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Solution Conformation of Wild-Type and Mutant IgG3 and IgG4 Immunoglobulins Using Crystallohydrodynamics: Possible Implications for Complement Activation

Yanling Lu ^*, Stephen E. Harding ^*, Terje E. Michaelsen , Emma Longman ^*, Kenneth G. Davis ^*, Álvaro Ortega , J. Günter Grossmann , Inger Sandlie ^¶ and José García de la Torre 

^* National Centre for Macromolecular Hydrodynamics, University of Nottingham, Sutton Bonington, England; Norwegian Institute of Public Health, Oslo, and Institute of Pharmacy, University of Oslo, Blindern, Oslo, Norway; Departamento de Quimica Fisica, Universidad de Murcia, Murcia, Spain; Molecular Biophysics Group, Science and Technology Facilities Council, Daresbury Laboratory, Daresbury Science and Innovation Campus, Warrington, Cheshire, United Kingdom; and ^¶ Institute of Molecular Bioscience, University of Oslo, Blindern, Oslo, Norway

Correspondence: Address reprint requests to S. E. Harding, E-mail: steve.harding{at}nottingham.ac.uk.

We have employed the recently described crystallohydrodynamic approach to compare the time-averaged domain orientation of human chimeric IgG3wt (wild-type) and IgG4wt as well as two hinge mutants of IgG3 and an IgG4S331P (mutation from serine to proline at position 331, EU numbering) mutant of IgG4. The approach involves combination of the known shape of the Fab and Fc regions from crystallography with hydrodynamic data for the Fab and Fc fragments and hydrodynamic and small angle x-ray scattering data for the intact IgG structures. In this way, ad hoc assumptions over hydration can be avoided and model degeneracy (uniqueness problems) can be minimized. The best fit model for the solution structure of IgG3wt demonstrated that the Fab regions are directed away from the plane of the Fc region and with a long extended hinge region in between. The best fit model of the IgG3m15 mutant with a short hinge (and enhanced complement activation activity) showed a more open, but asymmetric structure. The IgG3HM5 mutant devoid of a hinge region (and also devoid of complement-activation activity) could not be distinguished at the low-resolution level from the structure of the enhanced complement-activating mutant IgG3m15. The lack of inter-heavy-chain disulphide bond rather than a significantly different domain orientation may be the reason for the lack of complement-activating activity of the IgG3HM5 mutant. With IgG4, there are significant and interesting conformational differences between the wild-type IgG4, which shows a symmetric structure, and the IgG4S331P mutant, which shows a highly asymmetric structure. This structural difference may explain the ability of the IgG4S331P mutant to activate complement in stark contrast to the wild-type IgG4 molecule which is devoid of this activity.