The carboxy-terminal region of human interleukin-5 is essential for maintenance of tertiary structure but not for dimerization

J Protein Chem. 1996 Jul;15(5):491-9. doi: 10.1007/BF01886857.

Abstract

The C-terminal region of interleukin-5 has previously been suggested to be important for biological activity [Mackenzie et al., (1991), Mol. Immunol. 28, 155-158; Kodama et al. (1991), Biochem. Biophys. Res. Commun. 178, 514-519]. We have investigated this region by making a series of truncation mutants. The proteins were expressed in Escherichia coli, purified from inclusion bodies, and were able to refold with the disulfide homodimeric topology typical of interleukin-5. Analysis of the truncated carboxy-terminal proteins in an interleukin-5-dependent proliferation assay on TF-1 cells showed a rapid loss of activity as the C-terminal was shortened by more than two amino acids. This loss of biological activity correlated with a drop in binding affinity to both the alpha chain of the receptor and the high-affinity complex consisting of the alpha and beta subunits. Analysis of the proteins by 1H-NMR showed that the truncated mutants have higher exchange rates with solvent, indicating a less rigid structure. The carboxy-terminal region is therefore necessary to maintain the stability of the four-helix bundle and to orient correctly the important residues of the fourth helix. Inspection of the structure determined by X-ray crystallography shows that Trp-110 acts as the major residue in anchoring the fourth helix.

MeSH terms

  • Amino Acid Sequence
  • Cell Division
  • Crystallography, X-Ray
  • Dimerization
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Humans
  • Interleukin-5 / chemistry*
  • Interleukin-5 / genetics
  • Interleukin-5 / metabolism
  • Interleukin-5 / pharmacology
  • Magnetic Resonance Spectroscopy
  • Mutagenesis, Site-Directed
  • Protein Structure, Tertiary*
  • Receptors, Interleukin / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Recombinant Proteins / pharmacology
  • Sequence Deletion
  • Structure-Activity Relationship
  • Tumor Cells, Cultured

Substances

  • Interleukin-5
  • Receptors, Interleukin
  • Recombinant Proteins