Kinetic interaction analysis of human interleukin 5 receptor alpha mutants reveals a unique binding topology and charge distribution for cytokine recognition.
Human interleukin 5 receptor alpha (IL5Ralpha) comprises three fibronectin type III domains (D1, D2, and D3) in the extracellular region. Previous results have indicated that residues in the D1D2 domains are crucial for high affinity interaction with human interleukin 5 (IL5). Yet, it is the D2D3 domains that have sequence homology with the classic cytokine recognition motif that is generally assumed to be the minimum cytokine-recognizing unit. In the present study, we used kinetic interaction analysis of alanine-scanning mutational variants of IL5Ralpha to define the residues involved in IL5 recognition. Soluble forms of IL5Ralpha variants were expressed in S2 cells, selectively captured via their C-terminal V5 tag by anti-V5 tag antibody immobilized onto the sensor chip and examined for IL5 interaction by using a sandwich surface plasmon resonance biosensor method. Marked effects on the interaction kinetics were observed not only in D1 (Asp(55), Asp(56), and Glu(58)) and D2 (Lys(186) and Arg(188)) domains, but also in the D3 (Arg(297)) domain. Modeling of the tertiary structure of IL5Ralpha indicated that these binding residues fell into two clusters. The first cluster consists of D1 domain residues that form a negatively charged patch, whereas the second cluster consists of residues that form a positively charged patch at the interface of D2 and D3 domains. These results suggest that the IL5 x IL5Ralpha system adopts a unique binding topology, in which the cytokine is recognized by a D2D3 tandem domain combined with a D1 domain, to form an extended cytokine recognition interface.
Ishino
Tetsuya
T
Biochemistry Department and A. J. Drexel Institute of Basic and Applied Protein Science, College of Medicine, Drexel University, Philadelphia, Pennsylvania 19102, USA.
Pasut
Gianfranco
G
Scibek
Jeffery
J
Chaiken
Irwin
I
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D015374:Biosensing Techniques; D006801:Humans; D015848:Interleukin-5 / Q000378:metabolism*; D053666:Interleukin-5 Receptor alpha Subunit; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D025941:Protein Interaction Mapping; D018123:Receptors, Interleukin / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
C485918:IL5RA protein, human; D015848:Interleukin-5; D053666:Interleukin-5 Receptor alpha Subunit; D018123:Receptors, Interleukin