Saturation mutagenesis of the human interleukin 6 receptor-binding site: implications for its three-dimensional structure.
Interleukin 6 is a 184-aa polypeptide postulated to belong to the class of helical cytokines. We built a three-dimensional model of human interleukin 6 based on the similarity of its hydrophobicity pattern with that of other cytokines and on the x-ray structure of growth hormone, interleukin 2, interleukin 4, interferon beta, and granulocyte-macrophage colony-stimulating factor. The resulting model is a bundle of four alpha-helices and suggests possible alternative conformations for the 9 C-terminal amino acids; in this region, the importance of Arg-182 and Met-184 for biological activity has been demonstrated [Lutticken, C., Kruttgen, A., Moller, C., Heinrich, P.C. & Rose-John, S. (1991) FEBS Lett. 282, 265-267]. Therefore, we generated a large collection of single-amino acid variants in residues 175-181. Analysis of their biological activity in two systems and the receptor binding properties of a subset of the mutants indicates that the entire region is involved in forming the receptor binding surface and supports the hypothesis that this region does not assume an alpha-helical conformation. Remarkably, we also found a mutant with receptor affinity and biological activity much higher than wild type; the potential therapeutical value of this finding is discussed.
Savino
R
R
Instituto di Ricerche di Biologia Molecolare, P. Angeletti, Pomezia, Rome, Italy.
Lahm
A
A
Giorgio
M
M
Cabibbo
A
A
Tramontano
A
A
Ciliberto
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001681:Biological Assay; D016207:Cytokines / Q000737:chemistry / Q000235:genetics; D006801:Humans; D015850:Interleukin-6 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011971:Receptors, Immunologic / Q000378:metabolism*; D019947:Receptors, Interleukin-6
D016207:Cytokines; D015850:Interleukin-6; D011971:Receptors, Immunologic; D019947:Receptors, Interleukin-6