Estimating domain orientation of two human antibody IgG4 chimeras by crystallohydrodynamics

A modified crystallohydrodynamic approach introduced in 2001 is applied to two human IgG4 constructs from mouse IgG1. The constructs were point mutants of the chimeric antibody molecule cB72.3(γ4): cB72.3(γ4A), devoid of inter-chain disulfide bridging, and cB72.3(γ4P), which has full inter-chain bridging. As before, the known crystallographic structures for the Fab and Fc domains were combined with the measured translational frictional ratios to obtain an estimate for the apparent time-averaged hydration of the domains and hence for that of the intact molecule. The original approach was modified with the hydrated dimensions of the domains being applied, rather than the anhydrous crystallographic dimensions, for assessing the inter-domain orientations using the algorithms HYDROSUB and SOLPRO. Both chimeric IgG4 molecules were found to have open, rather than compact, structures, in agreement with the previous study on wild-type human IgG4. The insertion of a frictionless connector between the domains was necessary, however, for representing the cB72.3(γ4A) chimera. It therefore appears that the inter-chain disulfide bonds act as physical constraints in the cB72.3(γ4P) chimera, forcing the antibody domains together and producing a less elongated structure than that of cB72.3(γ4A). The open structures produced for the two IgG4 chimeras showed similarity to those structures identified for murine IgG1 and IgG2a molecules through X-ray crystallography.