. are supported by start-up funds provided by Auburn University, and D.L. acknowledges support from the Spanish Ministry of Science, Innovation and Universities for the Spanish State Research Agency Retos Grant RTI2018- 099318-B-I00, cofunded by the European Regional Development Fund.
Databáze: OpenAIRE
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