The solution structure of the heavy chain-only C5-Fc nanobody reveals exposed variable regions that are optimal for COVID-19 antigen interactions

J Biol Chem. 2023 Nov;299(11):105337. doi: 10.1016/j.jbc.2023.105337. Epub 2023 Oct 12.

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.

Keywords: analytical ultracentrifugation; antibody; atomistic modelling; molecular dynamics; nanobody; small angle X-ray scattering; small angle neutron scattering.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Antibodies, Viral* / chemistry
  • Humans
  • Immunoglobulin G / chemistry
  • Immunoglobulin Heavy Chains* / chemistry
  • Models, Molecular
  • Polysaccharides
  • SARS-CoV-2*
  • Single-Domain Antibodies / chemistry

Substances

  • Immunoglobulin G
  • Immunoglobulin Heavy Chains
  • Polysaccharides
  • Antibodies, Viral
  • Single-Domain Antibodies