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. 1993 May 1;90(9):4067–4071. doi: 10.1073/pnas.90.9.4067

Saturation mutagenesis of the human interleukin 6 receptor-binding site: implications for its three-dimensional structure.

R Savino 1, A Lahm 1, M Giorgio 1, A Cabibbo 1, A Tramontano 1, G Ciliberto 1
PMCID: PMC46447  PMID: 8483922

Abstract

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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abdel-Meguid S. S., Shieh H. S., Smith W. W., Dayringer H. E., Violand B. N., Bentle L. A. Three-dimensional structure of a genetically engineered variant of porcine growth hormone. Proc Natl Acad Sci U S A. 1987 Sep;84(18):6434–6437. doi: 10.1073/pnas.84.18.6434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arcone R., Pucci P., Zappacosta F., Fontaine V., Malorni A., Marino G., Ciliberto G. Single-step purification and structural characterization of human interleukin-6 produced in Escherichia coli from a T7 RNA polymerase expression vector. Eur J Biochem. 1991 Jun 15;198(3):541–547. doi: 10.1111/j.1432-1033.1991.tb16048.x. [DOI] [PubMed] [Google Scholar]
  3. Bazan J. F. Haemopoietic receptors and helical cytokines. Immunol Today. 1990 Oct;11(10):350–354. doi: 10.1016/0167-5699(90)90139-z. [DOI] [PubMed] [Google Scholar]
  4. Bazan J. F. Neuropoietic cytokines in the hematopoietic fold. Neuron. 1991 Aug;7(2):197–208. doi: 10.1016/0896-6273(91)90258-2. [DOI] [PubMed] [Google Scholar]
  5. Bernstein F. C., Koetzle T. F., Williams G. J., Meyer E. F., Jr, Brice M. D., Rodgers J. R., Kennard O., Shimanouchi T., Tasumi M. The Protein Data Bank: a computer-based archival file for macromolecular structures. J Mol Biol. 1977 May 25;112(3):535–542. doi: 10.1016/s0022-2836(77)80200-3. [DOI] [PubMed] [Google Scholar]
  6. Brakenhoff J. P., Hart M., De Groot E. R., Di Padova F., Aarden L. A. Structure-function analysis of human IL-6. Epitope mapping of neutralizing monoclonal antibodies with amino- and carboxyl-terminal deletion mutants. J Immunol. 1990 Jul 15;145(2):561–568. [PubMed] [Google Scholar]
  7. Cunningham B. C., Henner D. J., Wells J. A. Engineering human prolactin to bind to the human growth hormone receptor. Science. 1990 Mar 23;247(4949 Pt 1):1461–1465. doi: 10.1126/science.247.4949.1461. [DOI] [PubMed] [Google Scholar]
  8. Cunningham B. C., Wells J. A. High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis. Science. 1989 Jun 2;244(4908):1081–1085. doi: 10.1126/science.2471267. [DOI] [PubMed] [Google Scholar]
  9. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Diederichs K., Boone T., Karplus P. A. Novel fold and putative receptor binding site of granulocyte-macrophage colony-stimulating factor. Science. 1991 Dec 20;254(5039):1779–1782. doi: 10.1126/science.1837174. [DOI] [PubMed] [Google Scholar]
  11. Fiorillo M. T., Cabibbo A., Iacopetti P., Fattori E., Ciliberto G. Analysis of human/mouse interleukin-6 hybrid proteins: both amino and carboxy termini of human interleukin-6 are required for in vitro receptor binding. Eur J Immunol. 1992 Oct;22(10):2609–2615. doi: 10.1002/eji.1830221021. [DOI] [PubMed] [Google Scholar]
  12. Fontaine V., Savino R., Arcone R., de Wit L., Brakenhoff J. P., Content J., Ciliberto G. Involvement of the Arg179 in the active site of human IL-6. Eur J Biochem. 1993 Feb 1;211(3):749–755. doi: 10.1111/j.1432-1033.1993.tb17605.x. [DOI] [PubMed] [Google Scholar]
  13. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  14. Hibi M., Murakami M., Saito M., Hirano T., Taga T., Kishimoto T. Molecular cloning and expression of an IL-6 signal transducer, gp130. Cell. 1990 Dec 21;63(6):1149–1157. doi: 10.1016/0092-8674(90)90411-7. [DOI] [PubMed] [Google Scholar]
  15. Hirano T. Interleukin 6 (IL-6) and its receptor: their role in plasma cell neoplasias. Int J Cell Cloning. 1991 May;9(3):166–184. doi: 10.1002/stem.5530090303. [DOI] [PubMed] [Google Scholar]
  16. Krüttgen A., Rose-John S., Dufhues G., Bender S., Lütticken C., Freyer P., Heinrich P. C. The three carboxy-terminal amino acids of human interleukin-6 are essential for its biological activity. FEBS Lett. 1990 Oct 29;273(1-2):95–98. doi: 10.1016/0014-5793(90)81059-w. [DOI] [PubMed] [Google Scholar]
  17. Leebeek F. W., Kariya K., Schwabe M., Fowlkes D. M. Identification of a receptor binding site in the carboxyl terminus of human interleukin-6. J Biol Chem. 1992 Jul 25;267(21):14832–14838. [PubMed] [Google Scholar]
  18. Lütticken C., Krüttgen A., Möller C., Heinrich P. C., Rose-John S. Evidence for the importance of a positive charge and an alpha-helical structure of the C-terminus for biological activity of human IL-6. FEBS Lett. 1991 May 6;282(2):265–267. doi: 10.1016/0014-5793(91)80491-k. [DOI] [PubMed] [Google Scholar]
  19. MacArthur M. W., Thornton J. M. Influence of proline residues on protein conformation. J Mol Biol. 1991 Mar 20;218(2):397–412. doi: 10.1016/0022-2836(91)90721-h. [DOI] [PubMed] [Google Scholar]
  20. McKay D. B. Response. Science. 1992 Jul 17;257(5068):412–413. doi: 10.1126/science.257.5068.412. [DOI] [PubMed] [Google Scholar]
  21. McKenzie A. N., Barry S. C., Strath M., Sanderson C. J. Structure-function analysis of interleukin-5 utilizing mouse/human chimeric molecules. EMBO J. 1991 May;10(5):1193–1199. doi: 10.1002/j.1460-2075.1991.tb08060.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Morrone G., Ciliberto G., Oliviero S., Arcone R., Dente L., Content J., Cortese R. Recombinant interleukin 6 regulates the transcriptional activation of a set of human acute phase genes. J Biol Chem. 1988 Sep 5;263(25):12554–12558. [PubMed] [Google Scholar]
  23. Sehgal P. B. Interleukin 6 in infection and cancer. Proc Soc Exp Biol Med. 1990 Nov;195(2):183–191. doi: 10.3181/00379727-195-43129d. [DOI] [PubMed] [Google Scholar]
  24. Shanafelt A. B., Kastelein R. A. Identification of critical regions in mouse granulocyte-macrophage colony-stimulating factor by scanning-deletion analysis. Proc Natl Acad Sci U S A. 1989 Jul;86(13):4872–4876. doi: 10.1073/pnas.86.13.4872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Smith L. J., Redfield C., Boyd J., Lawrence G. M., Edwards R. G., Smith R. A., Dobson C. M. Human interleukin 4. The solution structure of a four-helix bundle protein. J Mol Biol. 1992 Apr 20;224(4):899–904. doi: 10.1016/0022-2836(92)90457-u. [DOI] [PubMed] [Google Scholar]
  26. Snouwaert J. N., Leebeek F. W., Fowlkes D. M. Role of disulfide bonds in biologic activity of human interleukin-6. J Biol Chem. 1991 Dec 5;266(34):23097–23102. [PubMed] [Google Scholar]
  27. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  28. Van Snick J., Cayphas S., Vink A., Uyttenhove C., Coulie P. G., Rubira M. R., Simpson R. J. Purification and NH2-terminal amino acid sequence of a T-cell-derived lymphokine with growth factor activity for B-cell hybridomas. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9679–9683. doi: 10.1073/pnas.83.24.9679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Van Snick J. Interleukin-6: an overview. Annu Rev Immunol. 1990;8:253–278. doi: 10.1146/annurev.iy.08.040190.001345. [DOI] [PubMed] [Google Scholar]
  30. Yamasaki K., Taga T., Hirata Y., Yawata H., Kawanishi Y., Seed B., Taniguchi T., Hirano T., Kishimoto T. Cloning and expression of the human interleukin-6 (BSF-2/IFN beta 2) receptor. Science. 1988 Aug 12;241(4867):825–828. doi: 10.1126/science.3136546. [DOI] [PubMed] [Google Scholar]
  31. Zurawski S. M., Zurawski G. Identification of three critical regions within mouse interleukin 2 by fine structural deletion analysis. EMBO J. 1988 Apr;7(4):1061–1069. doi: 10.1002/j.1460-2075.1988.tb02914.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. de Vos A. M., Ultsch M., Kossiakoff A. A. Human growth hormone and extracellular domain of its receptor: crystal structure of the complex. Science. 1992 Jan 17;255(5042):306–312. doi: 10.1126/science.1549776. [DOI] [PubMed] [Google Scholar]

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