A discontinuous eight-amino acid epitope in human interleukin-3 binds the alpha-chain of its receptor.
We have previously reported that, within the first helix of human interleukin (IL)-3, residues Asp21 and Glu22 are important for interaction with the alpha- and beta-chains of the IL-3 receptor, respectively. In order to define more precisely the sites of interaction with the receptor, we have performed molecular modeling of the helical core of IL-3 and single amino acid substitution mutagenesis of residues predicted to lie on the surfaces of the A, C, and D helices. The resulting analogues were characterized for their abilities to stimulate proliferation of TF-l cells and for binding to the high affinity (alpha- and beta-chain; IL-3Ralpha/Rbeta) or low affinity (alpha-chain alone; IL-3Ralpha) IL-3 receptor. We found that in addition to Asp21, residues Ser17, Asn18, and Thr25 within the A helix and Arg108, Phe113, Lys116, and Glu119 within the D helix of IL-3 were important for biological activity. Analysis of their binding characteristics revealed that these analogues were deficient in binding to both the IL-3Ralpha/Rbeta and the IL-3Ralpha forms of the receptor, consistent with a selective impairment of interaction with IL-3Ralpha. Molecular modeling suggests that these eight amino acid residues are adjacent in the tertiary structure, consistent with a discontinuous epitope interacting selectively with IL-3Ralpha. On the other hand, Glu22 of IL-3 was found to interact preferentially with the beta-chain with bulky and positively charged substitutions causing greater than 10,000-fold reduction in biological activity. These results show fundamental differences between IL-3 and granulocyte-macrophage colony-stimulating factor in the structural basis for recognition of their receptors that has implications for the construction of novel analogues and our understanding of receptor activation.
Bagley
C J
CJ
Division of Human Immunology, Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, Frome Road, Adelaide, South Australia 5001, Australia.
Phillips
J
J
Cambareri
B
B
Vadas
M A
MA
Lopez
A F
AF
D000595:Amino Acid Sequence; D001665:Binding Sites; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007377:Interleukin-3 / Q000737:chemistry* / Q000276:immunology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D016185:Receptors, Interleukin-3 / Q000378:metabolism*; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
D000939:Epitopes; D007377:Interleukin-3; D016185:Receptors, Interleukin-3; D016178:Granulocyte-Macrophage Colony-Stimulating Factor