Antibody-protein binding and conformational changes

I came across a recent paper on the antibody-protein binding and conformational changes. As I work mainly on the binding site/Fv regions of antibodies, I am intrigued to see the role of the constant domains in the overall antibody function.

The authors started by curating eight pairs of anti-protein antibodies: one version bound to the antigen, and the other antigen-free. The metrics that they used to quantify the structural differences were:

Domain orientation changes

They looked at the variable and the first constant regions of the heavy or light chain (VH-CH1 and VL-CL), selected the conserved cysteines (C) and VH/CH1 or VL/CL linkers (S,R,Q), and quantify the distances and angles as shown in this figure: 

Illustration of how the authors used the conserved Cysteines in VH/CH1 or VL/CL, and the conserved linkers (Serine, Arginine and Glutamine) to calculate the angles. The distances were measured between the conserved Cysteines in VH/CH1 or VL/CL.


They mainly looked at the angle that encodes for the aspect ratio of the diamond shape in the Fab region, and used the distances as a secondary confirmation. The greater the angular change between the bound and free antibodies, the greater the overall conformational change. They put pairs with 16-37 degrees of change into the B1 class.

Root-Mean-Square Deviation (RMSD)

The authors then inspected the overall RMSD of the bound and free antibody structures. Surprisingly, the constant domains tend to have larger RMSD values than the variable domains. They hypothesised that a high movement of the constant domains might be correlated to signal propagation.

Root-Mean-Square Fluctuations (RMSF)

RMSF is a per-atom distance between counterparts. They found that if they took the globally-optimal alignment to calculate the RMSF in B1, variations were larger than when the locally-optimal alignment was used, i.e. there was a “hinge” movement, consistent with the angular changes analysis earlier.

Apart from the CDRs in the antigen-binding site, the authors also described C_Loop1-3 which are the loops on the CH and CL. Interestingly, in a different study, Sela-Culang and co-authors found that C_Loop1 could move as much as CDRH3 upon antigen binding. This loop is also at the interface of the CH and CL, and is involved in complement binding. The structural changes in this loop seems to suggest an allosteric signal transduction, when an antibody engages an antigen.

The RMSF of the C_Loop1 region is the determinant for whether the pair of antibodies fall within B2 (high) and B3 (low) classes. In both B2 and B3, the “hinge” angular changes were small, and we would expect the overall RMSF to be small. However, the couples in the B2 class have a large change in the C_Loop1 region of the CH domain, in stark contrast to the pairs in B3. 

In summary, the paper proposed an alternative measurement for the orientation angle and established three classes that describe the conformational changes of antibodies upon antigen binding. Despite the small number of samples, it provides a starting framework for understanding signal transduction in antibody function.

Paper: Al Qaraghuli, M.M., Kubiak-Ossowska, K., Ferro, V.A. and Mulheran, P.A. (2020) Antibody-protein binding and conformational changes: identifying allosteric signalling pathways to engineer a better effector response. Scientific Reports, 10(1), 1-10.

Reference: Sela-Culang, I., Alon, S. and Ofran, Y. (2012) A systematic comparison of free and bound antibodies reveals binding-related conformational changes. Journal of Immunology, 1950(189), 4890-4899.

Author