Structure-function analysis of the C-terminal segment of human interleukin-6.

It has been hypothesized that interleukin-6 (IL-6) and granulocyte-colony-stimulating factor (G-CSF) may fold as four-alpha-helix bundle proteins. To probe the functional role of the putative fourth helical segment of IL-6 (D-helix), a chimeric IL-6/G-CSF analog containing the predicted D-helix of G-CSF as well as a panel of IL-6 D-helix point mutants were analyzed for their respective secondary structure, antigenicity, and receptor binding and biological activities. The putative D-helix of IL-6 could not be replaced by its G-CSF counterpart in spite of their high degree of similarity and thus is indispensable for the antigenic and functional integrity of the IL-6 receptor binding site. Conversely, the grafting of the G-CSF D-helix did not confer any G-CSF activity to IL-6. A synthetic helical peptide containing the IL-6 D-helix was inactive, even when mixed with or linked to a peptide from the A-helix known to be involved in the active site. However, the conserved residues F173, R179, and R182 found in the D-helices of both IL-6 and G-CSF critically contribute to the architecture of the IL-6 active site. Indeed, mutation of F173 or R179 markedly affected IL-6 receptor binding and biological activities, but not the conformation of a major neutralization epitope. Furthermore, substitution of R182 resulted in a significant unfolding of the D-helix accompanied by a drastic loss in IL-6 antigenicity and functional activities. Nevertheless, residues other than F173, R179, and R182 also contribute to IL-6 specificity.