Mutagenesis in the C-terminal region of human interleukin 5 reveals a central patch for receptor alpha chain recognition.
Cassette mutagenesis was used to identify side chains in human interleukin 5 (hIL-5) that mediate binding to hIL-5 receptor alpha chain (hIL-5R alpha). A series of single alanine substitutions was introduced into a stretch of residues in the C-terminal region, including helix D, which previously had been implicated in receptor alpha chain recognition and which is aligned on the IL-5 surface so as to allow the topography of receptor binding residues to be examined. hIL-5 and single site mutants were expressed in COS cells, their interactions with hIL-5R alpha were measured by a sandwich surface plasmon resonance biosensor method, and their biological activities were measured by an IL-5-dependent cell proliferation assay. A pattern of mutagenesis effects was observed, with greatest impact near the interface between the two four-helix bundles of IL-5, in particular at residues Glu-110 and Trp-111, and least at the distal ends of the D helices. This pattern suggests the possibility that residues near the interface of the two four-helix bundles in hIL-5 comprise a central patch or hot spot, which constitutes an energetically important alpha chain recognition site. This hypothesis suggests a structural explanation for the 1:1 stoichiometry observed for the complex of hIL-5 with hIL-5R alpha.
D000595:Amino Acid Sequence; D000818:Animals; D002522:Chlorocebus aethiops; D006801:Humans; D015848:Interleukin-5 / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D017510:Protein Folding; D018123:Receptors, Interleukin / Q000737:chemistry*; D053649:Receptors, Interleukin-5; D011994:Recombinant Proteins; D015398:Signal Transduction; D013329:Structure-Activity Relationship
D015848:Interleukin-5; D018123:Receptors, Interleukin; D053649:Receptors, Interleukin-5; D011994:Recombinant Proteins