title: Dynamics of inter-heavy chain interactions in human immunoglobulin G (IgG) subclasses studied by kinetic Fab arm exchange.
abstract: Interdomain interactions between the CH3 domains of antibody heavy chains are the first step in antibody assembly and are of prime importance for maintaining the native structure of IgG. For human IgG4 it was shown that CH3-CH3 interactions are weak, resulting in the potential for half-molecule exchange ("Fab arm exchange"). Here we systematically investigated non-covalent interchain interactions for CH3 domains in the other human subclasses, including polymorphisms (allotypes), using real-time monitoring of Fab arm exchange with a FRET-based kinetic assay. We identified structural variation between human IgG subclasses and allotypes at three amino acid positions (Lys/Asn-392, Val/Met-397, Lys/Arg-409) to alter the strength of inter-domain interactions by >6 orders of magnitude. Each substitution affected the interactions independent from the other substitutions in terms of affinity, but the enthalpic and entropic contributions were non-additive, suggesting a complex interplay. Allotypic variation in IgG3 resulted in widely different CH3 interaction strengths that were even weaker for IgG3 than for IgG4 in the case of allotype G3m(c3c5*/6,24*), whereas G3m(s*/15*) was equally stable to IgG1. These interactions are sufficiently strong to maintain the structural integrity of IgG1 during its normal life span; for IgG2 and IgG3 the inter-heavy chain disulfide bonds are essential to prevent half-molecule dissociation, whereas the labile hinge disulfide bonds favor half-molecule exchange in vivo for IgG4.
authors.affiliation: From Sanquin Research, 1066 CX Amsterdam, The Netherlands, and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, 1105 AZ, The Netherlands.
authors.forename: Theo
authors.initials: T
authors.lastname: Rispens
authors.forename: Anna M
authors.initials: AM
authors.lastname: Davies
authors.forename: Pleuni
authors.initials: P
authors.lastname: Ooijevaar-de Heer
authors.forename: Samira
authors.initials: S
authors.lastname: Absalah
authors.forename: Onno
authors.initials: O
authors.lastname: Bende
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Sutton
authors.forename: Gestur
authors.initials: G
authors.lastname: Vidarsson
authors.forename: Rob C
authors.initials: RC
authors.lastname: Aalberse
mesh_terms: D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000235:genetics; D007074:Immunoglobulin G / Q000737:chemistry* / Q000235:genetics; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000235:genetics
chemical_list: D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains
keywords: Antibodies; Antibody Engineering; Protein Structure; Structural Biology; Sulfhydryl; Thermodynamics