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Modulation of SARS-CoV-2 spike binding to ACE2 through conformational selection

Nature Nanotechnology volume 20, pages 926–934 (2025)Cite this article

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Abstract

The first step of SARS-CoV-2 infection involves the interaction between the viral trimeric spike protein (S) and the host angiotensin-converting enzyme 2 (ACE2). The receptor-binding domain (RBD) of S adopts two conformations: open and closed, respectively accessible and inaccessible to ACE2. Although these changes surely affect ACE2 binding, a quantitative description of the underlying mechanisms has remained elusive. Here we visualize RBD opening and closing using high-speed atomic force microscopy, gaining access to the corresponding transition rates. We also probe the S/ACE2 interaction at the ensemble level with biolayer interferometry and at the single-molecule level with atomic force microscopy and magnetic tweezers, evidencing that RBD dynamics hinder ACE2 binding but have no effect on unbinding. The resulting modulation is quantitatively predicted by a conformational selection model in which each S protomer behaves independently. Our work thus reveals a molecular mechanism by which RBD accessibility and binding strength can be tuned separately, providing hints to better understand the joint evolution of immune evasion and infectivity.

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Fig. 1: Single-molecule real-time visualization of the conformational dynamics of RBD opening and closing within S trimers.
Fig. 2: Ensemble measurements of the kinetic and thermodynamic parameters for ACE2 binding to RBD, either isolated or embedded within an S trimer.
Fig. 3: Single-molecule measurements of the dissociation kinetics and force response for ACE2 bound to the RBD, either isolated or embedded within an S trimer.
Fig. 4: Single-molecule measurements of the kinetic and thermodynamic parameters for ACE2 binding to the RBD, either isolated or embedded within an S trimer.

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Data availability

The data reported in this study are available within the article and its Supplementary Materials file. Raw data are available from the corresponding authors upon reasonable request. Source data are provided with this paper.

Code availability

The code used for image processing and analysis is available on the GitHub repository (source code: https://github.com/centuri-engineering/ProtruDe/).

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Acknowledgements

We thank M. Backovic (UVS, Institut Pasteur, Paris) for logistics; M. A. Nash (Chemistry Department, University of Basel) for the gift of the Sfp plasmid; P. England and the Biophysics Facility (Institut Pasteur) for access to the BLI; D. Joshi (RCAS, Academia Sinica, Taipei) for advice on PyMOL; P.-H. Puech and L. Limozin (LAI, Aix-Marseille University) for insightful discussions; and J. Reguera (AFMB, Aix-Marseille University) for critical reading of the paper. This project received funding from the Human Frontier Science Program (HFSP, grant number RGP0056/2018 to F.R.), the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement number 772257 to F.R.), the European Union’s Horizon 2020 research and innovation programme (Marie Skłodowska-Curie grant number 895819 to C.V.), the Turing Centre for Living Systems (Centuri), PSL-Valorisation (grant J-DNA 2 to C.G. and T.S.), Labex IBEID (grant ANR-10-LABX-62-IBEID to F.A.R.), ANRS-MIE project EMERGEN (grant ANRS0151 to F.A.R.) and the Pasteur Coronavirus Task-Force (grants Allospike and TooLab to F.A.R.). The Molecular Motors and Machines team at IBENS has received a ‘Coup d’élan’ from the Fondation Bettencourt Schueller and is also an ‘Equipe labellisée’ by the Ligue Nationale Contre le Cancer. D.K. was supported by the PSL Institut de Convergence QLife. F. Stransky benefited from a doctoral fellowship from the Ministère de l’Enseignement Supérieur et de la Recherche. The project leading to this publication received funding from France 2030, the French Government programme managed by the French National Research Agency (ANR-16-CONV-0001), and from Excellence Initiative of Aix-Marseille University—A*MIDEX.

Author information

Author notes

  1. Thomas Boudier

    Present address: Centrale Mediterranée, Marseilles, France

  2. These authors contributed equally: Prithwidip Saha, Ignacio Fernandez, Fidan Sumbul, Claire Valotteau, Dorota Kostrz.

Authors and Affiliations

  1. Aix-Marseille Univ., INSERM, DyNaMo, Turing Centre for Living Systems, Marseilles, France

    Prithwidip Saha, Fidan Sumbul, Claire Valotteau & Felix Rico

  2. Institut Pasteur, Université Paris-Cité, Structural Virology Unit, CNRS UMR3569, Paris, France

    Ignacio Fernandez, Annalisa Meola, Eduard Baquero, Arvind Sharma, Pablo Guardado-Calvo & Felix A. Rey

  3. Institut de Biologie de l’Ecole Normale Supérieure, ENS, CNRS, INSERM, PSL Research University, Paris, France

    Dorota Kostrz, James R. Portman, François Stransky, Charlie Gosse & Terence Strick

  4. Aix-Marseille Univ., Multiengineering Platform, Turing Centre for Living Systems, Marseilles, France

    Thomas Boudier

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Contributions

C.G., T.S., F.A.R. and F.R. supervised the research. I.F., A.M., E.B. and A.S. designed and produced the ACE2, RBD and spike proteins. P.S. and F. Sumbul acquired the HS-AFM videos. P.S. and T.B. developed and applied the ImageJ macros to extract the conformational trajectories from HS-AFM videos. F.R. analysed the conformational trajectories. C.G. and F.R. developed the conformational selection model and implemented it for data analysis. I.F., E.B. and P.G.-C. conducted the BLI measurements. C.V. performed the AFM and HS-AFM SMFS experiments and analysed the data with F.R. D.K. synthesized J-DNA and coupled them to the molecular partners. D.K. and J.R.P. realized the MT measurements. F. Stransky developed the software for analysing the constant-force MT experiments. P.S., C.G., C.V. and F.R. wrote the paper with the contributions of all the authors.

Corresponding authors

Correspondence to Charlie Gosse, Terence Strick, Felix A. Rey or Felix Rico.

Ethics declarations

Competing interests

PSL-Valorisation has submitted a patent related to the J-DNA forceps (PCT FR2018/053533) with D.K., T.S. and C.G. among the inventors. The other authors declare no competing interests.

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Nature Nanotechnology thanks Jan Lipfert and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Real-time RBD conformations in \({\mathbf{S}}_{\mathbf{Wuhan}}^{\mathbf{f-}}\).

Consecutive images from an HS-AFM video depicting S trimer in various states, as indicated in the bottom left corner of the images. The white arrowhead indicates the stalk region of the spike, while the yellow arrowheads indicate the opening of RBDs. The acquisition rate of the video was 2.5 fps. These images were processed with a set of user-written macros (see Methods).

Extended Data Fig. 2 Conformational dynamics of RBD opening and closing in \({\mathbf{S}}_{\mathbf{Wuhan}}^{\mathbf{f-,2C,2P}}\).

(a) Relative occupancy histogram (mean ± s.e.m., blue bars) and associated fits to the binomial distribution (red lines) for the four \({S}_{i}\) states, i indicating the number of opened RBDs. (b) Transition density plot (TDP) showing the fraction of transition events between the different states. (c) Distributions of the \({\Delta t}_{i,\;j}\) dwell times for the four most populated \({S}_{i}\) to \({S}_{j}\) transitions. Red lines show the global exponential fits used to extract the \({\tau }_{i,\;j}\) decays (see Extended Data Table 1 for results and Supplementary Table 2 for statistics).

Source data

Extended Data Fig. 3 Real-time RBD conformations in \({\mathbf{S}}_{\mathbf{Wuhan}}^{\mathbf{f-,6P}}\).

Consecutive images from an HS-AFM video depicting S trimer in various states, as indicated in the bottom left corner of the images. The acquisition rate of the video was 1.0 fps. Timestamps are displayed in the top right corner of the image. The white arrowhead indicates the stalk region of the spike, while the yellow arrowheads indicate the opening of RBDs. These images were processed with a set of user-written macros (see Methods).

Extended Data Fig. 4 Real-time RBD conformations in \({\mathbf{S}}_{\mathbf{Omicron}}^{\mathbf{f-,6P}}\).

Consecutive images from an HS-AFM video depicting S trimer in various states, as indicated in the bottom left corner of the images. The white arrowhead indicates the stalk region of the spike, while the yellow arrowheads indicate the opening of RBDs. The acquisition rate of the video was 1.0 fps. These images were processed with a set of user-written macros (see Methods).

Extended Data Fig. 5 Effect of the temperature correction on the rate constants measured in this study for the dissociation of ACE2 from the wild-type RBD.

Experiments involved either RBD alone or spike trimers (see Supplementary Table 5 for numerical values). (a) Data issued from acquisitions realized at various temperatures \({T}_{{meas}}\) (in linear scale, ± s.e.). (b) Same data extrapolated at 22 °C using the Arrhenius law and an activation energy equal to 90 kJ mol−1. (c) Same extrapolation but with an activation energy equal to 125 kJ mol−1.

Extended Data Fig. 6 Effect of the temperature correction on the rate constants reported in the single-molecule literature for the dissociation of ACE2 from the wild-type RBD.

Experiments involved either RBD alone, S1 alone, or spike trimers (see Table Supplementary Table 6 for numerical values). (a) Data issued from acquisitions realized at various temperatures \({T}_{{meas}}\) (in both logarithmic and linear scales, ± s.e. for the Saha et al. data). (b) Same data extrapolated at 22 °C using the Arrhenius law and an activation energy equal to 90 kJ mol−1. (c) Same extrapolation but with an activation energy equal to 125 kJ mol−1.

Extended Data Fig. 7 Representative images of an HS-AFM video showing binding of an ACE2 to S trimer \({\mathbf{S}}_{\mathbf{Wuhan}}^{\mathbf{f-}}\).

Free ACE2 is indicated by a cyan arrowhead and bound ACE2 (that cannot be distinguished from the RBD to which it is bound) by green ones. The spike stalk region is indicated by white and open RBDs by yellow ones. Timestamps are displayed in the top right corner of each image. On the left, a simulated AFM image is generated from the cryo-EM structure of one ACE2 bound to one S trimer (PDB: 7knb) to illustrate the relative positioning of ACE2 and RBD in the experimental observation.

Extended Data Fig. 8 Representative images of an HS-AFM video showing ACE2 association and dissociation from an S trimer \({\mathbf{S}}_{\mathbf{Wuhan}}^{\mathbf{f-}}\) already interacting with two other ACE2.

Unbound ACE2 are indicated by cyan arrowheads, bound ACE2 by green arrowheads, the stalk region by a white arrowhead, and open RBDs by yellow arrowheads. Timestamps are displayed in the top right corner of each image. The relative orientation of the ACE2 to the RBD causes the RBD to appear brighter compared to the other RBDs without ACE2 binding. On the left, a simulated AFM image is generated from the cryo-EM structure of three ACE2 bound to one S trimer (PDB: 7kms) to illustrate the relative positioning of ACE2 and RBD in the experimental observation.

Extended Data Table 1 Populations of the different RBD conformations of S trimers from cryo-EM literature and from HS-AFM (this work)
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Extended Data Table 2 Energy landscape parameters for the dissociation reaction between the ACE2 and RBD, either isolated or embedded within a S trimer
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Supplementary information

Supplementary Information

Supplementary Methods 1–3, Texts 1–5, Figs. 1–12, Tables 1–6 and video information.

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Saha, P., Fernandez, I., Sumbul, F. et al. Modulation of SARS-CoV-2 spike binding to ACE2 through conformational selection. Nat. Nanotechnol. 20, 926–934 (2025). https://doi.org/10.1038/s41565-025-01908-1

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