The solution structures of two human IgG1 antibodies show conformational stability and accommodate their C1q and Fc{gamma}R ligands [Molecular Biophysics]

February 6th, 2015 by Rayner, L. E., Hui, G. K., Gor, J., Heenan, R. K., Dalby, P. A., Perkins, S. J.

The human IgG1 antibody subclass shows distinct properties compared to the IgG2, IgG3 and IgG4 subclasses, and is the most exploited subclass in therapeutic antibodies. It is the most abundant subclass, has a half-life as long as that of IgG2 and IgG4, binds the FcγR receptor, and activates complement. There is limited structural information on full-length human IgG1 because of the challenges of crystallisation. To rectify this, we have studied the solution structures of two human IgG1 6a and 19a monoclonal antibodies in different buffers at different temperatures. Analytical ultracentrifugation showed that both antibodies were predominantly monomeric, with sedimentation coefficients s020,w of 6.3 S - 6.4 S. Only a minor dimer peak was observed, and the amount was not dependent on buffer conditions. Solution scattering showed that the X-ray radius of gyration Rg increased with salt concentration, while the neutron Rg values remained unchanged with temperature. The X ray and neutron distance distribution curves P(r) revealed two peaks, M1 and M2, whose positions were unchanged in different buffers to indicate conformational stability. Constrained atomistic scattering modelling revealed predominantly asymmetric solution structures for both antibodies with extended hinge structures. Both structures were similar to the only known crystal structure of full-length human IgG1. The Fab conformations in both structures were suitably positioned to permit the Fc region to bind readily to its FcγR and C1q ligands without steric clashes, unlike human IgG4. Our molecular models for human IgG1 explain its immune activities, and we discuss its stability and function for therapeutic applications.
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The Fab conformations in the solution structure of human IgG4 restricts access to its Fc region: implications for functional activity [Protein Structure and Folding]

May 29th, 2014 by Rayner, L. E., Hui, G. K., Gor, J., Heenan, R. K., Dalby, P. A., Perkins, S. J.

Human IgG4 antibody shows therapeutically-useful properties compared to the IgG1, IgG2 and IgG3 subclasses. Thus IgG4 does not activate complement, and shows conformational variability. These properties are attributable to its hinge region, which is the shortest of the four IgG subclasses. Using high throughput scattering methods, we have studied the solution structure of wild-type IgG4(Ser222) and a hinge mutant IgG4(Pro222) in different buffers and temperatures, where the proline substitution suppresses the formation of half-antibody. Analytical ultracentrifugation showed that both IgG4 forms were principally monomeric with sedimentation coefficients s020,w of 6.6-6.8 S. A monomer-dimer equilibrium was observed in heavy water buffer at low temperature. Scattering showed that the X-ray radius of gyration RG was unchanged with concentration in 50-250 mM NaCl buffers, while the neutron RG values showed a concentration-dependent increase as the temperature decreased in heavy water buffers. The distance distribution curves P(r) revealed two peaks, M1 and M2 that shifted below 2 mg/ml to indicate concentration-dependent IgG4 structures, in addition to IgG4 dimer formation at high concentration in heavy water. Constrained X-ray and neutron scattering modelling revealed asymmetric solution structures for IgG4(Ser222) with extended hinge structures. The IgG4(Pro222) structure was similar. Both IgG4 structures showed that their Fab regions were positioned close enough to the Fc region to restrict C1q binding. Our new molecular models for IgG4 explain its inability to activate complement, and clarifies aspects of its stability and function for therapeutic applications.
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