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Small Angle X-Ray and Neutron Scattering (SAXS/SANS)
Equipment, Experiments and Modelling
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Molecular IgG3 structure paves the way for new applications of antibodies

In humans, immunoglobulin (IgG) is the most common type of antibody found in blood circulation. IgG molecules are created by plasma B cells, and there are four subclasses called IgG1 to IgG4. Of the four, IgG3 is the least understood. It has a uniquely long hinge region separating its Fab antigen-binding and Fc receptor-binding regions. The presence of this elongated hinge makes it challenging to perform structural studies, for example, with X-ray crystallography. Our recent work used a combination of methods to provide the first experimentally determined molecularÌýstructural modelÌýfor a full-length IgG3 antibody.

Small angle X-ray scattering (SAXS) and analytical ultracentrifugation (AUC) showed that IgG3 is elongated compared to IgG1, IgG2 and IgG4. SAXS also showed that IgG3 has a moreÌýextended centralÌýhinge than IgG1 and IgG2Ìýthat links its three globular regions together. ÌýUsing atomistic modellingÌýbased on computationalÌýmolecularÌýdynamics and Monte Carlo methods to fit the scattering curves, the SAXS and SANS data sets were successfully modelled as molecular structures.ÌýThe results of this joint AUC-SAXS approach confirm that the IgG3 molecule has a semi-rigid long hinge with little disorder, and results in a wide separation betweenÌýtheÌýglobularÌýFab and Fc regions.

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Spiteri VA et al. . Journal of Biological Chemistry, 100995 (2021). DOI: 10.1016/j.jbc.2021.100995.Ìý

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Journal Front Cover. The new IgG3 structure is shown in red, and IgG1, IgG2 and IgG4 are shown in green, yellow and orange.

Sugar-free antibodies - Atomistic scattering modelling of human IgG1 reveals conformational changes on glycan removal

Antibodies are an important biotherapeutic tool: they can be tailored to treating different diseases, and can direct toxic drugs to the area in the body where they are needed. The computational modelling of antibodies alongside Small Angle Neutron and X-ray Scattering (SANS and SAXS) data determined the effect of bound sugars on their structural behaviour. We looked at the most common class of human antibodies called IgG1. These antibodies are shaped like a ‘Y’, and act as a bridge: the top two regions, the Fab regions, interact with the foreign material, and the bottom, known as the Fc region and decorated with sugars, interacts with your immune system via immune receptors. We measured the antibodies with and without sugars on the Sans2D instrument at ISIS, the B21 instrument at Diamond Light Source, and analytical ultracentrifuges at »Ê¼Ò»ªÈË. Small differences were detected between the two forms.

Combining the experimental data with a detailed computational modelling study, we produced 100 best-fit structures for each of the two IgG1 forms, using , an online web server which streamlines the atomistic modelling of SAS data. These best-fit models demonstrated conformational differences of the Fc region following glycan removal that accounted for changes in the scattering data, and that the glycans stabilised the structure of the Fc region in intact IgG1.

Our combination of analytical ultracentrifugation, small angle X-ray, neutron scattering, and the atomistic modelling of these data sets has now been demonstrated to be powerful enough in probing more subtle structural changes such as glycan removal in antibodies. This methodology can be extended to other systems.

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Spiteri, V. A., Doutch, J., Rambo, R. P., Gor, J., Dalby, P. A. & Perkins, S. J. (2021) Solution structure of deglycosylated human IgG1 shows the role of C_H2 glycans in its conformation. Biophysical J. 200, 1814-1834. Ìý

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Journal Front Cover. The ribbon cartoon images of the 16 IgG1 structures on the cover of the May 4 issue of Biophysical Journal were selected at random from the 100 and 100 best-fit structures for glycosylated and deglycosylated IgG1