Folding topologies of human interleukin-6 and its mutants as studied by NMR spectroscopy

To understand the structure-function relationship in the human interleukin-6 (IL-6) system, comparative studies were performed on the basis of NMR data obtained using the wild-type IL-6 and six mutants. In each of the six mutants, either Leu152, Leu159, Leu166, Leu168, Leu175, or Leu182, which exist in the C-terminal receptor-binding region, was substituted with Val. The resonance assignments of Val, Ile, Leu, and Phe residues were made by using specific double-labeling and site-specific mutagenesis strategies. On the basis of chemical shift and NOE data collected for six IL-6 mutants and those for the wild-type IL-6, we analyzed the structural changes induced by the substitution of each of the six Leu residues. The NMR data showed that substitution of Leu182 with Val (L182V) induced no structural change in IL-6, suggesting that Leu182 is located on the surface of the IL-6 molecule. A significant decrease in receptor-binding activity was observed in the L182V mutant. It was concluded that the side chain of Leu182 is directly involved in receptor binding. Substitution of Leu175 with Val (L175V) was shown to induce a significant structural change in IL-6. The NMR data are discussed on the basis of the location of four helix elements and an up-up-down-down helix topology of the predicted structure of IL-6 [Bazan, J.F. (1991) Neuron 7, 197-208]. It is possible that helix D bent more sharply toward helix B in the L175V mutant than in the wild-type IL-6 to maintain a closely packed and solvent-inaccessible core formed in the mutated region. It is suggested that the kink of helix D is related to the decrease in receptor-binding activity in the L175V mutant. On the basis of the observed NOE network, the folding topology of IL-6 was analyzed. A comparison of the folding topology of IL-6 with that of human granulocyte colony-stimulating factor determined by X-ray crystallography [Hill, C. P., Osslund, T. D., & Eisenberg, D. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 5167-5171] indicated that IL-6 has a significant similarity of folding topology to that of human granulocyte colony-stimulating factor.