Background:The human IgG1 antibody subclass is the most abundant one and is widely used in therapeutic applications. Results: Ultracentrifugation and x-ray/neutron scattering, together with atomistic modeling, revealed asymmetric concentration-independent IgG1 solution structures. Conclusion:The complement and receptor Fc binding sites are not hindered by the Fab regions, explaining its full activity. Significance: These solution structures clarify IgG1 activity and its therapeutic applications.

Background: The human IgG4 antibody subclass does not activate complement and forms half-antibodies.Results: Ultracentrifugation and x-ray/neutron scattering together with atomistic modeling revealed asymmetric concentration-dependent IgG4 solution structures.Conclusion: The complement and receptor Fc binding sites are hindered by the Fab regions, explaining loss of activity.Significance: These solution structures clarify IgG4 function and its therapeutic applications.

Detailed analytical ultracentrifugation and X-ray/neutron scattering data and a new atomistic modelling approach revealed asymmetric extended solution structures for human IgA1 that account for its receptor-binding function. IgA1 with different hinge O-galactosylation patterns showed similar structures.

Human IgG2 antibody displays distinct therapeutically-useful properties compared with the IgG1, IgG3 and IgG4 antibody subclasses. IgG2 is the second most abundant IgG subclass, being able to bind human FcγRII/FcγRIII, but not to FcγRI or complement C1q. Structural information on IgG2 is limited by the absence of a fulllength crystal structure for this. To this end, we determined the solution structure of human myeloma IgG2 by atomistic X-ray and neutron scattering modelling. Analytical ultracentrifugation disclosed that IgG2 is monomeric with a sedimentation coefficient s 0 20,w of 7.2 S. IgG2 dimer formation was ≤ 5% and independent of the buffer conditions. Small-angle X-ray scattering in a range of NaCl concentrations and in light and heavy water revealed that the X-ray radius of gyration Rg is 5.2-5.4 nm, after allowing for radiation damage at higher concentrations, and that the neutron Rg value of 5.0 nm remained unchanged in all conditions. The X-ray and neutron distance distribution curves P(r) revealed two peaks, M1 and M2, that were unchanged in different buffers. The creation of ˃123,000

Small angle x-ray and neutron scattering are techniques that give solution structures for large macromolecules. The creation of physically realistic atomistic models from known high-resolution structures to determine joint x-ray and neutron scattering best-fit structures offers a, to our knowledge, new method that significantly enhances the utility of scattering. To validate this approach, we determined scattering curves for two human antibody subclasses, immunoglobulin G (IgG) 1 and IgG4, on five different x-ray and neutron instruments to show that these were reproducible, then we modeled these by Monte Carlo simulations. The two antibodies have different hinge lengths that connect their antigen-binding Fab and effector-binding Fc regions. Starting from 231,492 and 190,437 acceptable conformations for IgG1 and IgG4, respectively, joint x-ray and neutron scattering curve fits gave low goodness-of-fit R factors for 28 IgG1 and 2748 IgG4 structures that satisfied the disulphide connectivity in their hinges. These joint best-fit structures showed that the best-fit IgG1 models had a greater separation between the centers of their Fab regions than those for IgG4, in agreement with their hinge lengths of 15 and 12 residues, respectively. The resulting asymmetric IgG1 solution structures resembled its crystal structure. Both symmetric and asymmetric solution structures were determined for IgG4. Docking simulations with our best-fit IgG4 structures showed greater steric clashes with its receptor to explain its weaker FcgRI receptor binding compared to our best-fit IgG1 structures with fewer clashes and stronger receptor binding. Compared to earlier approaches for fitting molecular antibody structures by solution scattering, we conclude that this joint fit approach based on x-ray and neutron scattering data, combined with Monte Carlo simulations, significantly improved our understanding of antibody solution structures. The atomistic nature of the output extended our understanding of known functional differences in Fc receptor binding between IgG1 and IgG4.