Coiled coil miniprotein randomization on phage leads to charge pattern mimicry of the receptor recognition determinant of interleukin 5.
Phage display was used to identify sequences that mimic structural determinants in interleukin5 (IL5) for IL5 receptor recognition. A coiled coil stem loop (CCSL) miniprotein scaffold library was constructed with its turn region randomized and panned for binding variants against human IL5 receptor alpha chain (IL5Ralpha). Competition enzyme-linked immunosorbent assays identified CCSL-phage selectants for which binding to IL5Ralpha was competed by IL5. The most frequently selected and IL5-competed CCSL-phage contain charged residues Arg and Glu in their turn sequences, in this regard resembling a beta strand sequence in the 'CD turn' region, of IL5, that has been proposed to present a key determinant for IL5 receptor alpha chain recognition. The most dominant CCSL-phage selectant sequence, PVEGRV, contains a negative/positive charge pattern similar to that seen in the original CD turn. To test the relatedness of CCSL-phage selectant sequences to the IL5 receptor recognition epitope, PVEGRV was grafted into the sequence 87--92 of a monomeric IL5. The resulting IL5 variant, [(87)PVEGRV(92)]GM1, was able to bind to IL5Ralpha in biosensor assays, to elicit TF-1 cell proliferation and to induce STAT5 phosphorylation in TF-1 cells. The results help discern sequence patterns in the IL5 CD turn region which are key in driving receptor recognition and demonstrate the utility of CCSL miniprotein scaffold phage display to identify local IL5 mimetic sequence arrangements that may ultimately lead to IL5 antagonists.
Li
Chuanzhao
C
Department of Medicine, University of Pennsylvania School of Medicine, 909 Stellar Chance Laboratories, 422 Curie Blvd, Philadelphia PA 19104, USA.
Plugariu
Carmela G
CG
Bajgier
Joanna
J
White
John R
JR
Liefer
Kristin M
KM
Wu
Sheng-Jiun
SJ
Chaiken
Irwin
I
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D006801:Humans; D015848:Interleukin-5 / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D019151:Peptide Library; D011485:Protein Binding; D011487:Protein Conformation; D018123:Receptors, Interleukin / Q000737:chemistry* / Q000235:genetics; D053649:Receptors, Interleukin-5; D015398:Signal Transduction
D015848:Interleukin-5; D019151:Peptide Library; D018123:Receptors, Interleukin; D053649:Receptors, Interleukin-5