Mapping the CD4 binding site for human immunodeficiency virus by alanine-scanning mutagenesis

Area of Science:

• Virology
• Immunology
• Structural Biology

Background:

• Human immunodeficiency virus (HIV) infects mononuclear cells by binding its gp120 envelope glycoprotein to the CD4 receptor.
• The first extracellular domain of CD4 (V1) is sufficient for HIV binding and contains sequences similar to immunoglobulin complementarity-determining regions (CDRs).
• Previous studies indicated only the CDR2-like region and flanking amino acids of CD4 V1 were involved in gp120 binding.

Purpose of the Study:

• To investigate regions within CD4 V1 beyond the CDR2-like sequence that participate in gp120 binding.
• To identify amino acids critical for CD4 V1 conformation.
• To explore potential for enhancing CD4-based HIV-blocking molecules.

Main Methods:

• Alanine scanning mutagenesis of 64 amino acids in the CD4 V1 domain, including hydrophilic residues.
• Assessment of gp120 binding affinity to mutated CD4 V1 variants.
• Probing V1 structure using conformationally sensitive monoclonal antibodies.

Main Results:

• Mutations at four specific locations outside the CDR2-like sequence (amino acids 29, 59-64, 77-81, and 85) significantly impacted gp120 binding without altering V1 structure.
• The gp120-binding site on CD4 V1 is composed of discontinuous segments, not solely the CDR2-like sequence.
• Three amino acid substitutions were identified that individually increased gp120 binding affinity 1.7- to 2-fold, and 4.2-fold when combined.

Conclusions:

• The gp120-binding site on CD4 V1 involves multiple discontinuous regions, extending beyond the previously identified CDR2-like sequence.
• Specific amino acids are critical for maintaining the conformation of CD4 V1, with implications for domain folding.
• Enhancing the gp120 binding affinity of CD4-based molecules through targeted amino acid substitutions is a viable strategy for improving HIV-blocking capabilities.