Roles of disulfide bonds in recombinant human interleukin 6 conformation.
Human IL-6 has two disulfide bonds linking Cys45 to Cys51 and Cys74 to Cys84, respectively. Previous site-directed mutagenesis studies have demonstrated that the Cys74-Cys84 bond is essential for full biological and receptor binding activities. To address the structural importance of these disulfide bonds in the formation and stabilization of IL-6 secondary and tertiary structures, we have generated a panel of disulfide bond-deficient rIL-6 analogs both by chemical reduction and alkylation as well as by site-directed mutagenesis. Conformational changes affecting these rIL-6 analogs were probed by circular dichroism spectroscopy, as well as reactivity with monoclonal antibodies, and correlated with changes in biological activities. We have shown that the first disulfide bridge (Cys45-Cys51) is highly sensitive to reduction and, therefore, more solvent-exposed or less thermodynamically stable. Contrary to previous reports, this bridge contributes, although minimally, to the full biological activity of the cytokine. However, no significant changes in secondary or tertiary structures were observed upon removal of this bond. In marked contrast, analogs lacking the disulfide bridge between Cys74 and Cys84 exhibited as little as 0.5% and 0.05% wild-type biological and receptor binding activities, respectively. These dramatic changes correlated with a slight reduction in alpha-helical content and a decreased reactivity with the neutralizing monoclonal antibody mAb8 which recognizes a conformational epitope associated with the active site. Our results suggest that the second disulfide bridge plays a critical role in maintaining the spatial relationship between the putative IL-6 A and D helices.
Rock
F L
FL
Department of Biochemistry, University of Toronto, Ontario, Canada.
Li
X
X
Chong
P
P
Ida
N
N
Klein
M
M
D000478:Alkylation; D000595:Amino Acid Sequence; D001402:B-Lymphocytes / Q000187:drug effects / Q000276:immunology; D001681:Biological Assay; D002460:Cell Line; D002942:Circular Dichroism; D004220:Disulfides; D005822:Genetic Vectors; D019791:Guanidine; D006146:Guanidines; D006801:Humans; D015850:Interleukin-6 / Q000031:analogs & derivatives / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D007700:Kinetics; D008213:Lymphocyte Activation; D008961:Models, Structural; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D015183:Restriction Mapping; D013816:Thermodynamics
D004220:Disulfides; D006146:Guanidines; D015850:Interleukin-6; D010446:Peptide Fragments; D011994:Recombinant Proteins; D019791:Guanidine