Abstract
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 FcγRI 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.
Highlights
Antibodies are glycoproteins that protect the host by identifying and neutralizing pathogens
For IgG4 B72.3, one new x-ray data set from instrument BM29 and one new neutron data set from instrument D22 were obtained
Analytical ultracentrifugation showed that IgG1 and IgG4 were unaffected by protein aggregation but showed minor reversible dimerization [15,16]
Summary
Antibodies are glycoproteins that protect the host by identifying and neutralizing pathogens. They mediate highly specific antigen binding to a specific epitope through their two Fab regions, followed by their effector binding to other components of the immune system through its Fc region (Fig. 1). Immunoglobulin G (IgG) is the most abundant of the five human antibody classes. In IgG, the heavy and light chains are paired to form two Fab regions that are joined by two polypeptide hinges linked by interchain disulphide bonds to the Fc region that is formed from two heavy chains (Fig. 1). The variable domains (VH and VL) mediate antigen binding, whereas the Fc constant domains (CH2 and CH3) perform effector functions [1,2].
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
