The human CD4 molecule binds both human immunodeficiency virus envelope protein gpl20 and class H major histocompatibility complex (MHC) molecules. We have studied a series of mutants in the region of amino acids 42-49 of CD4 for their ability to bind gpl20, to interact with class II MHC, to enhance T-cell activation, and to bind a panel of anti-CD4 antibodies. The mutation Q40P (Gln4O -Pro) and the deletion d42-49 were found to disrupt most antibody epitopes in the V1 domain of CD4, suggesting major conformational changes, whereas mutants F43L, G47R, and P48S retained the binding of most of the anti-CD4 antibodies tested. The mutants d42-49, Q40P, F43L, and G47R lost both gpl20 and class II MHC binding as well as the ability to enhance T-cell activation. In contrast, the mutation P48S affected neither gpl20 binding, nor class H MHC binding, nor T-ceUl activation. We conclude that within this region the binding sites for gpl20 and for class II MHC molecules overlap and that amino acids Phe43 and Gly47 comprise an intimate part ofboth binding sites.These observations are consistent with a three-dimensional model of the V1 domain of CD4 that was developed in order to understand the structural basis for binding to CD4.The CD4 molecule is a surface glycoprotein, expressed in T cells, that associates with the class II major histocompatibility complex (MHC) molecules on the surface of antigenpresenting cells (1-3). When the T-cell receptor (TCR) recognizes antigen in association with class II MHC molecules, the CD4 molecule binds the class II MHC molecule, enhancing a sometimes weak immune interaction (1, 2). The CD4 glycoprotein is also the receptor for the human immunodeficiency virus (HIV), binding the viral envelope glycoprotein gpl20 (4).The CD4 molecule is a single-chain 55-kDa protein with a 372-amino acid extracellular domain consisting of four immunoglobulin-like VJ (variable-joining) or CJ (constantjoining) regions, a 23-amino acid transmembrane domain, and a 38-amino acid cytoplasmic domain (5). The VJ-like (V1) domain of CD4 is 35% homologous to immunoglobulin K light-chain variable regions (6). Soluble forms of the V1 domain appear to block HIV infectivity and syncytium formation in vitro (7-9). Furthermore, epitope-loss mutants of CD4 identified amino acids in the area of residues 42-49 as important for syncytium formation (10). Homolog scanning mutational analysis identified a region between residues 42 and 55 that affects gpl20 binding (7,11). In addition, a much broader region was found to be involved in class II MHC binding compared to that seen affecting gp120 (12,13).Here we report a series of epitope-loss mutants of CD4, involving amino acids 42-49. These mutants were expressed in a T-cell hybridoma that is dependent on CD4 for efficient immune function. Binding of a panel of anti-CD4 antibodies was carried out to determine the effect on the conformation of the molecule. The effect of mutations on the binding of gp120 and class II MHC-bearing B cells, and on the ability of the mutant CD4 m...