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

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Abstract
The C-terminal region of interleukin-5 has previously been suggested to be important for biological activity [Mackenzieet al., (1991),Mol. Immunol. 28, 155–158; Kodamaet 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 inEscherichia 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 theα chain of the receptor and the high-affinity complex consisting of theα andβ subunits. Analysis of the proteins by1H-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.