Rayner, LE;
Hui, GK;
Gor, J;
Heenan, RK;
Dalby, PA;
Perkins, SJ;
(2014)
The Fab conformations in the solution structure of human IgG4 restricts access to its Fc region: implications for functional activity.
J Biol Chem
, 289
(30)
pp. 20740-20756.
10.1074/jbc.M114.572404.
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Abstract
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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