Estimating domain orientation of two human antibody IgG4 chimeras by crystallohydrodynamics

Abstract

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(gamma4): cB72.3(gamma4A), devoid of inter-chain disulfide bridging, and cB72.3(gamma4P), 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(gamma4A) chimera. It therefore appears that the inter-chain disulfide bonds act as physical constraints in the cB72.3(gamma4P) chimera, forcing the antibody domains together and producing a less elongated structure than that of cB72.3(gamma4A). 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.

The Fab Conformations in the Solution Structure of Human Immunoglobulin G4 (IgG4) Restrict Access to Its Fc Region

“…A major monomer peak was observed at s 20, w 0 values of 6.8 S for IgG4(Ser 222 ) and 6.6 S for IgG4(Pro 222 ). These s 20, w 0 values are consistent with the range of values (6.2–6.9 S) reported previously for human IgG4 (20, 45–48). Accordingly both IgG4(Ser 222 ) and IgG4(Pro 222 ) were predominantly monomeric in solution and were accompanied by a minor dimer peak.…”

“…1). In contrast, earlier studies reported few scattering and ultracentrifugation runs, which limited the evaluation of potential buffer-dependent IgG4 conformations (45–48). …”

Hydrodynamic modelling of protein conformation in solution: ELLIPS and HYDRO

“…Obviously, many classes of molecule cannot be reasonably represented by a smooth symmetrical shape—antibodies are a good example—this type of whole-body modelling is not applicable and bead-approaches need to be employed. Even here, however, ellipsoidal representations of the major domains (Fab, Fc) have helped in the bead modelling of the intact assembly (Carrasco et al 1999, 2001; Longman et al 2003; Lu et al 2006, 2007). …”

Molecular perspective of antibody aggregates and their adsorption on Protein A resin

“…They are especially useful for the characterization of antibodies, antibody aggregates, and complexes because obtaining the full-length structures of these large proteins at the atomic-level is difficult if solely based on experimental approaches. Crystallohydrodynamics modeling was used to determine IgG domain orientation and solution conformation [30,31]. Using molecular dynamics simulation, an all-atom model for trastuzumab was established for representative structure in aqueous solution from the crystal structures of antibody fragments [18].…”