Human myeloma IgG4 reveals relatively rigid asymmetric Y-like structure with different conformational stability of C H 2 domains.

Human IgG4 (hIgG4) has weak pro-inflammatory activity. The structural basis for this is still unclear. Here a 3D model of myeloma hIgG4 was created at ∼3nm resolution using electron microscopy (EM) with negative staining and single-particle 3D reconstruction. The hIgG4 model reveals relatively rigid asymmetric Y-like structure. The model shows that one Fab subunit is closer to the upper portion of the Fc subunit (CH 2 domain) than the other Fab. This is in agreement with X-ray crystallography and X-ray/neutron scattering, recently published by others. The same hIgG4 sample was studied with differential scanning calorimetry (DSC) and fluorescence. The thermodynamics and fluorescence observations indicate that one CH 2 domain displays less conformational stability than the other. This finding is consistent with the flipping of one CH 2 domain, observed in pembrolizumab (recombinant hIgG4) by X-ray crystallography. The specific feature of hIgG4 CH 2 domains together with relatively rigid asymmetric Y-like structure, in which one Fab subunit is closer to the upper portion of the Fc subunit (CH 2 domain) than the other Fab, can explain the unique biological properties of hIgG4, such as its weak pro-inflammatory activity.