Removal of a Conserved Disulfide Bond Does Not Compromise Mechanical Stability of a VHH Antibody Complex

Abstract

Single-domain VHH antibodies are promising reagents for medical therapy. A conserved disulfide bond within the VHH framework region is known to be critical for thermal stability, however, no prior studies have investigated its influence on the stability of VHH antibody-antigen complexes under mechanical load. Here, we used single-molecule force spectroscopy to test the influence of a VHH domain's conserved disulfide bond on the mechanical strength of the interaction with its antigen mCherry. We found that although removal of the disulfide bond through cysteine-to-alanine mutagenesis significantly lowered VHH domain denaturation temperature, it had no significant impact on the mechanical strength of the VHH:mCherry interaction with complex rupture occurring at ∼60 pN at 10³-10⁴ pN/sec regardless of disulfide bond state. These results demonstrate that mechanostable binding interactions can be built on molecular scaffolds that may be thermodynamically compromised at equilibrium.

Keywords: Antibodies; biophysics; disulfide bond; nanobody; single-molecule force spectroscopy.

Figure 1

AFM-SMFS on VHH/mCherry complexes. (a) Homology model of VHH domain. The disulfide bond is shown as surface mesh in blue and yellow. Modeling was carried out using the ABodyBuilder server. (b) Measurement setup with mCherry-FIVAR attached to the cantilever and VHH-ddFLN4 attached to the substrate. (c) Typical force versus extension trace showing unfolding of FIVAR (cyan) and ddFLN4 (orange) marker domains, followed by complex rupture. (d) Contour length histogram of the data in panel (c). Increments between peaks were used to make domain assignments to unfolding events.

Figure 2

Dynamic force spectroscopy on VHH(WT)/mCherry complexes. (a) VHH(WT)/mCherry complex rupture events detected at four pulling speeds from 400 to 3 200 nm/s. Colored points represent rupture events of individual molecular complexes. Open circle markers represent the most probable rupture force and average loading rate at each pulling speed. The dashed line represents the Bell-Evans fit to the open circle. (b) Rupture force histograms at each pulling speed. (c) Lifetime plot obtained using histogram transformation based on the DHS model with v = 0.5. The dashed line represents the fit to the DHS model.

Figure 3

SMFS experiments on VHH(WT) and disulfide bond knockout mutants C24A, C98A, and C24A/C98A. (a) Dynamic force spectra of VHH library members obtained from four pulling speeds from 400 to 3, 200 nm/s. Points shown represent the most probable rupture force versus average loading rate at a given pulling speed. The dashed line represents the Bell-Evans model fit. (b) Rupture force histograms obtained at each pulling speed. (c) Data fitting with the DHS model was used to extract energy landscape parameters shown in Table 2.