between the center of mass of the oxygens in the acidic side chain and the center of mass of the nitrogens in the basic side chain is within the cut-off distance of 3.5 Å.

**Angles.** We measured the angle formed between 1) the Fc domain and the M1 protein, 2) Fc and Fab domains, and 3) Fab1 and Fab2 domains. The vectors used to calculate each angle are defined here:

* *between the Fc domain and the M1 protein:* i) the center of mass of the Fc domain and center of mass of the M1 protein, ii) the center of mass and the N-terminal region of the M1 protein.

* *between the Fc domain and Fab domains:* i) the center of mass of the Fc domain and center of mass of the hinge region, ii) the center of mass of the Fab domain and center of mass of the hinge region.

* *between the Fab1 and Fab2 domains:* the center of mass of the hinge region and center of mass of each Fab domain.

* *Fab2 domain:* the center of mass of the hinge region and center of mass of the Fab2.

The angles were then defined based on the lines that best fit these segments. The results are reported in two scenarios: for all trajectories (Supplementary Fig. 5A–C) and in dual-Fab binding conformations where the distance between residues at the interface of Fab1 and the M1 protein is less than 30 Å (Supplementary Fig. 5D–F). The second scenario resulted in 20,074 angles for M1-IgG1, 10,871 angles for M1-IgG3, and 11,306 angles for M1-IgG<sub>62</sub> simulations.

## Clustering analysis

To characterize representative conformations, we performed a cluster analysis of the MD trajectories using backbone RMSD as the similarity metrics by the GROMOS clustering approach<sup>68</sup> with a cutoff equal to 0.7 nm. The trajectories of replicates were first concatenated, clustering was then performed, and finally, the population of each cluster was calculated. The same analysis was performed only considering the replicates where the dual-Fab binding was observed (replicates 1 and 3 for M1-IgG1, replicate 3 for M1-IgG3, and replicate 1 for IgG<sub>62</sub> simulations). Clusters with a population of at least 3000 conformations were colored in shades of green on the plot (Supplementary Fig. 6A). The results are reported for all MD trajectories of M1-IgG1, M1-IgG3 and IgG<sub>62</sub> systems (30000, 30000 and 20000 conformations, respectively) and only for dual-Fab binding conformations (20000, 10000 and 10000 conformations, respectively). The three highly populated clusters of IgG1 consist of 9071, 4852, and 3239 conformations. And the top three highly populated clusters of IgG3 consist of 5363, 4195, and 4031 conformations. The representative conformations of the highly populated clusters are shown when dual-Fab binding was observed (Supplementary Fig. 6B–E).

## Binding free energy and alanine scanning

The AnalyseComplex in FoldX version 4<sup>69</sup> was used to estimate the binding free energy between the Fab1 and M1 protein, and between the Fab2 and M1 protein (Supplementary Fig. 7). The AlaScan in FoldX version 4 was used to identify relevant residues involved in the interactions between the IgGs and M1 protein. For each system we identified Fab residues that are at the interface with M1 protein, as those representing an average distance of at most 5 Å with the closest M1 residue over the course of the MD simulations. Then we reported the ΔΔG values for interface Fab residues only for the replicates when the average distance is less than or equal to 5 Å. The results of alanine scanning are reported for the interface residues of Fab1 (Supplementary Fig. 8) and Fab2 (Supplementary Fig. 9). The values in blue at the top of each box show number of replicates from which the ΔΔG values were reported (i.e. number of replicates in which the average distance between the corresponding Fab residue and the M1 protein is at most 5 Å).

## Crystallization data analysis

The Ab25 Fab sample was concentrated to 6.75 mg/mL in the buffer of 20 mM HEPES(2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane-1-sulfonic acid), 150 mM NaCl, pH 7.0. A crystallization experiment set up against commercially available screens was performed with a Mosquito crystallization robot (SPT Labtech) using sitting drops with a total volume of 300 nL and different ratios between the protein sample and reservoir solution. Optimization of initial hits gave crystals appearing within a week in 27.50% (w/v) PEG8000, 0.1 M CHES pH 8.5. After growing to full size (50 x 100 x 250 µm), the crystals were harvested, cryoprotected in reservoir solution supplemented with 20% (v/v) glycerol, and flash frozen in liquid nitrogen. Testing for diffraction and data collection was performed at the BioMAX beamline at the MAX IV Laboratory (Lund, Sweden). Diffraction data were collected by fine-slicing with an oscillation range of 0.1°, 360° total. The data were auto-processed with the software EDNA<sup>70</sup> to a resolution of 1.88 Å, and the structure was solved by molecular replacement with PHASER of the PHENIX software suite<sup>71</sup>, using PDB entry 6JC2.pdb as a search model for both the heavy and light chain. Electron and difference density maps were manually inspected, and the model was improved using Coot<sup>72</sup> and several rounds of refinement with PHENIX<sup>71</sup>. The calculation of Rfree used 5.04% of the data. Statistics over data and refinement can be seen in Supplementary Table 1. The crystallographic data have been deposited to the Protein Data Bank (PDB) with the accession ID: 8C67.

## IgG pulldowns

IgGs were purified in a 96-well plate setup using the Protein G Assay-MAP Bravo (Agilent) technology, according to the manufacturer's instructions. Briefly, 10 µl plasma was diluted with PBS to a final volume of 100 µL and applied to pre-equilibrated Protein G columns. Columns were washed with PBS and eluted in 0.1 M glycine (pH 2). The final pH was neutralized with 1 M Tris and saved until further use.

## Trypsin digestion and peptide desalting

Protein samples were resuspended in 8 M urea and reduced with 5 mM Tris(2-carboxyethyl)phosphine hydrochloride, pH 7.0 for 45 min at 37 °C, and alkylated with 25 mM iodoacetamide (Sigma) for 30 min at RT, followed by dilution with 100 mM ammonium bicarbonate to a final urea concentration below 1.5 M. Proteins were digested by incubation with trypsin (1/100, w/w, Sequencing Grade Modified Trypsin, Porcine; Promega) overnight. Digestion was stopped using 10% trifluoracetic acid (Sigma) to pH 2 to 3. Peptide clean-up was performed by C-18 AssayMAP Bravo (Agilent), according to the manufacturer's guideline. Samples were lyophilized and resuspended in 50 µl HPLC-water (Fisher Chemical) with 2% acetonitrile and 0.2% formic acid (Sigma).

## LC-MS/MS and bioinformatic analysis

Mass spectrometry analysis was performed on a Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific) connected to an EASY-nLC 1200 ultra-HPLC system (Thermo Fisher Scientific). Peptides were trapped on pre-column (PepMap100 C18 3 µm; 75 µm x 2 cm; Thermo Fisher Scientific) and separated on an EASY-Spray column (ES903, column temperature 45 °C; Thermo Fisher Scientific). Equilibrations of columns and sample loading were performed per the manufacturer's guidelines. Mobile phases of solvent A (0.1% formic acid) and solvent B (0.1% formic acid, 80% acetonitrile) were used to run a linear gradient from 5% to 38% over 90 min at a flow rate of 350 nl/min. Complete MS analysis was conducted at a resolution of 120000, the automatic gain control (AGC) was 3e6, and the injection time (IT) was set to 50 ms. The MS2 acquisition was performed in a data-dependent mode, where the 15 most intense ions were fragmented and analyzed at a resolution of 60000, AGC 1e5, 120 ms of IT, and a normalized collision energy (NCE) of 25. Raw files were searched using Proteome Discoverer 2.5 and
against a mouse UniProt database, including the Fasta sequences of Ab25 antibodies. The precursor and fragment mass tolerances were set to 10 ppm and 0.02 Da, respectively. Methionine oxidation was set as a dynamic modification. Carbamidomethylation of cysteines was included as a static modification.

## Statistical analysis

Statistical analysis was performed with a one-way ANOVA multiple comparisons test followed by a post-hoc correction with Dunnette's correction test for in vitro data or RM one-way ANOVA with multiple comparisons test with Dunnette's for ex vivo data. Kruskall-Wallis test was performed with multiple comparisons corrected with Dunn's post-hoc test for statistical comparisons for the in vivo experiments. Mann-Whitney U two-tailed test was performed when comparing two treatments only. All tests were done with GraphPad Prism.

## Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

## Data availability

The in vitro, in silico and in vivo data shown in the graphs in Figs. 1–8 are provided in the source data file provided with the manuscript. The structural data generated in this manuscript has been deposited in the Worldwide protein data bank (wwPDB) PDB ID: 8C67. The mass spectrometry data generated in this study have been deposited to MassIVE, under the accession: MSV000094287 and is publicly available. Source data are provided with this paper.

## References

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10. Bruhns, P. et al. Specificity and affinity of human Fcγ receptors and their polymorphic variants for human IgG subclasses. *Blood. **113***, 3716–3725 (2009).

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27. Woof, J. M. & Burton, D. R. Human antibody-Fc receptor interactions illuminated by crystal structures. *Nature Reviews Immunology. **4***, 89–99 (2004).

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30. Karlsson, C. A. Q. et al. Streptococcus pyogenes Infection and the Human Proteome with a Special Focus on the Immunoglobulin G-cleaving Enzyme IdeS. *Mol Cell Proteomics. **17***, 1097–1111 (2018).

31. Crowley, A. R. et al. Evidence of variable human Fcγ receptor-Fc affinities across differentially-complexed IgG. *mAbs **15***, 2231128 (2023).

32. Sanderson-Smith, M., De Oliveira, D. M. P., Guglielmini, J., McMillan, D. J. & Vu, T. A systematic and functional classification of streptococcus pyogenes that serves as a new tool for molecular typing and vaccine development. *J Infect Dis **210***, 1325–1338 (2014).
against a mouse UniProt database, including the Fasta sequences of Ab25 antibodies. The precursor and fragment mass tolerances were set to 10 ppm and 0.02 Da, respectively. Methionine oxidation was set as a dynamic modification. Carbamidomethylation of cysteines was included as a static modification.

## Statistical analysis

Statistical analysis was performed with a one-way ANOVA multiple comparisons test followed by a post-hoc correction with Dunnette's correction test for in vitro data or RM one-way ANOVA with multiple comparisons test with Dunnette's for ex vivo data. Kruskall-Wallis test was performed with multiple comparisons corrected with Dunn's post-hoc test for statistical comparisons for the in vivo experiments. Mann-Whitney U two-tailed test was performed when comparing two treatments only. All tests were done with GraphPad Prism.

## Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

## Data availability

The in vitro, in silico and in vivo data shown in the graphs in Figs. 1–8 are provided in the source data file provided with the manuscript. The structural data generated in this manuscript has been deposited in the Worldwide protein data bank (wwPDB) PDB ID: 8C67. The mass spectrometry data generated in this study have been deposited to MassIVE, under the accession: MSV000094287 and is publicly available. Source data are provided with this paper.

## References

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4. Torres, M. & Casadevall, A. The immunoglobulin constant region contributes to affinity and specificity. *Trends in Immunology. **29***, 91–97 (2008).

5. Janda, A., Bowen, A., Greenspan, N. S. & Casadevall, A. Ig constant region effects on variable region structure and function. *Frontiers in Microbiology. **7***, 1–10 (2016).

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## Acknowledgements

This work was funded by the Swedish Research Council (Vetenskapsrådet, JM, PN (VR-2020-01511), the Knut and Alice Wallenberg Foundation (JM, OS, LM, PN), the Institut Pasteur (Pasteur-Cantarini-Roux fellowship to Y.K.), and the Alfred Österlunds Foundation (JM, LM, PN). L.M. is funded by the Swedish Research Council (Vetenskapsrådet, VR) (VR-2020-02419). PN and OS are funded by the Mats Paulsson Foundation. This work was granted access to the HPC resources of IDRIS under the allocation 2021- A0110711500 made by GENCI (granted to Y.K. and M.N.). HK was supported by the French Agence Nationale de la Recherche (ANR) under grant ANR-22-CPJ2-0075-01. We thank Anki Mossberg for technical assistance. Al was funded by the Royal Physiographic Society (43972) and Lars Hiertas Memory (FO2023-0534).

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## Acknowledgements

This work was funded by the Swedish Research Council (Vetenskapsrådet, JM, PN (VR-2020-01511), the Knut and Alice Wallenberg Foundation (JM, OS, LM, PN), the Institut Pasteur (Pasteur-Cantarini-Roux fellowship to Y.K.), and the Alfred Österlunds Foundation (JM, LM, PN). L.M. is funded by the Swedish Research Council (Vetenskapsrådet, VR) (VR-2020-02419). PN and OS are funded by the Mats Paulsson Foundation. This work was granted access to the HPC resources of IDRIS under the allocation 2021- A0110711500 made by GENCI (granted to Y.K. and M.N.). HK was supported by the French Agence Nationale de la Recherche (ANR) under grant ANR-22-CPJ2-0075-01. We thank Anki Mossberg for technical assistance. Al was funded by the Royal Physiographic Society (43972) and Lars Hiertas Memory (FO2023-0534).

## Author contributions

A.I., Y.K., H.K., J.M., W.B., O.S., L.M., M.O., M.N. and P.N. designed research; A.I., Y.K., E.B., S.W., H.K., M.N.Y., B.O., L.H., M.S., M.G., T.S., Y.C.,
A.G.T. and D.T., performed research; A.I., Y.K., E.B., H.K., M.N.y., M.G., A.G.T., L.H. and P.N. analyzed data; A.I., Y.K. and P.N. wrote the paper. All authors contributed to reading and editing the final manuscript.

## Funding

Open access funding provided by Lund University.

## Competing interests

AI, HK, WB, LH, OS, JM, LM, and PN have filed patents related to the mAbs described in this manuscript. The remaining authors declare no competing interests.

## Additional information

**Supplementary information** The online version contains supplementary material available at https://doi.org/10.1038/s41467-024-47928-8.

**Correspondence** and requests for materials should be addressed to Pontus Nordenfelt.

**Peer review information** *Nature Communications* thanks Eric J. Sundberg, Michael Good and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

**Reprints and permissions information** is available at http://www.nature.com/reprints

**Publisher's note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Open Access** This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

© The Author(s) 2024

<figure><img src="image_78.png" /></figure>

e-mail: pontus.nordenfelt@med.lu.se

<footnote n="1">¹Department of Clinical Sciences Lund, Infection Medicine, Faculty of Medicine, Lund University, Lund, Sweden. ²Université de Lorraine, CNRS, Inria, LORIA, F-54000 Nancy, France. ³Institut Pasteur, Université Paris cite, CNRS UMR3528, Structural Bioinformatics Unit, Department of Structural Biology and Chemistry, F-75015 Paris, France. ⁴Department of Biology & Lund Protein Production Platform (LP3), Lund University, Lund, Sweden. ⁵Department of Immunotechnology and SciLifeLab Drug Discovery and Development Platform, Lund University, Lund, Sweden. ⁶Department of Clinical Sciences Lund, Division of Pediatrics, Faculty of Medicine, Lund University, Lund, Sweden. ⁷Section for Oral Biology and Pathology, Faculty of Odontology, Malmö University, Malmö, Sweden. ⁸Department of Laboratory Medicine, Clinical Microbiology, Skåne University Hospital Lund, Lund University, Lund, Sweden.</footnote>
A.G.T. and D.T., performed research; A.I., Y.K., E.B., H.K., M.N.y., M.G., A.G.T., L.H. and P.N. analyzed data; A.I., Y.K. and P.N. wrote the paper. All authors contributed to reading and editing the final manuscript.

## Funding

Open access funding provided by Lund University.

## Competing interests

AI, HK, WB, LH, OS, JM, LM, and PN have filed patents related to the mAbs described in this manuscript. The remaining authors declare no competing interests.

## Additional information

**Supplementary information** The online version contains supplementary material available at https://doi.org/10.1038/s41467-024-47928-8.

**Correspondence** and requests for materials should be addressed to Pontus Nordenfelt.

**Peer review information** *Nature Communications* thanks Eric J. Sundberg, Michael Good and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

**Reprints and permissions information** is available at http://www.nature.com/reprints

**Publisher's note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Open Access** This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

© The Author(s) 2024

<figure><img src="image_80.png" /></figure>

e-mail: pontus.nordenfelt@med.lu.se

<footnote n="1">¹Department of Clinical Sciences Lund, Infection Medicine, Faculty of Medicine, Lund University, Lund, Sweden. ²Université de Lorraine, CNRS, Inria, LORIA, F-54000 Nancy, France. ³Institut Pasteur, Université Paris cite, CNRS UMR3528, Structural Bioinformatics Unit, Department of Structural Biology and Chemistry, F-75015 Paris, France. ⁴Department of Biology & Lund Protein Production Platform (LP3), Lund University, Lund, Sweden. ⁵Department of Immunotechnology and SciLifeLab Drug Discovery and Development Platform, Lund University, Lund, Sweden. ⁶Department of Clinical Sciences Lund, Division of Pediatrics, Faculty of Medicine, Lund University, Lund, Sweden. ⁷Section for Oral Biology and Pathology, Faculty of Odontology, Malmö University, Malmö, Sweden. ⁸Department of Laboratory Medicine, Clinical Microbiology, Skåne University Hospital Lund, Lund University, Lund, Sweden.</footnote>