Analysis of the contact sites on the CD4 molecule with class II MHC molecule: co-ligand versus co-receptor function

The CD4 molecule interacts with the alpha2 and beta2 domains of the MHC class II molecules. However, the class II contact sites on CD4 are less clear. Involvement of different regions throughout D1, D2, and D3 domains have been suggested. To further delineate the class II MHC contact sites on CD4, a crystal structure-informed mutagenesis was performed. Alanine scan mutants were generated for exposed residues located throughout D1 and the FG loop of D2, and in the "hinge-like" region, a short and flexible region between D2 and D3. Mutants were tested in a co-ligand (Dd stimulation) and a co-receptor (staphylococcal enterotoxin B stimulation) assay. In the co-ligand assay, TCR and CD4 interact with two distinct ligands (Dd or HLA-DR), while in the co-receptor assay both molecules interact with the same ligand, namely HLA-DR. Results show that residues from both lateral faces of D1 and the FG loop of D2 are implicated in interaction with class II, although a bigger surface of CD4 was involved in co-receptor compared with co-ligand function. The potential involvement of residues on both the top and two side faces of CD4 is consistent with a bivalent model, which involves the interaction between a single CD4 and two class II molecules. Alternatively, our results can be interpreted with a model of a specifically organized CD4 and/or class II oligomerization event. Finally, results from the hinge region mutants revealed a very important role in maintaining the overall structural integrity of CD4, its topology, and function.