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.
Li
X
X
Connaught Centre for Biotechnology Research, Willowdale, Ontario, Canada.
Rock
F
F
Chong
P
P
Cockle
S
S
Keating
A
A
Ziltener
H
H
Klein
M
M
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001665:Binding Sites; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002942:Circular Dichroism; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry; D006801:Humans; D007075:Immunoglobulin M / Q000378:metabolism; D015850:Interleukin-6 / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D017354:Point Mutation; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D018123:Receptors, Interleukin / Q000378:metabolism; D019947:Receptors, Interleukin-6; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D007075:Immunoglobulin M; D015850:Interleukin-6; D010446:Peptide Fragments; D018123:Receptors, Interleukin; D019947:Receptors, Interleukin-6; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor