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Heavy-chain-only antibodies can offer advantages of higher binding affinities, reduced sizes, and higher stabilities compared to conventional antibodies. To address the challenge of SARS-CoV-2, a llama-derived single-domain nanobody C5 was developed previously that has high COVID-19 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 characterised the solution arrangement of the molecule. Two 1,443 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 radius of gyration RG values, which provide information about structural elongation, were similar at 4.1-4.2 nm for glycosylated and deglycosylated C5-Fc. To explain these RG values, atomistic scattering modelling 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 publication




Journal article


The Journal of biological chemistry

Publication Date



Department of Structural and Molecular Biology, Division of Biosciences, Darwin Building, University College London, Gower Street, London WC1E 6BT, U.K.