ABSTRACT
The interaction between interleukin-6 (IL-6) and IL-6 receptor (IL-6R) is the initial and most specific step in the IL-6 signaling pathway. Understanding its mechanism at the amino acid level is the basis for developing small IL-6-inhibiting molecules. We studied the human IL-6 (hIL-6)/hIL-6R binding interface by a combination of molecular modelling and site-directed mutagenesis. Our model suggests that the center of the interface between the two molecules consists of hydrophobic contacts predicted to account for most of the binding-free energy. These contacts can be regarded as a hydrophobic core shielded by hydrophilic residues that are also needed for recognition. Following this hypothesis, we altered in hIL-6 and hIL-6R residues predicted to reside in the contact region and to interact with each other. We studied the capacity of these mutants to form an IL-6/IL-6R complex and their ability to transduce the signal. This combined approach has led to the identification of certain residue-clusters in the binding interface and to a rational explanation of their specific interactions, suggesting therein a likely mechanism of complex formation. The results confirm the predictive model and strongly support our hypothesis. Comparison with other cytokines and their alpha-subunit receptors suggests that the structural location of certain binding sites are conserved.
Subject(s)
Antigens, CD/chemistry , Interleukin-6/chemistry , Protein Conformation , Receptors, Interleukin/chemistry , Amino Acid Sequence , Antibodies, Monoclonal/immunology , Antigens, CD/genetics , Antigens, CD/metabolism , Binding Sites , Chemical Phenomena , Chemistry, Physical , Humans , Interleukin-6/genetics , Interleukin-6/metabolism , Melanoma/pathology , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Binding , Receptors, Cell Surface/chemistry , Receptors, Interleukin/genetics , Receptors, Interleukin/metabolism , Receptors, Interleukin-6 , Sequence Alignment , Sequence Homology, Amino Acid , Signal Transduction , Structure-Activity Relationship , Tumor Cells, CulturedABSTRACT
We have designed a ribozyme (Rz) that cleaves human interleukin-6 (IL-6) mRNA in vivo. This Rz was tested in vitro, and was found to give expected size fragments. It was then incorporated into a mammalian expression vector containing the constitutive cytomegalovirus (CMV) immediate early promoter and transfected into human U amniotic cells (UAC). Cell clones that stably express this catalytic RNA have been obtained. Some of them displayed a marked reduction of tumor necrosis factor (TNF)-induced IL-6 production. Their reduced ability to express IL-6 was related to the amount of Rz they produced and to the extent of IL-6 mRNA cleavage as observed by a ribonuclease protection assay. These data provide a method to study further the role of IL-6 production in various biologic situations, and suggest the feasibility of developing Rzs directed against various cytokines to study their biologic role and mechanism of action.
