Poster: CD4 cis-dimer protein significantly enhances binding

The CD4 molecule on the T-cell surface can present as monomer and dimer forms. Dimeric CD4 can be important for T-cell activation. As the primary receptor of HIV, the interaction between CD4 and HIV envelope glycoprotein (Env) plays a critical role in mediating HIV infection. Env binds to the CD4 ectodomain (ECD) which contains 4 Ig-like subdomains (4D). To better mimic the native dimer conformation and quaternary structure, we designed a novel soluble CD4 cis-dimer with ECD-4D fused to a dimer motif at the C-terminus. The CD4 dimer protein was expressed/purified from HEK293T cells and evaluated by multiple tests. CD4 dimer binding potency to Env proteins from various HIV-1 subtypes increased on average by >10 fold as measured by ELISA and had stronger binding affinity and broader dynamic range as measured by BLI, compared to the CD4 monomer protein. The CD4 dimer also bound well to the CD4 binding site (CD4bs) resurfaced core RSC3. The gp120-D368R CD4bs mutation diminished the binding to the CD4 dimer. The CD4 dimer could also bind to a CD4-specific therapeutic mAb and IgG in patient samples, with better activities compared to the monomer. AlphaFold 2 predicted CD4 cisdimer structure is well-superimposed with known CD4 crystal structures. These findings imply that the soluble CD4 cis-dimer protein is in the desirable conformation, resulting in an increased binding to HIV-1 Env. This novel CD4 dimer can be a very useful molecule for HIV and immunology research.