Folding and association of the antibody domain CH3: prolyl isomerization preceeds dimerization.
The simplest naturally occurring model system for studying immunoglobulin folding and assembly is the non-covalent homodimer formed by the C-terminal domains (CH3) of the heavy chains of IgG. Here, we describe the structure of recombinant CH3 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 beta-sandwich structure is the rate-limiting step. beta-Sheet formation of CH3 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 CH3 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 CH3 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 beta-sandwich formation and the stage at which prolyl isomerization occurs during the folding process can be completely different.
Thies
M J
MJ
Institut für Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstrasse 4, Garching, 85747, Germany.
Mayer
J
J
Augustine
J G
JG
Frederick
C A
CA
Lilie
H
H
Buchner
J
J
D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002942:Circular Dichroism; D019281:Dimerization; D005470:Fluorometry; D007536:Isomerism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019696:Peptidylprolyl Isomerase; D011392:Proline / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry
D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D014364:Tryptophan; D011392:Proline; D019696:Peptidylprolyl Isomerase