Structure-guided design of a potent Clostridioides difficile toxin A inhibitor
Crystal structures of camelid heavy-chain antibody variable domains (VHHs) bound to fragments of the combined repetitive oligopeptides domain of Clostridioides difficile toxin A (TcdA) reveal that the C-terminus of VHH A20 was located 30 Å away from the N-terminus of VHH A26. Based on this observati...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2023-01-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2023.1110541/full |
| _version_ | 1797855933842325504 |
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| author | Greg Hussack Martin A. Rossotti Henk van Faassen Tomohiko Murase Luiz Eugenio Joseph D. Schrag Kenneth K.-S. Ng Kenneth K.-S. Ng Jamshid Tanha Jamshid Tanha |
| author_facet | Greg Hussack Martin A. Rossotti Henk van Faassen Tomohiko Murase Luiz Eugenio Joseph D. Schrag Kenneth K.-S. Ng Kenneth K.-S. Ng Jamshid Tanha Jamshid Tanha |
| author_sort | Greg Hussack |
| collection | DOAJ |
| description | Crystal structures of camelid heavy-chain antibody variable domains (VHHs) bound to fragments of the combined repetitive oligopeptides domain of Clostridioides difficile toxin A (TcdA) reveal that the C-terminus of VHH A20 was located 30 Å away from the N-terminus of VHH A26. Based on this observation, we generated a biparatopic fusion protein with A20 at the N-terminus, followed by a (GS)6 linker and A26 at the C-terminus. This A20-A26 fusion protein shows an improvement in binding affinity and a dramatic increase in TcdA neutralization potency (>330-fold [IC50]; ≥2,700-fold [IC99]) when compared to the unfused A20 and A26 VHHs. A20-A26 also shows much higher binding affinity and neutralization potency when compared to a series of control antibody constructs that include fusions of two A20 VHHs, fusions of two A26 VHHs, a biparatopic fusion with A26 at the N-terminus and A20 at the C-terminus (A26-A20), and actoxumab. In particular, A20-A26 displays a 310-fold (IC50) to 29,000-fold (IC99) higher neutralization potency than A26-A20. Size-exclusion chromatography-multiangle light scattering (SEC-MALS) analyses further reveal that A20-A26 binds to TcdA with 1:1 stoichiometry and simultaneous engagement of both A20 and A26 epitopes as expected based on the biparatopic design inspired by the crystal structures of TcdA bound to A20 and A26. In contrast, the control constructs show varied and heterogeneous binding modes. These results highlight the importance of molecular geometric constraints in generating highly potent antibody-based reagents capable of exploiting the simultaneous binding of more than one paratope to an antigen. |
| first_indexed | 2024-04-09T20:32:12Z |
| format | Article |
| id | doaj.art-40e3d0c986f54e86b2d0109a94a5ac34 |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-04-09T20:32:12Z |
| publishDate | 2023-01-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Microbiology |
| spelling | doaj.art-40e3d0c986f54e86b2d0109a94a5ac342023-03-30T14:54:13ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2023-01-011410.3389/fmicb.2023.11105411110541Structure-guided design of a potent Clostridioides difficile toxin A inhibitorGreg Hussack0Martin A. Rossotti1Henk van Faassen2Tomohiko Murase3Luiz Eugenio4Joseph D. Schrag5Kenneth K.-S. Ng6Kenneth K.-S. Ng7Jamshid Tanha8Jamshid Tanha9Life Sciences Division, Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, ON, CanadaLife Sciences Division, Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, ON, CanadaLife Sciences Division, Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, ON, CanadaDepartment of Biological Sciences, University of Calgary, Calgary, AB, CanadaDepartment of Biological Sciences, University of Calgary, Calgary, AB, CanadaLife Sciences Division, Human Health Therapeutics Research Centre, National Research Council Canada, Montréal, QC, CanadaDepartment of Biological Sciences, University of Calgary, Calgary, AB, CanadaDepartment of Chemistry and Biochemistry, University of Windsor, Windsor, ON, CanadaLife Sciences Division, Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, ON, CanadaDepartment of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, CanadaCrystal structures of camelid heavy-chain antibody variable domains (VHHs) bound to fragments of the combined repetitive oligopeptides domain of Clostridioides difficile toxin A (TcdA) reveal that the C-terminus of VHH A20 was located 30 Å away from the N-terminus of VHH A26. Based on this observation, we generated a biparatopic fusion protein with A20 at the N-terminus, followed by a (GS)6 linker and A26 at the C-terminus. This A20-A26 fusion protein shows an improvement in binding affinity and a dramatic increase in TcdA neutralization potency (>330-fold [IC50]; ≥2,700-fold [IC99]) when compared to the unfused A20 and A26 VHHs. A20-A26 also shows much higher binding affinity and neutralization potency when compared to a series of control antibody constructs that include fusions of two A20 VHHs, fusions of two A26 VHHs, a biparatopic fusion with A26 at the N-terminus and A20 at the C-terminus (A26-A20), and actoxumab. In particular, A20-A26 displays a 310-fold (IC50) to 29,000-fold (IC99) higher neutralization potency than A26-A20. Size-exclusion chromatography-multiangle light scattering (SEC-MALS) analyses further reveal that A20-A26 binds to TcdA with 1:1 stoichiometry and simultaneous engagement of both A20 and A26 epitopes as expected based on the biparatopic design inspired by the crystal structures of TcdA bound to A20 and A26. In contrast, the control constructs show varied and heterogeneous binding modes. These results highlight the importance of molecular geometric constraints in generating highly potent antibody-based reagents capable of exploiting the simultaneous binding of more than one paratope to an antigen.https://www.frontiersin.org/articles/10.3389/fmicb.2023.1110541/fullbiparatopicClostridioides difficileinhibitornanobodysingle-domain antibodytoxin |
| spellingShingle | Greg Hussack Martin A. Rossotti Henk van Faassen Tomohiko Murase Luiz Eugenio Joseph D. Schrag Kenneth K.-S. Ng Kenneth K.-S. Ng Jamshid Tanha Jamshid Tanha Structure-guided design of a potent Clostridioides difficile toxin A inhibitor Frontiers in Microbiology biparatopic Clostridioides difficile inhibitor nanobody single-domain antibody toxin |
| title | Structure-guided design of a potent Clostridioides difficile toxin A inhibitor |
| title_full | Structure-guided design of a potent Clostridioides difficile toxin A inhibitor |
| title_fullStr | Structure-guided design of a potent Clostridioides difficile toxin A inhibitor |
| title_full_unstemmed | Structure-guided design of a potent Clostridioides difficile toxin A inhibitor |
| title_short | Structure-guided design of a potent Clostridioides difficile toxin A inhibitor |
| title_sort | structure guided design of a potent clostridioides difficile toxin a inhibitor |
| topic | biparatopic Clostridioides difficile inhibitor nanobody single-domain antibody toxin |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2023.1110541/full |
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