β-turn Phe in HIV-1 env binding site of CD4 and CD4 mimetic miniprotein enhances env binding affinity but is not required for activation of co-receptor/17b site

HIV-1 enters a host cell after an initial interaction between viral envelope glycoprotein gp120 and cell surface receptor CD4, followed by a second interaction between gp120 and a cell surface chemokine receptor. CD4 residue Phe43 makes a significant contribution to the high-affinity interaction between CD4 and env. We and others have used scorpion toxin scaffolds to display and examine CD4 epitopes used for gp120 recognition. These peptides, which have a β-turn Phe that acts as a Phe43 surrogate, compete with CD4 for gp120 binding and enhance the binding of gpl20 to 17b, an antibody that binds near the co-receptor-binding site. In the current study, a scyllatoxin-scaffolded peptide, identified via phage epitope randomization and lacking a β-turn Phe (indeed, containing no aromatic residues), was shown to behave in a distinctly CD4-like manner. This peptide, denoted [₂₀EGLV₂₃]ST, not only competed with CD4 for gpl20 binding, but also enhanced the binding of gpl20 to 17b. Quantitatively, an [₂₀EGLV₂₃]ST-gp120 complex exhibited the same 17b binding on-rate as a complex of gpl20 with [₂₀AGSF₂₃]ST, a scyllatoxin-based CD4 mimetic peptide containing a β-turn Phe. In view of this result, we examined the role of Phe43 in CD4 itself by comparing F43V D1D2 sCD4 versus D1D2 sCD4 Like the peptides, a close similarity was observed for both Phe43 and Phe43-1ess D1D2 sCD4s in enhancing gpl20 binding to 17b. Further, when examined for their ability to enhance binding of gpl20 to CCR5⁺ cells, [₂₀EGLV₂₃]ST and [₂₀AGSF₂₃]ST were found to have the same efficacy, after correcting for the difference in their gpl20 affinities. These results show that, although Phe43 is important in maintaining high affinity in gp120 ligands, the aromatic residue is not necessary for triggering the conformational isomerization in gpl20 that results in formation or exposure of the binding sites for the 17b antibody and the CCR5 receptor.