Unraveling the effect of changes in conformation and compactness at the antibody VL-VH interface upon antigen binding

Abstract

We have analyzed conformational changes that occur at the interface between the light (V(L)) and heavy (V(H)) chains in antibody variable fragments upon binding to antigens. We wrote and applied the Tiny Probe program that computes the buried atomic contact surface area of three-dimensional structures to evaluate changes in compactness of the V(L)-V(H) interface between bound and unbound antibodies. We found three categories of these changes, which correlated with the size of the antigen. Upon binding, medium-sized nonprotein antigens cause an opening of the V(L)-V(H) interface (less compact), small antigens or haptens cause a closure of the interface (more compact), whereas large protein antigens have little effect on the compactness of the V(L)-V(H) interface. The largest changes in the atomic buried contact surface area at the V(L)-V(H) interface occur in residue pairs providing two 'shock absorbers' between the edge beta-strands of the V(L) and V(H) beta-sheets forming the antibody binding site. Importantly, the correlation between the size of antigens and conformational changes indicates that the V(L)-V(H) interface in antibodies plays a significant role in the antigen binding process. Furthermore, as the energy involved in such a motion is significant (up to 3 kcal/mol), these results provide a general mechanism for how residues distant from the combining site can significantly alter the affinity of an antibody for its antigen. Thus, mutations introduced at the V(L)-V(H) interface can be used to change antibody binding affinity with antigens. Due to the tightly packed V(L)-V(H) interface, the introduction of random mutations is not advisable. Rather our analysis suggests that concerted mutations of residues preceding CDRL2 and following CDRH3 or residues preceding CDRH2 and at the end of CDRL3 are most likely to alter or improve antigen binding affinity.

A Systematic Comparison of Free and Bound Antibodies Reveals Binding-Related Conformational Changes

“…Experimental and structural studies suggest that Abs are no different, showing some flexibility upon Ag binding (4). This flexibility was suggested to be essential to their ability to bind multiple Ags (3).…”

“…A possible mechanism for such observations was suggested by Torres and Casadevall (10), in which electrostatic and hydrophobic interactions resulting from differences in the microenvironment of CH domains (e.g., pH, ionic strength) may affect the Ag binding site. Additionally, the arrangement of the Fab constant domains relative to the variable domains and to each other may increase the probability of an appropriate VH-variable light (VL) relative orientation (28), which, in turn, can shape the Ag binding site (4,29,30).…”

“…This included reports of very small overall changes, side-chain movements, large rearrangement of one or more of the CDRs, change in the VH-VL relative orientation, and, in some cases, combinations of some or all of the above. Recent studies used newer structurecomparison methods (4,36) and used the growing number of available structures (37)(38)(39)(40) to perform comparative research to identify new and more general characteristics of Ab structures. However, many of these studies did not compare free and bound structures of the same Ab (4,37,40), focused only on the VH-VL relative orientation (4,37,38,40), or included only a limited number of structures (4,36,39).…”

Recovering Antibody Secretion Using a Hapten Ligand as a Chemical Chaperone

“…In addition, the thermal denaturation of peptide-filled, purified K b class I molecules results in simultaneous loss of peptide and dissociation of the heavy chain and ␤ 2 -microglobulin (42). The structural consequences of ligand binding have been investigated by comparison of crystal structures of ligand-bound and unbound antibodies (43). These computational comparisons have identified that small antigens or haptens compact the V H -V L interface in the McPC603, 28B4, N1G9, and DB3 antibodies.…”

“…to stability, representing a 10-fold improvement in binding affinity between the V H and V L domains (43). An improvement in affinity of this magnitude, occurring between the H and L chains in the mutant PCG1-1 antibodies, may be sufficient to offset the potentially destabilizing effect of the CDR2 mutation(s).…”