The central objective of this research is to use structure-based mimetics to elucidate the topology of receptor recognition surfaces in interleukin 5 (IL5) and to advance mechanistic understanding of IL5 specifically and 4-helix bundle growth factor proteins generally. IL-5 is the major hematopoietin which stimulates the proliferation, migration and activation of eosinophils and has been implicated in the pathogenesis of diseases such as asthma. IL5 is a homodimeric protein, dominated by two 4-helix bundle conformational domains. It functions through a cell- surface receptor that contains an alpha chain that binds IL5 selectively and a more 'public' Beta chain for signal transduction. Available data for IL5 provide an important opportunity to use recognition site mimetics to decipher basic structural principles about how the 4-helix bundle framework in IL5 displays receptor binding epitopes for alpha and Beta chains. This project has several specific aims: (1) based on the topology of receptor recognition residues in hIL5, design chimeric cytokine hybrids of IL5, using both a 2-bundle framework (IL5 type) and 1-bundle framework (GM-CSF type), and compare their binding and structural properties, including testing the ability of IL5 to fold and function as a one 4- helix bundle monomer vs the naturally occuring 2-bundle dimer; (2) design and characterize de novo mimetics of IL5 by transplanting key helical and loop surfaces, as defined by topological mapping and hybrids, into structure-selected scaffolds including the recently developed coiled coil stem loop miniprotein; (3) design contact residue topology mimetics from CDRs of neutralizing anti-[IL5 receptor] monoclonal antibodies; (4) perform followup mutagenesis of hIL5, using a newly designed, biologically active single chain construct of hIL5 (scIL5), in order to verify the importance of residues or residue clusters determined to be important for receptor recognition by cytokine hybrids and de novo mimetics and also to construct advanced receptor binding site topology maps. Long term, this project will lead to a fundamental understanding of recognition epitopes within the structural architecture of IL5. It will yield models for how variations within the basic 4-helix bundle architecture, common to many growth factor proteins, trigger selective receptor recognition. And, it will provide mimetic strategies useful to design 4-helix bundle cytokine antagonists.
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