Folding and association of the antibody domain C H3: prolyl isomerization preceeds dimerization

Abstract

The simplest naturally occuring model system for studying immunoglobulin folding and assembly is the non-covalent homodimer formed by the C-terminal domains (C H3) of the heavy chains of IgG. Here, we describe the structure of recombinant C H3 dimer as determined by X-ray crystallography and an analysis of the folding pathway of this protein. Under conditions where prolyl isomerization does not contribute to the folding kinetics, formation of the β-sandwich structure is the rate-limiting step. β-Sheet formation of C H3 is a slow process, even compared to other antibody domains, while the subsequent association of the folded monomers is fast. After long-time denaturation, the majority of the unfolded C H3 molecules reaches the native state in two serial reactions, involving the re-isomerization of the Pro35-peptide bond to the cis configuration. The species with the wrong isomer accumulate as a monomeric intermediate. Importantly, the isomerization to the correct cis configuration is the prerequisite for dimerization of the C H3 domain. In contrast, in the Fab fragment of the same antibody, prolyl isomerization occurs after dimerization demonstrating that within one protein, comprised of highly homologous domains, both the kinetics of β-sandwich formation and the stage at which prolyl isomerization occurs during the folding process can be completely different.