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(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.
Longman
Emma
E
National Centre for Macromolecular Hydrodynamics, School of Biosciences, University of Nottingham, Sutton Bonington, LE12 5RD, UK. sbxejl@nottingham.ac.uk
Kreusel
Katja
K
Tendler
Saul B
SB
Fiebrig
Immo
I
King
Kevin
K
Adair
John
J
O'Shea
Paul
P
Ortega
Alvaro
A
Garcia de la Torre
Jose
J
Harding
Stephen E
SE
D000818:Animals; D000906:Antibodies / Q000032:analysis / Q000737:chemistry; D002499:Centrifugation, Density Gradient / Q000379:methods*; D002678:Chimera; D003198:Computer Simulation; D018360:Crystallography, X-Ray / Q000379:methods*; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis* / Q000737:chemistry*; D051379:Mice; D008958:Models, Molecular; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013045:Species Specificity
D000906:Antibodies; D007074:Immunoglobulin G