Mechanical Stability of the Antibody Domain C_H3 Homodimer in Different Oxidation States

The C_H3 homodimer at the C-terminal end of the antibody heavy chain is the key noncovalent interaction stabilizing antibody proteins. Here, we use single-molecule force spectroscopy to investigate the dissociation mechanics of C_H3 as a proxy for antibody mechanical stability. We find the C_H3 homodimer to be a highly stable complex, and its dissociation force of >150 pN at a loading rate of ≈5500 pN/s exceeds the stability of most protein–protein interactions studied to date. Separated C_H3 monomers, on the other hand, are mechanically labile and only short-lived. Each C_H3 monomer contains a conserved buried disulfide bridge, and we find that the successive reduction of one or both disulfide bridges in the dimer results in a stepwise decrease of the dissociation force. This suggests a structural role of the disulfide bridges helping to mold the high-affinity domain–domain interface, even though they are neither required for nor directly involved in dimerization. Taken together, our results set a limit on how much force a single antibody can bear and reveal the C_H3 homodimer as a mechanical fastener that prevents antibody dissociation

Retention profiles of the IgG1 glycovariants obtained by analytical FcγRIIIa affinity chromatography.

<p>The chromatograms, obtained by monitoring signal at 280 nm, show two peaks corresponding to fucosylated and afucosylated antibody fractions (deglycosylated IgG is not shown).</p

Retention profiles of IgG1 glycovariants obtained by analytical FcRn affinity chromatography.

<p>Retention profiles of IgG1 glycovariants obtained by analytical FcRn affinity chromatography.</p

Interaction of target- or F(ab’)₂ -bound IgG with FcγRIIIa.

<p>Prior to interaction analysis IgG was incubated with its monomeric target (A, B) or with F(ab’)₂ (C, D) and loaded onto affinity column (A, C) and examined by SPR (B, D). The IgG: ligand ratio: 1:0 (red), 1:1 (green), 1:2 (blue) and 1:4 (pink).</p

Binding of glycovariants to FcγRIIIa, expressed on living cells.

<p>Unlabeled glycovariants compete for binding to receptor with acceptor-labeled antibody, resulting in decrease of FRET signal: deglycosylated (red diamonds), WT (black circles), G0 (black triangles), G1(black inverted triangles), G2 (black squares). Initial signal was normalized to 1. (For remaining glycovariants refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143520#pone.0143520.s003" target="_blank">S3 Fig</a>).</p

Thermal transitions of IgG1 glycovariants obtained by Thermofluor Stability Assay (TSA).

<p>Melting curves: deglycosylated (black), M3 (blue), G0 (green), G1 (pink), G2 (orange), G1+1SA (light blue), G2+1SA (yellow), G2+2SA (purple), WT (red).</p

Structures of IgG N-glycans attached to the Asn297 in the Fc domain.

<p>The core (G0) heptasaccharide is highlighted in light blue. GlcNAc, N-acetylglucosamine; Fuc, fucose; Man, mannose; Gal, galactose; NeuAc, N-acetyl neuraminic (sialic) acid.</p

SPR analysis of interaction of IgG1 glycovariants with FcγRs.

<p>Anti His-antibody immobilized on the chip, glycovariants were injected as analytes, after capturing of the respective receptors: A. FcγRI, B. FcγRIIa, C. FcγRIIb and D. FcγRIIIa. Binding of WT antibody was set as 100%. Each graph represents results from at least three independent experiments; data are given as means ± SD.</p

Interaction of IgG1 glycovariants with FcRn.

<p>Comparison of different SPR experimental set-ups: FcRn receptor was captured by Anti-6xHis antibody, followed by the application of IgG glycovariants (black); FcRn was directly immobilized onto the chip surface, subsequently IgG glycovariants were loaded (grey); glycovariants were captured by the immobilized protein L, followed by the application of FcRn (dark grey); glycovariants were bound onto the immobilized target, then the receptor was applied (light grey).</p

SPR analysis of the interaction of target-bound or F(ab’)₂

<p>Glycovariants were injected to bind antigen (A) or F(ab’)₂ (B), immobilized on the chip, followed by the application of the respective receptors: FcγRI (black), FcγRIIa (grey), FcγRIIb (dark grey) and FcγRIIIa (light grey). Binding of WT antibody was set as 100% Each graph represents results from at least three independent experiments; data are given as means ± SD.</p