ROLES OF DISULFIDE BONDS IN RECOMBINANT HUMAN INTERLEUKIN-6 CONFORMATION

Human IL-6 has two disulfide bonds linking Cys(45) to Cys(51) and Cys( 74) to Cys(84), respectively. Previous site-directed mutagenesis studi es have demonstrated that the Cys(74)-Cys(84) bond is essential for fu ll biological and receptor binding activities. To address the structur al importance of these disulfide bonds in the formation and stabilizat ion of IL-6 secondary and tertiary structures, we have generated a pan el of disulfide bond-deficient rIL-6 analogs both by chemical reductio n and alkylation as well as by site-directed mutagenesis. Conformation al changes affecting these rIL-6 analogs were probed by circular dichr oism spectroscopy, as well as reactivity with monoclonal antibodies, a nd correlated with changes in biological activities. We have shown tha t the first disulfide bridge (Cys(45)-Cys(51)) is highly sensitive to reduction and, therefore, more solvent-exposed or less thermodynamical ly stable. Contrary to previous reports, this bridge contributes, alth ough minimally, to the full biological activity of the cytokine. Howev er, no significant changes in secondary or tertiary structures were ob served upon removal of this bond. In marked contrast, analogs lacking the disulfide bridge between Cys(74) and Cys(84) exhibited as little a s 0.5% and 0.05% wild-type biological and receptor binding activities, respectively. These dramatic changes correlated with a slight reducti on in alpha-helical content and a decreased reactivity with the neutra lizing monoclonal antibody mAb8 which recognizes a conformational epit ope associated with the active site. Our results suggest that the seco nd disulfide bridge plays a critical role in maintaining the spatial r elationship between the putative IL-6 A and D helices.