Identification and structural analysis of residues in the V1 region of CD4 involved in interaction with human immunodeficiency virus envelope glycoprotein gp120 and class II major histocompatibility complex molecules.

The human CD4 molecule binds both human immunodeficiency virus envelope protein gp120 and class II 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 gp120, to interact with class II MHC, to enhance T-cell activation, and to bind a panel of anti-CD4 antibodies. The mutation Q40P (Gln40----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 gp120 and class II MHC binding as well as the ability to enhance T-cell activation. In contrast, the mutation P48S affected neither gp120 binding, nor class II MHC binding, nor T-cell activation. We conclude that within this region the binding sites for gp120 and for class II MHC molecules overlap and that amino acids Phe43 and Gly47 comprise an intimate part of both 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.

Overproduction and dissection of proteins by the expression-cassette polymerase chain reaction.

We report an efficient, general approach for the construction of protein-overproducing strains of Escherichia coli. The method, expression-cassette polymerase chain reaction (ECPCR), allows the insertion of virtually any contiguous coding sequence between sequences that direct high-level protein biosynthesis in E. coli. The gene expression cassettes obtained by ECPCR are inserted into a regulated overexpression plasmid, and the resulting construct is used to transform E. coli. By effecting simultaneous 5' and 3' modification of a coding sequence, ECPCR permits the facile generation of mutant proteins having N- and/or C-terminal truncations. The method is a highly efficient way to dissect a multidomain protein into its component domains. The efficiency of the ECPCR approach is demonstrated in this study by construction of permuted overexpression vectors for the first two extracellular domains of the human CD4 protein.