SATURATION MUTAGENESIS OF THE HUMAN INTERLEUKIN-6 RECEPTOR-BINDING SITE - IMPLICATIONS FOR ITS 3-DIMENSIONAL STRUCTURE

Interleukin 6 is a 184-aa polypeptide postulated to belong to the clas s of helical cytokines. We built a three-dimensional model of human in terleukin 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-macrop hage colony-stimulating factor. The resulting model is a bundle of fou r alpha-helices and suggests possible alternative conformations for th e 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) F EBS Lett. 282, 265-267]. Therefore, we generated a large collection of single-amino acid variants in residues 175-181. Analysis of their bio logical 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 th at this region does not assume an alpha-helical conformation. Remarkab ly, we also found a mutant with receptor affinity and biological activ ity much higher than wild type; the potential therapeutical value of t his finding is discussed.