Role of the C‐terminus in the activity, conformation, and stability of interleukin‐6

Larry D. Ward, Annet Hammacher, Jian‐Guo ‐G Zhang, Craig J. Morton, Richard J. Simpson, Janet Weinstock, Kiyoshi Yasukawa, Raymond S. Norton

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Abstract

Two murine interleukin‐6 (mIL‐6) variants were constructed using the polymerase chain reaction (PCR), one lacking the last five residues (183–187) at the C‐terminus (pMC5) and another with the last five residues of mIL‐6 substituted by the corresponding residues of human IL‐6 (pMC5H). The growth stimulatory activity of pMC5 on the mouse hybridoma cell line 7TD1 was <0.05% of mIL‐6, whereas pMC5H and mIL‐6 were equipotent. The loss of biological activity of pMC5 correlated with its negligible receptor binding affinity on 7TD1 cells, while the binding of pMC5H was comparable to that of mIL‐6. Both pMC5 and pMC5H, like mIL‐6, failed to interact with recombinant soluble human IL‐6 receptor when assayed by surface plasmon resonance‐based biosensor analysis. These studies suggest that the C‐terminal seven amino acids of human IL‐6, alone, do not define species specificity for receptor binding. A variety of biophysical techniques, as well as the binding of a conformational‐specific monoclonal antibody, indicated that the global fold of the mIL‐6 variants was similar to that of mIL‐6, although small changes in the NMR spectra, particularly for pMC5, were observed. Some of these changes involved residues widely separated in the primary structure. For instance, interactions involving Tyr‐22 were influenced by the C‐terminal amino acids suggesting that the N‐ and C‐termini of mIL‐6 are in close proximity. Equilibrium unfolding experiments indicated that pMC5 was 0.8 kcal/mol less stable than mIL‐6, whereas pMC5H was 1.4 kcal/mol more stable. These studies emphasize the structural importance of the C‐terminal amino acids of IL‐6 and suggest that truncation or mutation of this region could lead to small but significant alterations in other regions of the molecule.

Original languageEnglish
Pages (from-to)1472-1481
Number of pages10
JournalProtein Science
Volume2
Issue number9
DOIs
Publication statusPublished - 1993
Externally publishedYes

Keywords

  • biosensor
  • CD
  • interleukin‐6
  • mutants
  • NMR
  • receptor binding activity

Cite this

Ward, L. D., Hammacher, A., Zhang, JG. G., Morton, C. J., Simpson, R. J., Weinstock, J., Yasukawa, K., & Norton, R. S. (1993). Role of the C‐terminus in the activity, conformation, and stability of interleukin‐6. Protein Science, 2(9), 1472-1481. https://doi.org/10.1002/pro.5560020911