Title: Identification of lectin receptors for conserved SARS‐CoV‐2 glycosylation sites
Abstract: Article23 August 2021Open Access Transparent process Identification of lectin receptors for conserved SARS-CoV-2 glycosylation sites David Hoffmann David Hoffmann IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria These authors contributed equally to this work Search for more papers by this author Stefan Mereiter Stefan Mereiter orcid.org/0000-0002-4832-3090 IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria These authors contributed equally to this work Search for more papers by this author Yoo Jin Oh Yoo Jin Oh orcid.org/0000-0002-9636-3329 Institute of Biophysics, Johannes Kepler University Linz, Linz, Austria Search for more papers by this author Vanessa Monteil Vanessa Monteil orcid.org/0000-0002-2652-5695 Department of Laboratory Medicine, Unit of Clinical Microbiology, Karolinska Institute and Karolinska University Hospital, Stockholm, Sweden Search for more papers by this author Elizabeth Elder Elizabeth Elder orcid.org/0000-0003-1615-2642 Public Health Agency of Sweden, Solna, Sweden Search for more papers by this author Rong Zhu Rong Zhu Institute of Biophysics, Johannes Kepler University Linz, Linz, Austria Search for more papers by this author Daniel Canena Daniel Canena Institute of Biophysics, Johannes Kepler University Linz, Linz, Austria Search for more papers by this author Lisa Hain Lisa Hain Institute of Biophysics, Johannes Kepler University Linz, Linz, Austria Search for more papers by this author Elisabeth Laurent Elisabeth Laurent orcid.org/0000-0002-5234-5524 Department of Biotechnology and BOKU Core Facility Biomolecular & Cellular Analysis, University of Natural Resources and Life Sciences, Vienna, Austria Search for more papers by this author Clemens Grünwald-Gruber Clemens Grünwald-Gruber Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria Search for more papers by this author Miriam Klausberger Miriam Klausberger orcid.org/0000-0001-8409-454X Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria Search for more papers by this author Gustav Jonsson Gustav Jonsson orcid.org/0000-0001-6692-8395 IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria Search for more papers by this author Max J Kellner Max J Kellner IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria Search for more papers by this author Maria Novatchkova Maria Novatchkova IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria Search for more papers by this author Melita Ticevic Melita Ticevic IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria Search for more papers by this author Antoine Chabloz Antoine Chabloz Department of Medical Genetics, Life Sciences Institute, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Gerald Wirnsberger Gerald Wirnsberger Apeiron Biologics, Vienna, Austria Search for more papers by this author Astrid Hagelkruys Astrid Hagelkruys orcid.org/0000-0003-3015-4038 IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria Search for more papers by this author Friedrich Altmann Friedrich Altmann Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria Search for more papers by this author Lukas Mach Lukas Mach orcid.org/0000-0001-9013-5408 Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria Search for more papers by this author Johannes Stadlmann Johannes Stadlmann IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria Search for more papers by this author Chris Oostenbrink Chris Oostenbrink Department for Material Sciences and Process Engineering, Institute for Molecular Modeling and Simulation, University of Natural Resources and Life Sciences, Vienna, Austria Search for more papers by this author Ali Mirazimi Ali Mirazimi orcid.org/0000-0003-2371-6055 Department of Laboratory Medicine, Unit of Clinical Microbiology, Karolinska Institute and Karolinska University Hospital, Stockholm, Sweden National Veterinary Institute, Uppsala, Sweden Search for more papers by this author Peter Hinterdorfer Peter Hinterdorfer orcid.org/0000-0003-2583-1305 Institute of Biophysics, Johannes Kepler University Linz, Linz, Austria Search for more papers by this author Josef M Penninger Corresponding Author Josef M Penninger [email protected] orcid.org/0000-0002-8194-3777 IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria Department of Medical Genetics, Life Sciences Institute, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author David Hoffmann David Hoffmann IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria These authors contributed equally to this work Search for more papers by this author Stefan Mereiter Stefan Mereiter orcid.org/0000-0002-4832-3090 IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria These authors contributed equally to this work Search for more papers by this author Yoo Jin Oh Yoo Jin Oh orcid.org/0000-0002-9636-3329 Institute of Biophysics, Johannes Kepler University Linz, Linz, Austria Search for more papers by this author Vanessa Monteil Vanessa Monteil orcid.org/0000-0002-2652-5695 Department of Laboratory Medicine, Unit of Clinical Microbiology, Karolinska Institute and Karolinska University Hospital, Stockholm, Sweden Search for more papers by this author Elizabeth Elder Elizabeth Elder orcid.org/0000-0003-1615-2642 Public Health Agency of Sweden, Solna, Sweden Search for more papers by this author Rong Zhu Rong Zhu Institute of Biophysics, Johannes Kepler University Linz, Linz, Austria Search for more papers by this author Daniel Canena Daniel Canena Institute of Biophysics, Johannes Kepler University Linz, Linz, Austria Search for more papers by this author Lisa Hain Lisa Hain Institute of Biophysics, Johannes Kepler University Linz, Linz, Austria Search for more papers by this author Elisabeth Laurent Elisabeth Laurent orcid.org/0000-0002-5234-5524 Department of Biotechnology and BOKU Core Facility Biomolecular & Cellular Analysis, University of Natural Resources and Life Sciences, Vienna, Austria Search for more papers by this author Clemens Grünwald-Gruber Clemens Grünwald-Gruber Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria Search for more papers by this author Miriam Klausberger Miriam Klausberger orcid.org/0000-0001-8409-454X Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria Search for more papers by this author Gustav Jonsson Gustav Jonsson orcid.org/0000-0001-6692-8395 IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria Search for more papers by this author Max J Kellner Max J Kellner IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria Search for more papers by this author Maria Novatchkova Maria Novatchkova IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria Search for more papers by this author Melita Ticevic Melita Ticevic IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria Search for more papers by this author Antoine Chabloz Antoine Chabloz Department of Medical Genetics, Life Sciences Institute, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Gerald Wirnsberger Gerald Wirnsberger Apeiron Biologics, Vienna, Austria Search for more papers by this author Astrid Hagelkruys Astrid Hagelkruys orcid.org/0000-0003-3015-4038 IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria Search for more papers by this author Friedrich Altmann Friedrich Altmann Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria Search for more papers by this author Lukas Mach Lukas Mach orcid.org/0000-0001-9013-5408 Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria Search for more papers by this author Johannes Stadlmann Johannes Stadlmann IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria Search for more papers by this author Chris Oostenbrink Chris Oostenbrink Department for Material Sciences and Process Engineering, Institute for Molecular Modeling and Simulation, University of Natural Resources and Life Sciences, Vienna, Austria Search for more papers by this author Ali Mirazimi Ali Mirazimi orcid.org/0000-0003-2371-6055 Department of Laboratory Medicine, Unit of Clinical Microbiology, Karolinska Institute and Karolinska University Hospital, Stockholm, Sweden National Veterinary Institute, Uppsala, Sweden Search for more papers by this author Peter Hinterdorfer Peter Hinterdorfer orcid.org/0000-0003-2583-1305 Institute of Biophysics, Johannes Kepler University Linz, Linz, Austria Search for more papers by this author Josef M Penninger Corresponding Author Josef M Penninger [email protected] orcid.org/0000-0002-8194-3777 IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria Department of Medical Genetics, Life Sciences Institute, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Author Information David Hoffmann1, Stefan Mereiter1, Yoo Jin Oh2, Vanessa Monteil3, Elizabeth Elder4, Rong Zhu2, Daniel Canena2, Lisa Hain2, Elisabeth Laurent5, Clemens Grünwald-Gruber6, Miriam Klausberger7, Gustav Jonsson1, Max J Kellner1, Maria Novatchkova1, Melita Ticevic1, Antoine Chabloz8, Gerald Wirnsberger9, Astrid Hagelkruys1, Friedrich Altmann6, Lukas Mach10, Johannes Stadlmann1,6, Chris Oostenbrink11, Ali Mirazimi3,12, Peter Hinterdorfer2 and Josef M Penninger *,1,8 1IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria 2Institute of Biophysics, Johannes Kepler University Linz, Linz, Austria 3Department of Laboratory Medicine, Unit of Clinical Microbiology, Karolinska Institute and Karolinska University Hospital, Stockholm, Sweden 4Public Health Agency of Sweden, Solna, Sweden 5Department of Biotechnology and BOKU Core Facility Biomolecular & Cellular Analysis, University of Natural Resources and Life Sciences, Vienna, Austria 6Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria 7Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria 8Department of Medical Genetics, Life Sciences Institute, University of British Columbia, Vancouver, BC, Canada 9Apeiron Biologics, Vienna, Austria 10Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria 11Department for Material Sciences and Process Engineering, Institute for Molecular Modeling and Simulation, University of Natural Resources and Life Sciences, Vienna, Austria 12National Veterinary Institute, Uppsala, Sweden *Corresponding author. Tel: +43 1790 44; E-mail: [email protected] The EMBO Journal (2021)40:e108375https://doi.org/10.15252/embj.2021108375 [The copyright line of this article was changed on 4 October 2021 after original online publication.] PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract New SARS-CoV-2 variants are continuously emerging with critical implications for therapies or vaccinations. The 22 N-glycan sites of Spike remain highly conserved among SARS-CoV-2 variants, opening an avenue for robust therapeutic intervention. Here we used a comprehensive library of mammalian carbohydrate-binding proteins (lectins) to probe critical sugar residues on the full-length trimeric Spike and the receptor binding domain (RBD) of SARS-CoV-2. Two lectins, Clec4g and CD209c, were identified to strongly bind to Spike. Clec4g and CD209c binding to Spike was dissected and visualized in real time and at single-molecule resolution using atomic force microscopy. 3D modelling showed that both lectins can bind to a glycan within the RBD-ACE2 interface and thus interferes with Spike binding to cell surfaces. Importantly, Clec4g and CD209c significantly reduced SARS-CoV-2 infections. These data report the first extensive map and 3D structural modelling of lectin-Spike interactions and uncovers candidate receptors involved in Spike binding and SARS-CoV-2 infections. The capacity of CLEC4G and mCD209c lectins to block SARS-CoV-2 viral entry holds promise for pan-variant therapeutic interventions. SYNOPSIS SARS-CoV-2 Spike harbors 22 conserved N-glycan sites. Here we report the first extensive map of lectin-Spike interactions and assess the Spike-lectins associations at single molecule resolution. A lectin library screen identified Clec4g and CD209c to bind the SARS-CoV-2 spike protein. The lectin-spike interaction was visualized and quantified using atomic force microscopy. A binding site of CLEC4G is located within the RBD-ACE2 interface. The identified lectins block SARS-CoV-2 viral entry. Introduction COVID-19 caused by SARS-CoV-2 infections has triggered a pandemic massively disrupting health care, social and economic life. SARS-CoV-2 main entry route into target cells is mediated by the viral Spike protein, which binds to angiotensin converting enzyme 2 (ACE2) expressed on host cells (Monteil et al, 2020). The Spike protein is divided into two subunits, S1 and S2. The S1 subunits comprises the receptor binding domain (RBD) which confers ACE2 binding activity. The S2 subunit mediates virus fusion with the cell membrane following proteolytic cleavage (Hoffmann et al, 2020; Shang et al, 2020; Walls et al, 2020). Cryo-electron microscopy studies have shown that the Spike protein forms a highly flexible homotrimer containing 22 N-glycosylation sites each, 18 of which are conserved with the closely related SARS-CoV which caused the 2002/03 SARS epidemic (Ke et al, 2020; Walls et al, 2020). Point mutations removing glycosylation sites of the SARS-CoV-2 Spike protein were found to yield less infectious pseudo-typed viruses (Li et al, 2020). As Spike and RBD glycosylation affect ACE2 binding and SARS-CoV-2 infections, targeting virus-specific glycosylation could be a novel means for therapeutic intervention. Glycosylation of viral proteins ensures proper folding and shields antigenic viral epitopes from immune recognition (Watanabe et al, 2019; Watanabe et al, 2020b). To create this glycan shield, the virus hijacks the host glycosylation machinery and thereby ensures the presentation of self-associated glycan epitopes. Apart from shielding epitopes from antibody recognition, glycans can be ligands for lectin receptors. For instance, mannose-specific mammalian lectins, such as DC-SIGN (CD209) or its homolog L-SIGN (CD299), are well known to bind to viruses like HIV-1 and also SARS-CoV (Van Breedam et al, 2014). Lectin receptors are often expressed on immune and endothelial cells and serve as pattern recognition receptors involved in virus internalization and transmission (Osorio & Reis e Sousa, 2011). Recent studies have characterized the recognition of the SARS-CoV-2 Spike by previously known virus-binding lectins, such as DC-SIGN, L-SIGN, MGL, and MR (preprint: Gao et al, 2020). Given that SARS-CoV-2 relies less on oligo-mannose-type glycosylation, as compared to for instance HIV-1, and displays more complex-type glycosylation, it is unknown if additional lectin receptors are capable of binding the Spike protein and whether such interactions might have functional relevance in SARS-CoV-2 infections. Results Preparation of the first near genome-wide lectin library to screen for novel binders of Spike glycosylation To systematically identify lectins that bind to the trimeric Spike protein and RBD of SARS-CoV-2, we searched for all annotated carbohydrate recognition domains (CRDs) of mouse C-type lectins, Galectins and Siglecs. Of 168 annotated CRDs, we were able to clone, express and purify 143 lectin-CRDs as IgG2a-Fc fusion proteins from human HEK293F cells (Fig 1A, Dataset EV1). The resulting dimeric lectin-Fc fusion proteins (hereafter referred to as lectins) showed a high degree of purity (Fig 1B). This collection of lectins is, to our knowledge, the first comprehensive library of mammalian CRDs. Figure 1. Lectin library and SARS-CoV-2 Spike and RBD glycosylation Schematic overview of cloning, expression and purification of 143 carbohydrate recognition domain (CRD)—mouse IgG2a-Fc fusion proteins, from 168 annotated murine CRD containing proteins. The constructs were expressed in HEK293F cells, and secreted Fc-fusion proteins were purified using protein A columns. See Dataset EV1 for full list of expressed CRDs. Exemplified SDS–PAGE of purified Clec7a and Mgl2 stained with Coomassie blue. Glycosylation map of the SARS-CoV-2 Spike and RBD. The most prominent glycan structures are represented for each site, with at least 15% relative abundance. * marks highly variable glycosylation sites in which no single glycan structure accounted for > 15% relative abundance. The different monosaccharides are indicated using standardized nomenclature. NTD, n-terminal domain; RBD, receptor binding domain; S1/S2 and S2′, proteolytic cleavage sites; HR1 and HR2, α-helical heptad repeat domains 1 and 2; GlcNAc, N-acetylglucosamine. Download figure Download PowerPoint We next recombinantly expressed monomeric RBD and stabilized full-length trimeric pre-fusion Spike protein (hereafter referred to as Spike protein) in human HEK293-6E cells. Using mass spectrometry, we characterized all 22 N-glycosylation sites on the full-length Spike protein and 2 N-glycosylation sites on the RBD (Fig 1C and Dataset EV2). Most of the identified structures were in accordance with previous studies using full-length Spike (Watanabe et al, 2020a) with the exception of N331, N603 and N1194, which presented a higher structural variability of the glycan branches (Fig 1C and Dataset EV2). Importantly, the N-glycan sites of Spike are highly conserved among the sequenced SARS-CoV-2 viruses including the emerging variants B.1.1.7, 501Y.V2 and P.1 (Fig EV1A and B). The detected N-glycan species ranged from poorly processed oligo-mannose structures to highly processed multi-antennary complex N-glycans in a site-dependent manner. This entailed also a large variety of terminal glycan epitopes, which could act as ligands for lectins. Notably, the two glycosylation sites N331 and N343 located in the RBD carried more extended glycans, including sialylated and di-fucosylated structures, when expressed as an independent construct as opposed to the full-length Spike protein (Fig 1C and Dataset EV2). These data underline the complex glycosylation of Spike and reveal that N-glycosylation of the RBD within the 3D context of full-length trimeric Spike is different from N-glycosylation of the RBD expressed as minimal ACE2 binding domain. Click here to expand this figure. Figure EV1. Mutation frequency of N-glycan sites on SARS-CoV-2 Spike Among the 1,273 amino acids of Spike the frequency of mutational amino acid conversion within N-glycan sequons is plotted against all other sites. The mutation frequency of all 1,273 amino acids of Spike is shown. N-glycan sequons and mutations harboured by the new variants B.1.1.7, 501Y.V2 and P.1 are highlighted. Data information: (A) Two-tailed Student's t-test, *P < 0.05. Download figure Download PowerPoint CD209c and Clec4g are novel high affinity binders of SARS-CoV-2 Spike We evaluated the reactivity of our murine lectin library against the trimeric Spike and monomeric RBD of SARS-CoV-2 using an ELISA assay (Fig EV2A). This screen revealed that CD209c (SIGNR2), Clec4g (LSECtin) and Reg1 exhibited pronounced binding to Spike, whereas Mgl2 and Asgr1 displayed elevated binding to the RBD (Fig 2A and B, Dataset EV3). Further, we investigated the reactivity of the lectin library against human recombinant soluble ACE2 (hrsACE2); none of the lectins bound to hrsACE2 (Fig EV2B). We excluded Reg1 from further studies due to inconsistent ELISA results, likely due to protein instability. Asgr1 was excluded because it bound only to RBD but not to the Spike trimer, in accordance with the differences in glycosylation of glycosites N331 and N343 between Spike and RBD (Fig 1C). This highlights the importance of using a full-length trimeric Spike protein for functional studies. To confirm that the observed interactions were independent of protein conformation, Spike was denatured prior to the ELISA assay; binding of CD209c and Clec4g to the unfolded Spike remained unaltered (Fig 2C). Importantly, enzymatic removal of N-glycans by PNGase F treatment reduced the binding of CD209c, Clec4g, and Mgl2 towards Spike (Figs 2D and EV2C), confirming N-glycans as ligands. Binding of ACE2, which relies on protein–protein interactions, was completely abrogated when Spike was denatured (Fig 2C). These data identify lectins that have the potential to bind to the RBD and trimeric Spike of SARS-CoV-2. Click here to expand this figure. Figure EV2. ELISA assays to detect lectin binding Schematic representation of the ELISA protocol, consisting of coating with trimeric full-length Spike or the monomeric receptor binding domain (RBD) followed by sequential incubation with lectin-Fc fusion proteins and secondary anti-IgG-HRP antibodies. The binding of lectin-Fc fusion proteins was quantified by peroxidase-dependent substrate conversion, measured by optical density (OD) at 490 nm and normalized against a BSA control. ELISA screen of the lectin-Fc library against human recombinant soluble ACE2 (hrsACE2). Results are shown as mean OD values of 2 replicates normalized against a BSA control and ranked by value. The 5 main lectin targets identified by Spike and RBD ELISA screen (Fig 2A and B) are labelled. SDS–Page of RBD and full-length trimeric Spike de-N-glycosylated with PNGase F and stained with Coomassie blue. A PNGase F control was added to display the size of the PNGase F protein. Download figure Download PowerPoint Figure 2. Identification of lectins that bind to Spike and RBD of SARS-CoV-2 A, B. ELISA screen of the lectin-Fc library against full-length trimeric SARS-CoV-2 Spike (A) or monomeric RBD (B). Results are shown as mean OD values of 2 replicates normalized against a BSA control and ranked by value. Lectin-Fc fusion proteins with a normalized OD > 0.5 in either (A) or (B) are indicated in both panels. See Dataset EV2 for primary ELISA data. C. Lectin-Fc and human ACE2-mIgG1 Fc-fusion protein (hACE2) binding to untreated or heat-denatured full-length SARS-CoV-2 Spike by ELISA. hACE2-mIgG1 was used as control for complete denaturation of Spike protein. Results are shown as mean OD values ± SD normalized to the BSA control (technical replicates, N = 3). D. Lectin-Fc binding to full-length SARS-CoV-2 Spike with or without de-N-glycosylation by PNGase F. "PNGase F only" denotes wells that were not coated with the Spike protein. Results are shown as mean OD values ± SD normalized to BSA controls (technical replicates, N = 3). E, F. Surface plasmon resonance (SPR) analysis with immobilized full-length trimeric Spike, probed with various concentrations of Clec4g-Fc (E) and CD209c-Fc (F). See Table 1 for kinetics values. Data information: (C) t-test with Holm–Sidak correction for multiple comparisons. (D) One-way ANOVA with Tukey's multiple comparisons; **P < 0.01; ***P < 0.001; ns: not significant. Download figure Download PowerPoint Based on the robust N-glycan dependent Spike binding, we focused our further studies on CD209c and Clec4g. We first used surface plasmon resonance (SPR) to determine the kinetic and equilibrium binding constants of these lectins to the trimeric Spike. The resulting experimental binding curves were fitted to the "bivalent analyte model" (Traxler et al, 2017) which assumes two-step binding of the lectin dimers to adjacent immobilized Spike trimer binding sites (Fig 2E and F). From these fits, we computed the kinetic association (ka,1), kinetic dissociation (kd,1) and equilibrium dissociation (Kd,1) binding constants of single lectin bonds (Table 1). The equilibrium dissociations (Kd,1) values were 1.6 μM and 1.0 μM for Clec4g and CD209c, respectively. Table 1. Values computed for surface plasmon resonance (SPR) and atomic force microscopy (AFM). SPR AFM ka,1 [M−1s−1] kd,1 [s−1] Kd,1 [M] koff [s−1] xβ [nm] Clec4g 6.17 × 104 0.0997 1.62 × 10−6 0.037 ± 0.007 0.55 ± 0.02 CD209c 1.61 × 104 0.0159 0.988 × 10−6 0.041 ± 0.025 0.76 ± 0.03 hCLEC4G 7.77 × 104 0.0201 0.259 × 10−6 0.007 ± 0.0004 0.64 ± 0.55 hCD209 1.32 × 104 0.0316 2.39 × 10−6 0.008 ± 0.001 0.73 ± 0.14 SPR. Kinetic association (ka,1), kinetic dissociation (kd,1) and equilibrium dissociation (Kd,1) constants of the first binding step fitted from the bivalent analyte model, assuming two-step binding and dissociation of the lectins to adjacent immobilized Spike trimer binding sites under spontaneous thermodynamic energy barriers; no reasonable fit was obtained with the simple 1:1 binding model. AFM. Kinetic off-rate constants (koff) and lengths of dissociation paths (xβ) of single lectin bonds, originating from force-induced unbinding in single-molecule force spectroscopy (SMFS) experiments and computed using Evans's (Bell, 1978; Evans & Ritchie, 1997) model, assuming a sharp single dissociation energy barrier. Multiple CD209c and Clec4g molecules bind simultaneously to SARS-CoV-2 Spike and form compact complexes To study Spike binding of these two lectins at the single-molecule level, we used atomic force microscopy (AFM) and performed single-molecule force spectroscopy (SMFS) experiments. To this end, we coupled trimeric Spike to the tip of the AFM cantilever and performed single-molecule force measurements (Hinterdorfer et al, 1996), by moving the Spike trimer-coupled tip towards the surface-bound lectins to allow for bond formations (Fig 3A). Unbinding was accomplished by pulling on the bonds, which resulted in characteristic downward deflection signals of the cantilever, whenever a bond was ruptured (Fig 3B). The magnitude of these vertical jumps reflects the unbinding forces, which were of typical strengths for specific molecular interactions (Rankl et al, 2008). Using this method (Rankl et al, 2008; Zhu et al, 2010), we quantified unbinding forces (Fig 3C) and calculated the binding probability and the number of bond ruptures between CD209c or Clec4g and trimeric Spike (Fig 3D). Both lectins showed a very high binding probability and could establish up to 3 strong bonds with accumulating interaction force strengths reaching 150 pN in total with trimeric Spike (Fig 3C), with the preference of single and dual bonds (Figs 3C and D, and EV3A and B, Table 1). Of note, multi-bond formation leads to stable complex formation, in which the number of formed bonds enhances the overall interaction strength and dynamic stability of the complexes. To assess dynamic interactions between single molecules of trimeric Spike and the lectins in real time we used high-speed AFM (Kodera et al, 2010; Preiner et al, 2014). Addition of Clec4g and CD209c led to a volume increase of the lectin/Spike complex in comparison with the trimeric Spike alone; based on the volumes we could calculate that on average 3.2 molecules of Clec4g and 5.2 molecules of CD209c were bound to one Spike trimer (Figs 3E and EV3C–F and Movies EV1–EV4). These data show, in real-time, at single-molecule resolution, that mouse Clec4g and CD209c can directly associate with trimeric Spike. Figure 3. Single molecule, real-time imaging of lectin-Spike binding Schematic overview of single-molecule force spectroscopy (SMFS) experiments using full-length trimeric Spike coupled to an atomic force microscopy (AFM) cantilever tip and surface coated murine Clec4g-Fc or CD209c-Fc. Arrow indicates pulling of cantilever. Representative force traces showing sequential bond ruptures in the SMFS experiments. Measured forces are shown in pico-Newtons (pN). Experimental probability density function (PDF) of unbinding forces (in pN) determined by SMFS (black line, measured data). The three distinct maxima fitted by a multi-Gaussian function reveal rupture of a single bond (blue dotted line), or simultaneous rupture of 2 (red dotted line) and 3 (green dotted line) bonds, respectively. SMFS-determined binding probability for the binding of trimeric Spike to Clec4g and CD209c. Data are shown as mean binding probability ± SD of single, double, triple or quadruple bonds (technical replicates, N = 4). High-speed AFM of single trimeric Spike visualizing the real-time interaction dynamics with lectins. Top panel shows 5 frames of trimeric Spike alone imaged on mica. Mi