An efficient phage plaque screen for the random mutational analysis of the interaction of HIV-1 gp120 with human CD4.
A lambda phage expression methodology was adapted to dissect protein/ligand interactions efficiently through the creation and rapid screening of large numbers of mutants. Here we describe the method and its specific application to the interaction between the external envelope glycoprotein of the human immunodeficiency virus (HIV-1), gp120, and the human cell surface protein CD4. Random substitutions were introduced throughout the gp120 binding region (amino acids 38-62) in the amino-terminal domain of CD4 by oligonucleotide mutagenesis. These mutations were expressed within phage plaques and directly screened for their effect on binding of gp120 using a modified phage plaque lift procedure. Plaques showing increased, decreased, and no effect on binding were identified and mutations were verified by sequence analysis. In this manner, 25 unique mutations were identified that altered CD4 binding to gp120. A new site was identified at which mutations reduced binding to gp120 and several novel amino acid substitutions were defined at sites previously implicated in binding. Of particular interest, this in vitro genetic approach identified a mutation which significantly increased binding to gp120. The phenotypes of several of these mutants were further characterized by quantitative measurement of their binding affinity. The results confirmed the accuracy of the phenotypic selection and demonstrated that the sensitivity of the system allowed detection of a 3-4-fold increase or decrease in affinity. In the context of the recently determined atomic structure of CD4, these results further implicate residues in the CDR2-like region and in an adjacent loop in recognition of gp120. This methodology should be generally applicable to other high affinity protein/ligand interactions that are compatible with expression in Escherichia coli.
Tsui
P
P
Biopharmaceuticals Research and Development, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406.
Sweet
R W
RW
Sathe
G
G
Rosenberg
M
M
D000595:Amino Acid Sequence; D010582:Bacteriophage lambda / Q000235:genetics*; D001667:Binding, Competitive; D015704:CD4 Antigens / Q000378:metabolism*; D005814:Genes, Viral; D005822:Genetic Vectors; D015699:HIV Envelope Protein gp120 / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism* / Q000502:physiology; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D011863:Radioimmunoassay; D010948:Viral Plaque Assay / Q000379:methods*
D015704:CD4 Antigens; D015699:HIV Envelope Protein gp120