Folding and association of the antibody domain C(H)3: Prolyl isomerizationpreceeds dimerization

The simplest naturally occuring model system for studying immunoglobulin fo lding and assembly is the non-covalent homodimer formed by the C-terminal d omains (C(H)3) of the heavy chains of IgG. Here, we describe the structure of recombinant C(H)3 dimer as determined by X-ray crystallography and an an alysis of the folding pathway of this protein. Under conditions where prolyl isomerization does not contribute to the fold ing kinetics, formation of the beta-sandwich structure is the rate-limiting step. beta-Sheet formation of C(H)3 is a slow process, even compared to ot her antibody domains, while the subsequent association of the folded monome rs is fast. After long-time denaturation, the majority of the unfolded C(H) 3 molecules reaches the native state in two serial reactions, involving the re-isomerization of the Pro35-peptide bond to the cis configuration. The s pecies with the wrong isomer accumulate as a monomeric intermediate. Import antly, the isomerization to the correct cis configuration is the prerequisi te for dimerization of the C(H)3 domain. In contrast, in the Fab fragment o f the same antibody, prolyl isomerization occurs after dimerization demonst rating that within one protein, comprised of highly homologous domains, bot h the kinetics of beta-sandwich formation and the stage at which prolyl iso merization occurs during the folding process can be completely different. ( C) 1999 Academic Press.