Design, expression and characterization of 2DLT. A) The schematic view of the interactions between the NHR and CHR of gp41 and C-peptides. The dashed lines between NHR and CHR indicate the interaction between the residues located at the e and g positions in the NHR and the a and d positions in the CHR. The sequences of peptide T20, C34 and T1144 are shown, and the pocket-forming sequence (aa 565–581) in the NHR and pocket-binding domain (aa 628–635) in the CHR are colored in red and green, respectively. B) Schematic view of the 2DLT and D1D2 molecules (not drawn to scale). The D1D2 of CD4 (aa 1–185) and T1144 are connected by a 35-mer of linker (GGGGS)7. C) A diagram showing the predicted structure of 2DLT interacting with gp120. The complex containing the gp120 core (yellow), the two-domain sCD4 (D1D2) (red) and the T1144 (green), was derived from the coordinates of the published X-ray crystallographic complex  using the Pymol program (http://pymol.sourceforge.net). D) The model of CD4-induced gp41 PFI. Soluble CD4 (sCD4) or CD4 D1D2 domains bind to HIV-1 Env surface subunit gp120, resulting in the formation of the gp41 PFI with the exposed grooves on the NHR-trimer, which is a target for HIV-1 inactivator. E) Characterization of 2DLT. The soluble recombinant proteins 2DLT and D1D2 were expressed in E. coli using the PDI-chaperone expression system and analyzed by SDS-PAGE (a); Western blot using anti-CD4 polyclonal antibody (b); anti-T1144 polyclonal antibody (c); and by ELISA using anti-CD4 pAb T4-4, a conformation-dependent mAb Sim.4 and anti-T1144 pAbs F). The data are representative of results from three similar experiments performed in triplicate (means ± SD).