Abstract
Heavy chain–only antibodies can offer advantages of higher binding affinities, reduced sizes, and higher stabilities than conventional antibodies. To address the challenge of SARS-CoV-2 coronavirus, a llama-derived single-domain nanobody C5 was developed previously that has high COVID-19 virus neutralization potency. The fusion protein C5-Fc comprises two C5 domains attached to a glycosylated Fc region of a human IgG1 antibody and shows therapeutic efficacy in vivo. Here, we have characterized the solution arrangement of the molecule. Two 1443 Da N-linked glycans seen in the mass spectra of C5-Fc were removed and the glycosylated and deglycosylated structures were evaluated. Reduction of C5-Fc with 2-mercaptoethylamine indicated three interchain Cys–Cys disulfide bridges within the hinge. The X-ray and neutron Guinier RG values, which provide information about structural elongation, were similar at 4.1 to 4.2 nm for glycosylated and deglycosylated C5-Fc. To explain these RG values, atomistic scattering modeling based on Monte Carlo simulations resulted in 72,737 and 56,749 physically realistic trial X-ray and neutron structures, respectively. From these, the top 100 best-fit X-ray and neutron models were identified as representative asymmetric solution structures, similar to that of human IgG1, with good R-factors below 2.00%. Both C5 domains were solvent exposed, consistent with the functional effectiveness of C5-Fc. Greater disorder occurred in the Fc region after deglycosylation. Our results clarify the importance of variable and exposed C5 conformations in the therapeutic function of C5-Fc, while the glycans in the Fc region are key for conformational stability in C5-Fc.
Original language | English |
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Article number | 105337 |
Number of pages | 17 |
Journal | Journal of Biological Chemistry |
Volume | 299 |
Issue number | 11 |
Early online date | 12 Oct 2023 |
DOIs | |
Publication status | Published - Nov 2023 |
Bibliographical note
Funding Information:We thank Dr Katsuaki Inoue for excellent user support on Instrument B21 at Diamond and Dr James Doutch likewise on the Instrument SANS2D at ISIS. We thank Dr Kersti Karu for the mass measurements in the UCL Department of Chemistry Mass Spectrometry Facility. X. G. and J. G. investigation; X. G. and J. W. T. formal analysis; X. G. and S. J. P. writing-original draft; J. H. N. R. J. O. and S. J. P. project administration; S. J. P. conceptualization; S. J. P. supervision; R. J. O. resources. S. J. P. was supported by the CCP-SAS project, a joint EPSRC (EP/K039121/1) and NSF (CHE-1265821) grant. R. J. O. and J. W. T. were supported by the Rosalind Franklin Institute (funding delivery partner EPSRC), and Wellcome Trust (223733/Z/21/Z) and BBSRC (BB/V018523/1) grants.
Publisher Copyright:
© 2023 The Authors
Keywords
- analytical ultracentrifugation
- antibody
- atomistic modelling
- molecular dynamics
- nanobody
- small angle neutron scattering
- small angle X-ray scattering
ASJC Scopus subject areas
- Biochemistry
- Molecular Biology
- Cell Biology