Solution Conformation of Wild-Type and Mutant IgG3 and IgG4 Immunoglobulins Using Crystallohydrodynamics: Possible Implications for Complement Activation

Abstract

We have employed the recently described crystallohydrodynamic approach to compare the time-averaged domain orientation of human chimeric IgG3wt (wild-type) and IgG4wt as well as two hinge mutants of IgG3 and an IgG4S331P (mutation from serine to proline at position 331, EU numbering) mutant of IgG4. The approach involves combination of the known shape of the Fab and Fc regions from crystallography with hydrodynamic data for the Fab and Fc fragments and hydrodynamic and small angle x-ray scattering data for the intact IgG structures. In this way, ad hoc assumptions over hydration can be avoided and model degeneracy (uniqueness problems) can be minimized. The best fit model for the solution structure of IgG3wt demonstrated that the Fab regions are directed away from the plane of the Fc region and with a long extended hinge region in between. The best fit model of the IgG3m15 mutant with a short hinge (and enhanced complement activation activity) showed a more open, but asymmetric structure. The IgG3HM5 mutant devoid of a hinge region (and also devoid of complement-activation activity) could not be distinguished at the low-resolution level from the structure of the enhanced complement-activating mutant IgG3m15. The lack of inter-heavy-chain disulphide bond rather than a significantly different domain orientation may be the reason for the lack of complement-activating activity of the IgG3HM5 mutant. With IgG4, there are significant and interesting conformational differences between the wild-type IgG4, which shows a symmetric structure, and the IgG4S331P mutant, which shows a highly asymmetric structure. This structural difference may explain the ability of the IgG4S331P mutant to activate complement in stark contrast to the wild-type IgG4 molecule which is devoid of this activity.

Global Shape and Ligand Binding Efficiency of the HIV-1-neutralizing Antibodies Differ from Those of Antibodies That Cannot Neutralize HIV-1

“…Additionally, reduced degrees of freedom in its hinges due to a high incidence of O-glycosylation and Pro residues, which rigidly position the Fab arms distally from the Fc, have been reported for human IgA1 (39). Partial rigidity paving the way for neutralizing potency could be seen in another recent report where, by introducing rigidity in the linker region, researchers could induce neutralizing potency in IgG3 mAbs (16). With the intention of studying SAXS-based change in global shape, we tried to alter the glycosylation pattern in a few gp120-reactive nmAbs by chemically removing N-linked glycans, but the experiments led to misfolded versions of the mAbs.…”

Molecular perspective of antibody aggregates and their adsorption on Protein A resin

“…They are especially useful for the characterization of antibodies, antibody aggregates, and complexes because obtaining the full-length structures of these large proteins at the atomic-level is difficult if solely based on experimental approaches. Crystallohydrodynamics modeling was used to determine IgG domain orientation and solution conformation [30,31]. Using molecular dynamics simulation, an all-atom model for trastuzumab was established for representative structure in aqueous solution from the crystal structures of antibody fragments [18].…”

On the hydrodynamic analysis of conformation in mixed biopolymer systems

“…Because of their flexibility on a small number with relatively short hinge regions have been crystallized and had their structures determined. In the case of the others it is possible to estimate conformation in terms of the orientation of the domains -which dictate the hydrodynamic properties of these substances [31][32][33]. To do this requires knowledge of the shape of the Fab and Fc domains (from x-ray crystallography), knowledge of the hydrodynamic properties of the domains and the hydrodynamic properties of the intact antibody.…”