The carboxy-terminal region of human interleukin-5 is essential for maintenance of tertiary structure but not for dimerization.
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.
Glaxo Institute for Molecular Biology, Geneva, Switzerland.
A E
AE
Proudfoot
S C
SC
Brown
P
P
Graber
F
F
Talabot
C Y
CY
Arod
M C
MC
Peitsch
M
M
Banks
M
M
McKinnon
R
R
Solari
T N
TN
Wells
D000595:Amino Acid Sequence; D002455:Cell Division; D018360:Crystallography, X-Ray; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015848:Interleukin-5 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D018123:Receptors, Interleukin / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
D015848:Interleukin-5; D018123:Receptors, Interleukin; D011994:Recombinant Proteins