Phage randomization in a charybdotoxin scaffold leads to CD4-mimetic recognition motifs that bind HIV-1 envelope through non-aromatic sequences.
Binding of HIV-1 gp120 to T-cell receptor CD4 initiates conformational changes in the viral envelope that trigger viral entry into host cells. Phage epitope randomization of a beta-turn loop of a charybdotoxin-based miniprotein scaffold was used to identify peptides that can bind gp120 and block the gp120-CD4 interaction. We describe here the display of the charybdotoxin scaffold on the filamentous phage fUSE5, its use to construct a beta-turn library, and miniprotein sequences identified through library panning with immobilized Env gp120. Competition enzyme-linked immunosorbent assay (ELISA) identified high-frequency phage selectants for which specific gp120 binding was competed by sCD4. Several of these selectants contain hydrophobic residues in place of the Phe that occurs in the gp120-binding beta-turns of both CD4 and previously identified scorpion toxin CD4 mimetics. One of these selectants, denoted TXM[24GQTL27], contains GQTL in place of the CD4 beta-turn sequence 40QGSF43. TXM[24GQTL27] peptide was prepared using solid-phase chemical synthesis, its binding to gp120 demonstrated by optical biosensor kinetics analysis and its affinity for the CD4 binding site of gp120 confirmed by competition ELISA. The results demonstrate that aromatic-less loop-containing CD4 recognition mimetics can be formed with detectable envelope protein binding within a beta-turn of the charybdotoxin miniprotein scaffold. The results of this work establish a methodology for phage display of a charybdotoxin miniprotein scaffold and point to the potential value of phage-based epitope randomization of this miniprotein for identifying novel CD4 mimetics. The latter are potentially useful in deconvoluting structural determinants of CD4-HIV envelope recognition and possibly in designing antagonists of viral entry.
Li
C
C
Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, USA.
Dowd
C S
CS
Zhang
W
W
Chaiken
I M
IM
D001483:Base Sequence; D001665:Binding Sites / Q000502:physiology; D015704:CD4 Antigens / Q000378:metabolism*; D018999:Charybdotoxin / Q000378:metabolism*; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000378:metabolism; D015699:HIV Envelope Protein gp120 / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism*; D019151:Peptide Library; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism
D015704:CD4 Antigens; D000939:Epitopes; D015699:HIV Envelope Protein gp120; D010446:Peptide Fragments; D019151:Peptide Library; D010455:Peptides; D018999:Charybdotoxin