Mapping the CD4 binding site for human immunodeficiency virus by alanine-scanning mutagenesis.
Infection of mononuclear cells by human immunodeficiency virus (HIV) begins with binding of the viral envelope glycoprotein, gp120, to its receptor, CD4. CD4 contains four extracellular immunoglobulin-like domains, the first of which (V1) is sufficient for HIV binding. V1 contains three sequences homologous to the antigen-complementarity-determining regions (CDR1 to -3) of immunoglobulin variable domains. While all three immunoglobulin CDRs are involved in antigen binding, only amino acids within and flanking the CDR2-like region of CD4 have been shown previously to be involved in gp120 binding. To investigate whether other regions in V1 take part in gp120 binding, we substituted alanine for each of 64 amino acids, including all of the hydrophilic residues in this domain. Mutations at four locations outside the CDR2-like sequence (amino acids 29, 59-64, 77-81, and 85) markedly affected gp120 binding, but not the overall structure of V1 as probed with eight conformationally sensitive monoclonal antibodies. Thus, the gp120-binding site of CD4 is not limited to the CDR2-like sequence and consists of several discontinuous segments. Several amino acids were identified that are critical for the conformation of V1; the importance of these residues suggests some differences in the folding of this domain compared to immunoglobulin variable domains. Three amino acid substitutions were found that increase the affinity for gp120 significantly (1.7- to 2-fold individually and 4.2-fold when combined), suggesting that it may be possible to improve the HIV-blocking ability of CD4-based molecules by increasing their gp120 binding affinity.
Department of Molecular Biology, Genentech, South San Francisco, CA 94080.
A
A
Ashkenazi
L G
LG
Presta
S A
SA
Marsters
T R
TR
Camerato
K A
KA
Rosenthal
B M
BM
Fendly
D J
DJ
Capon
D000409:Alanine; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D015704:CD4 Antigens / Q000235:genetics*; D002460:Cell Line; D003196:Computer Graphics; D006678:HIV / Q000276:immunology*; D015699:HIV Envelope Protein gp120 / Q000276:immunology; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D014162:Transfection
D000911:Antibodies, Monoclonal; D015704:CD4 Antigens; D015699:HIV Envelope Protein gp120; D007074:Immunoglobulin G; D000409:Alanine